research article construction and immunogenicity of dna...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 318698 9 pageshttpdxdoiorg1011552013318698

Research ArticleConstruction and Immunogenicity of DNA VaccinesEncoding Fusion Protein of Porcine IFN-1205821 and GP5 Gene ofPorcine Reproductive and Respiratory Syndrome Virus

Luping Du1234 Bin Li2345 Kongwang He2345 Haibin Zhang1

Kehe Huang1 and Shaobo Xiao6

1 College of Veterinary Medicine Nanjing Agricultural University No 1 Wei-gang Nanjing 210095 China2 Institute of Veterinary Medicine Jiangsu Academy of Agricultural Sciences Nanjing Jiangsu Province 210014 China3 Key Laboratory of Animal Diseases Diagnostics and Immunology Ministry of Agriculture Nanjing Jiangsu Province 210014 China4National Center for Engineering Research of Veterinary Bio-Products Nanjing Jiangsu Province 210014 China5 Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses YangzhouJiangsu Province 225009 China

6 State Key Laboratory of Agricultural Microbiology Division of Animal Infectious Diseases College of Veterinary MedicineHuazhong Agricultural University Wuhan 430070 China

Correspondence should be addressed to Kongwang He kwh2003263net and Kehe Huang khhuangnjaueducn

Received 28 October 2013 Accepted 29 November 2013

Academic Editor Dongwan Yoo

Copyright copy 2013 Luping Du et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the catastrophic economic lossesin pig industry worldwide The commercial vaccines only provide a limited protection against PRRSV infection Thus the focusand direction is to develop safer and more effective vaccines in the research field of PRRS The immune modulators are beingconsidered to enhance the effectiveness of PRRSV vaccines IFN-1205821 belongs to type III interferon a new interferon family IFN-1205821 is an important cytokine with multiple functions in innate and acquired immunity In this study porcine IFN-1205821 (PoIFN-1205821)was evaluated for its adjuvant effects on the immunity of a DNA vaccine carrying the GP5 gene of PRRSV Groups of mice wereimmunized twice at 2-week interval with 100120583g of the plasmid DNA vaccine pcDNA31-SynORF5 pcDNA31-PoIFN-1205821-SynORF5and the blank vector pcDNA31 respectivelyThe results showed that pcDNA31-PoIFN-1205821-SynORF5 can significantly enhanceGP5-specific ELISA antibody PRRSV-specific neutralizing antibody IFN-120574 level and lymphocyte proliferation rather than the responsesinduced by pcDNA31-SynORF5Therefore type III interferon PoIFN-1205821 could enhance the immune responses of DNA vaccine ofPRRSV highlighting the potential value of PoIFN-1205821 as a molecular adjuvant in the prevention of PRRSV infection

1 Introduction

Porcine reproductive and respiratory syndrome (PRRS)characterized by severe reproductive failure in sows andrespiratory distress in piglets and growing pigs is one ofthe most economically significant viral diseases of swine [1ndash5] Since firstly reported in the United States in 1987 andin Europe in 1990 [6 7] PRRS has been gaining graduallyincreased attention because of its large-scale outbreak andtremendous losses in the global swine industry

PRRSV the causative agent of PRRS is a small envelopedsingle-stranded positive-sense RNA virus belonging to thefamily Arteriviridae The PRRSV genome with a size ofapproximately 15 kb contains 9 open reading frames (ORFs)ORFs 1a and 1b encoded for nonstructural proteins and ORF2ndash7 encoded for structural proteins [8ndash10] Among them theORF5 that encoded major envelope glycoprotein (GP5) isone of the key immunogenic proteins of PRRSV and is theleading target for the development of the genetic engineeringvaccines against PRRS [11ndash20] The modified GP5 which

2 BioMed Research International

used three methods to modify the PRRSV GP5 exhibitedsignificantly enhanced immunogenicity particularly in theability to induce neutralizing antibody responses and cel-lular immune responses compared to the native GP5 [21]Consequently this modified GP5 may be useful to facilitatethe development of the new generation of vaccines such asDNA vaccines live attenuated chimeric virus vaccines andlive virus-vectored vaccines against the highly pathogenicPRRSV in the future

Type III interferon a new interferon family was firstlyreported in 2003 and different from the types I and IIinterferon including IFN-1205821 IFN-1205822 and IFN-1205823 IFN-120572and IFN-120573 belonging to type I interferon were confirmedto be adjuvants to improve the vaccinesrsquo immune responses[22ndash24] In addition previous studies have shown that typeIII interferon has almost the same biological activity ofother interferons such as anti-viral antitumor and immuneregulation but when compared with type I interferon itsside effects are obviously little Thus the research on type IIIinterferon will play a role in promoting the control of animaldiseases and medical treatment of human disease

In view of the above information in this study weconstructed the DNA construct units encoding pcDNA31-PoIFN-1205821-SynORF5 and find that pcDNA31-PoIFN-1205821-SynORF5 could induce stronger cellular and humoralimmune responses than the responses induced by pcDNA31-SynORF5 Therefore PoIFN-1205821 might be a promising candi-date molecular adjuvant to develop more effective vaccines

2 Material and Methods

21 Plasmids and Cells pcDNA31-SynORF5 which wasbased on the native ORF5 gene of highly pathogenic PRRSVstrain (constructed and kept in our lab) pcDNA31Hela cellsand Marc-145 cells were kept in our lab

22 Experimental Animals 6-week-old BALBc mice werepurchased from Yang Zhou University The mice were ran-domly divided into 3 groups and acclimated under controlledspecific pathogen-free (SPF) conditions for 1 week prior to thestart of the experiment

23 Cloning and Sequencing of PoIFN-1205821 Gene The primerswere designed for amplifying PoIFN-1205821 based on genesequence of porcine IFN-1205821 gene (GenBank accessionnumber FJ853390) PoIFN-1205821F 51015840-TTTGCTAGCGCCACC-ATGGCTACAGCTTGGATCGTGGTG-31015840 PoIFN-1205821RGAGGGTACCGCTACCACCACCACCGATGTGCAA-GTCTCCACTGGTAA-31015840 PCR reaction was performed inthe thermocycler with the following program denaturationat 95∘C for 5min 30 cycles were comprised of denaturationat 95∘C for 1min annealing at 60∘C for 1min and extensionat 72∘C for 1min and was ended with the final extension of10min at 72∘C PCR products obtained with primers PoIFN-1205821F and PoIFN-1205821R were inserted into vector pMD18-Tgenerating plasmids pMD18-T-PoIFN-1205821 cDNAs encodingPoIFN-1205821 were obtained subsequently by RT-PCR usingmRNAs from porcine peripheral blood mononuclear cells(PBMC) The sequence of the insert was confirmed bysequencing

24 Construction of pcDNA31-PoIFN-1205821-SynORF5 PlasmidsThe cloning product was inserted into pMD18-T vector andthen sequenced Based on the sequencing result the PCRproduction and pcDNA31-SynORF5 were digested with asimilar pair of restriction enzymes Nhe IKpn I then thecorresponding restriction fragments were linked using T4DNA ligase The standard molecular biological techniques toconstruct the pcDNA31-PoIFN-1205821-SynORF5 plasmid wereshown in Figure 1

25 Restriction Enzyme Digestion of the Plasmid DNA Therecombinant plasmids were purified by AxyPrep PlasmidMiniprep Kit (Axygen Biosciences Zhejiang China) Thenthe obtained plasmids were respectively digested with threepairs of restriction enzyme which included Nhe IKpn I NheIXho I and Kpn IXho I

26 Transfection andWestern Blotting Hela cells were seededat a concentration of 25 times 104 cellswell into 6-well tis-sue culture plate until the cells reached approximately 70ndash80 confluence Transfection was performed with Lipo-fectAMINE 2000 reagent (Invitrogen) as specified by themanufacturer The transfected cells were collected at 48 hafter transfection and lyzed in a buffer containing 10mMTris-HCl (pH 75) 150mMNaCl 1mM EDTA 1 NP-40 andprotease inhibitors cocktail (Roche) Protein quantificationwas carried out using a BCATM 223 protein assay kit(Pierce) Equal amounts of proteins were separated using10 SDS-PAGE and electroblotted onto a nitrocellulosemembrane The membrane was blocked with 5 nonfatmilk in phosphate-buffered saline (PBS) and incubated withGP5-specific monoclonal antibodies (kept in our lab) andsubsequently with HRP-conjugated goat anti-mouse IgG(Sigma) Signals were developed using SuperSignal West PioLuminol kit (Pierce)

27 Immunization of BALBcMice with Plasmid DNA Large-scale preparations of plasmid DNA including pcDNA31pcDNA31-SynORF5 and pcDNA-PoIFN-1205821-SynORF5 werepurified by EndoFree Maxi Plasmid Kit (TIANGEN BeijingON China) as instructed by themanufacturerThe plasmidsrespectively adjust to a final concentration of 1120583g120583L

Six-week-old BALBc mice were purchased from YangZhou University Twenty-one mice were randomly devidedinto three groups and mice were vaccinated intramus-cularly twice at 2-week intervals with pcDNA31-PoIFN-1205821-SynORF5 pcDNA31-SynORF5 and the empty vectorpcDNA31 (+) respectively Serum samples were collected 2and 4 weeks after primary inoculation for serological testsSix weeks after primary immunization mice were euthanizedand the sera were harvested for the detection of antibodiesagainst PRRSV and splenocytes were isolated as describedpreviously [25] for IFN-120574 assay and lymphocyte-proliferationassay

28 Serological Tests GP5-specific antibodies were deter-mined with an endpoint ELISA using the purified recom-binant GP5 as antigen as described previously [26] The

BioMed Research International 3

Ampicillin

BGH

pA

f1 or

i

SV40 oriNeomycin

SV40 pA

pUC ori

pMD18-T-PoIFN-1205821

pcDNA31-SynORF5

pcDNA31-SynORF5

pcDNA31-PoIFN-1205821-SynORF5

Restriction enzyme digestedNhe I + Kpn I


Nhe I

Kpn I

Xho I

PoIFN-1205821

PoIFN-1205821 SynORF5

PCM

V

pcDNA31 (+)5428bp

Figure 1 Schematic representation of DNA vaccine constructs Briefly PoIFN-1205821 in the pMD18-T vector was obtained using a pair ofrestriction enzymes Nhe I and Kpn I And the pcDNA31-SynORF5 was digested using the same restriction enzymes in order that PoIFN-1205821can be ligated to it using the T4 DNA ligase So the recombinant plasmids (pcDNA31-PoIFN-1205821-SynORF5) were constructed successfully

titers were expressed as the reciprocal of the highest dilutionof sera producing ratio values of 21 Serum neutralizationassays were essentially performed as described by Ostrowskiet al [27] The neutralization titers were expressed as thereciprocal of the highest serum dilution resulting in completeneutralization Each sample was run in triplicate

29 Lymphocytes Proliferation Assay Lymphocyte prolifera-tion assaywas performedusing the splenocytes of immunizedmice Six weeks after the primary inoculation splenocyteswere collected respectively Lymphocyte proliferation assayswere performed as described previously [25]The stimulationindex (SI) was calculated as the ratio of the average OD valueofwells containing antigen-stimulated cells to the averageODvalue of wells containing only cells with medium

210 IFN-120574 Release Assay The isolated splenocytes (1 times 106cellsmL) were cultured in 24-well plates at 37∘C in thepresence of 5 CO

2with or without the PRRSV inactived by

UV After 72 h incubation culture supernatant was harvestedand the presence of IFN-120574was tested with commercial mouseIFN-120574 immunoassay ELISA kits (Boster Biological Tech-nology LTD Wuhan China) according to manufacturerrsquosinstructionsThe concentrations of IFN-120574 in the samplesweredetermined based on the standard curves

211 Real-Time PCR Analysis of IFN-120574 mRNA ExpressionSplenocytes (1 times 106 cellsmL) were cultured in 24-well plates

for 18 h at 37∘C in the presence of 5 CO2 Total RNA

was extracted and 04 120583g of RNA was reverse transcribed ina 20120583L reaction mixture The cDNA product (05 120583L) wasamplified in a 25120583L reactionmixture containing SYBRGreenReal-time PCR Master Mix (ToYoBo) and 02120583M of eachof the forward and reverse gene-specific primers (Mouse-IFN-120574 TCAAGTGGCATAGATGTGGAAGAATGGCT-CTGCAGGATTTTCATG Mouse-120573-actin CACTGCCGC-ATCCTC-TTCCTCCCCAATAGTGATGACCTGGCCG-T) Each cDNA sample was performed in triplicate PCRamplifications were performed using an Applied Biosystems7500 Real-Time PCR System (ABI) Thermal cycling condi-tions were 2min at 50∘C 10min at 94∘C and 40 cycles of 15 sat 94∘C and 1min at 60∘C Gene expression was measured byrelative quantity as described previously [28]

212 Statistical Analysis Studentrsquos t-test was used to comparethe level of immune responses among the different groups 119875values of lt005 were considered statistically significant

3 Results

31 Cloning and Sequencing of the PoIFN-1205821 Gene FragmentA single PCR product of an estimated 576 bp of the PoIFN-1205821 gene (Figure 2) was amplified using the cDNAs whichwere obtained by RT-PCR with mRNAs of porcine PBMCas template The fragment was cloned into the pMD18-T


(bp) 1 M (bp)

576

2000

1000750

500

250

100

(a)

2000

1000750

500

250

100

576

2692

(bp) (bp)1 M

(b)

2000

(bp) (bp)1 2 3 4 5 6 7 8 9 M1 M2

5428

1000

500250

300040005000

1239663576

(c)

Figure 2 (a) Subcloning of the PoIFN-1205821 gene Lane 1 PCR products of PoIFN-1205821 laneM marker DL2000 (b) pMD18-T-PoIFN-1205821 digestedwith Nhe I and Kpn I Lane 1 pMD18-T-PoIFN-1205821 lane M Marker DL2000 (c) pcDNA31-PoIFN-1205821-SynORF5 pcDNA31-SynORF5 andthe empty vector pcDNA31 digested with Nhe IKpn I Kpn IXho I and Nhe IXho I respectively Lane 1ndash3 pcDNA31-PoIFN-1205821-SynORF5lane 4ndash6 pcDNA31-SynORF5 lane 7ndash9 pcDNA31 lane M1 marker DL (2000) and lane M2 Marker DL (5000)

vector and sequenced The nucleotide sequences for PoIFN-1205821 were 99 identical to published PoIFN-1205821 (Acc noFJ853390) sequences The predicted protein sequences forPoIFN-1205821 were 100 identical to published PoIFN-1205821 (Accno NP 001136309) sequences as determined by BLASTanalysis

32 Construction of Plasmids The gene fragment encodingPoIFN-1205821 (Figure 2(a)) was cloned into the cloning plas-mid vector pMD18-T PoIFN-1205821 was analyzed by restrictionendonuclease double digestion with Nhe I and Kpn I Thesize of the digested fragments was 576 bp and an estimated2692 bp pMD18-T vector band (Figure 2(b)) Eukaryoticexpression plasmids pcDNA31-PoIFN-1205821-SynORF5 werealso constructed as described (Figure 1) and analysed by threepairs of restriction endonuclease double digestion with NheIKpn IKpn IXho I andNhe IXho IThe size of the digestedfragments containing the inserted fragments was 576 663and 1239 bp respectively and an estimated 5428 bp pcDNA31vector band (Figure 2(c))

33 Western Blotting Detection of Recombinant ProteinsTo investigate whether the inserted gene fragment PoIFN-1205821 influences the in vitro expression and authenticity ofthe SynORF5 gene Hela cells were transiently transfectedwith pcDNA31-PoIFN-1205821-SynORF5 and Western blot wasperformed at 48 h after transfection The DNA construct

pcDNA31-SynORF5 only expressing the SynORF5 gene ofPRRSV strain NJGC was used as control As shown inFigure 3 the fusion protein bands with expected molecu-lar sizes could be detected in lysates of cells transfectedwith pcDNA31-PoIFN-1205821-SynORF5 (48KDa) and just GP5-specific protein bands with expected molecular sizes couldbe detected in lysates of cells transfected with pcDNA31-SynORF5 (25KDa) but there are no protein bands in lysatesof cells transfected with the empty vector So the resultsshowed that the inserted gene fragment PoIFN-1205821 did notinfluence the in vitro expression of SynORF5 gene

34 Humoral Immune Responses Induced in Mice Immu-nized with Different DNA Constructs To further comparethe ability of pcDNA31-SynORF5 and pcDNA31-PoIFN-1205821-SynORF5 to induce specific immune responses in vivothree groups of 6-week-old BALBc mice (seven miceper group) were injected twice at 2-week intervals intothe quadriceps muscle with 100 120583g of pcDNA31-PoIFN-1205821-SynORF5 pcDNA31-SynORF5 and the empty vectorpcDNA31 respectively Serum samples were collected at2 4 and 6 weeks after the primary immunization GP5-specific ELISA antibody was determined using the purifiedGP5 protein as the antigen As shown in Figure 4 2 weeksafter primary immunization the antibody titer reached adetectable level only in the group immunizedwith pcDNA31-PoIFN-1205821-SynORF5 and a further increase in antibody levels


1160

662450

350

250184144

(kDa)

1 2 3

Figure 3 Expression of the fusion protein which encoded bythe recombinant plasmid pcDNA31-PoIFN-1205821-SynORF5 in thetransfected cells Approximately 70ndash80 confluent Hela cells weretansfected with 2120583g of pcDNA31-PoIFN-1205821-SynORF5 (lane 3)pcDNA31-SynORF5 (lane 2) and control vector pcDNA31 (+) (lane1) respectively At 48 h after transfection the cells were collectedand subjected to Western blot as described in Section 2 Proteinstandards are indicated on left side of panel

was observed at 4 and 6 weeks after primary immuniza-tion Although a continuous increase in antibody levels wasobserved at 4 and 6 weeks after primary immunization inthe group immunized with pcDNA31-SynORF5 the wholeincreasing trend observed in the group immunized withpcDNA31-SynORF5 was not significant compared with thegroup immunized with pcDNA31-PoIFN-1205821-SynORF5

Serum samples were further evaluated for the ability toneutralize PRRSV strain NJGC in vitro using serum neutral-ization assays As shown in Figure 5 mice immunized withpcDNA31-PoIFN-1205821-SynORF5 developed higher PRRSV-specific neutralizing antibody titer (1 514) than that of micereceived pcDNA31-SynORF5 (1 467) at 2 weeks after pri-mary immunization (119875 lt 005) After boost immunizationthe neutralizing antibody levels went increasingly higher andreached up to 1 16 in group immunized with pcDNA31-PoIFN-1205821-SynORF5 at 6 weeks after primary immunizationin comparison to 1 971 in mice immunized with pcDNA31-SynORF5 No detectable neutralizing antibodies (lt1 4) wereobserved in the sera from mice immunized with the emptyvector during the experimental period

35 Cellular Immune Responses Induced in Mice Immunizedwith Different DNA Constructs The results presented aboveclearly demonstrated that PoIFN-1205821 could effectively enhancehumoral immune responses elicited by DNA vaccine Toinvestigate whether PoIFN-1205821 could also enhance cellularimmune responses elicited by DNA vaccine the lymphocyte-proliferative responses were analyzed at 6 weeks after pri-mary immunization As shown in Figure 6 the SI washigher (119875 lt 005) in mice immunized with pcDNA31-PoIFN-1205821-SynORF5 than that in those immunized with

0

500

1000

1500

2000

2500

3000

2 weeks 4 weeks 6 weeks

Endp

oint

dilu

tion

titer

pcDNA31pcDNA31-SynORF5pcDNA31-PoIFN1205821-SynORF5

Figure 4 The titers of GP5-specific ELISA antibody in miceimmunized with different DNA constructs Six-week-old BALBcmice (seven per group) were immunized intramuscularly with100120583g of pcDNA31-PoIFN-1205821-SynORF5 pcDNA31-SynORF5 andpcDNA31 (+) respectively at 0 and 2 weeks Serum samples werecollected at 2 4 and 6 weeks after primary immunization todetermine the GP5-specific ELISA antibody Data represent themean and SD for 7 mice per group

02468

1012141618


Neu

tral

izin

g an

tibod

ies t

iters

pcDNA31pcDNA31-SynORF5pcDNA31-PoIFN-1205821-SynORF5

Figure 5 The PRRSV-specific neutralizing antibodies in miceimmunized with different DNA constructs Mice were immunizedas in Figure 4 Serum sampleswere collected at 2 4 and 6weeks afterprimary immunization to determine neutralizing antibody Datarepresent the mean and SD for 7 mice per group

pcDNA31-SynORF5 These results indicated that PoIFN-1205821can also enhanceTh1-type immune response

To further characterize the cellular immune responses inmice immunized with pcDNA31-PoIFN-1205821-SynORF5 IFN-120574 secretion in splenocytes restimulated with PRRSV proteinwas measured by ELISA As shown in Figure 7 the meanIFN-120574 production of 3958 pgmL was detected in miceimmunized with pcDNA31-PoIFN-1205821-SynORF5 and was


0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5

Figure 6 Lymphocyte proliferative responses of mice immunizedwith differentDNAconstructsMicewere immunized as in Figure 4Mice splenocytes samples (119899 = 7) were collected at 6 weeksafter primary immunization and restimulated in vitro with purifiedPRRSV proteins (20120583gmL) Lymphocyte proliferative assay wasperformed as described in Section 2 Data are presented as themeanplusmn SD

significantly higher (119875 lt 005) than that in mice immunizedwith pcDNA31-ORF5 (2978 pgmL) Quantitative real-timeRT-PCR was also performed to analyze the level of IFN-120574mRNA expression in the restimulated splenocytes Similarlyto the results of IFN-120574 ELISA assay the highest IFN-120574mRNAexpression was found in restimulated splenocytes from miceimmunized with pcDNA31-PoIFN-1205821-SynORF5 (Figure 8)Themean relative IFN-120574mRNA expression in this group was342-fold higher than that in group empty vector and 199-foldhigher than that in group pcDNA31-SynORF5 respectively

4 Discussion

At present PRRS continues to be one of the most econom-ically significant viral diseases in the swine industry world-wide Though there are many commercial vaccination strate-gies they can provide only a limited protectionThus PRRSVgenetic engineered vaccines have recently been reportedincluding pseudorabies virus expressing GP5 [16] recom-binant fowlpox virus coexpressing GP5GP3 and swine IL-18 [20] recombinant adenoviruses expressing GP5GP4GP3[29] and mycobacterium bovis BCG expressing GP5 and M[18 30] In order to increase the efficiency of the vaccine analternative approach is to codeliver cytokines to upregulatethe immune response of PRRSV including HSP70 [31] IL-18 [30] GM-CSF [32] C3d-p28 [33] and interferon 120572120574 [34]In this study porcine IFN-1205821 was amplified and recombinantplasmid encoding PoIFN-1205821 and themodifiedGP5 of PRRSVwere constructed It was found that the porcine IFN-1205821can effectively increase the humoral and cellular immuneresponses of GP5 of PRRSV in mice GP5 protein is astructural PRRSV protein with the size of 25 KDa GP5 is

0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)

Figure 7 Concentrations (pgmL) of Th1-type cytokine of IFN-120574in the immunized mice Mice splenocytes samples (119899 = 7) werecollected 6 weeks after primary immunization and restimulatedin vitro with purified PRRSV proteins (20120583gmL) At 72 h thesupernatant was collected to determine the IFN-120574 content It wasperformed by using commercially available mice cytokine ELISAkits Data are presented as the mean plusmn SD

0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A

Figure 8 The level of IFN-120574 mRNA expression of immunizedmice Mice splenocytes samples (119899 = 7) were collected at 6weeks after primary immunization and restimulated in vitro withpurified PRRSV proteins (20120583gmL) At 18 h the total RNA wasextracted and subjected to amplification by real-time PCR IFN-120574quantitative RT-PCR was performed as described in Section 2 Dataare presented as the mean value of triplicate sample plusmn SD

the most important glycosylation of PRRSV involved in thegeneration of PRRSV-neutralizing antibodies and protectiveimmunity [12 35ndash38] So most vaccine research PRRSV isfocused on the GP5 In our previous research the DNAvaccine encoding the modified GP5 induced significantly


enhancedGP5-specific ELISA antibody PRRSV-specific neu-tralizing antibody IFN-120574 level and lymphocyte prolifera-tion response compared to native GP5 in the vaccinatedmice and piglets indicating that these modifications couldenhance the immunogenicity of GP5 And in an otherresearch the purified recombinant poIFN-1205821 exhibited sig-nificant antiviral effects against porcine reproductive andrespiratory syndrome virus (PRRSV) and pseudorabies virus(PRV) suggesting that poIFN-1205821 is a potential antiviralagent against swine infectious diseases [39] So in this studythe recombinant DNA units pcDNA31-PoIFN-1205821-SynORF5were constructed and the immune responses were detectedin mice in order to identify whether PoIFN-1205821 can furtherimprove the efficacy of the immune responses induced bypcDNA31-SynORF5 or not

Figures 3 and 4 showed that PoIFN-1205821 can effectivelyenhance humoral immune responses elicited by DNA vac-cine Although themechanisms of adaptive immune responsethat are responsible for mediating the vaccine inducedprotective immunity have not been fully understood it iswidely accepted that neutralizing antibodies could possiblyrepresent a valuable parameter to evaluate the efficacy ofa vaccine against PRRSV [40ndash42] Likewise cell-mediatedimmunity particularly the level of virus-specific IFN-120574 hasbeen another potential correlate of protective immunityagainst PRRSV [43ndash46] In previous study Jiang et alfound that mice immunized with recombinant adenovirusesexpressing GP5 with mutation in different glycosylation sitesdeveloped significantly enhancedneutralizing antibodies butnot in lymphocyte proliferation response [47] In Figures5 and 6 enhanced IFN-120574 level as well as lymphocyteproliferation response could be observed in pcDNA31-PoIFN-1205821-SynORF5-immunizedmice It is indicated that theenhanced cellular immune responses might be enhanced bythe adjuvant effect of the PoIFN-1205821 in mice In a word therecombinant construct containing PoIFN-1205821 and SynORF5were successfully constructed then the grouped mice werevaccinated with different plasmids Results showed that sig-nificantly enhanced GP5-specific ELISA antibody PRRSV-specific neutralizing antibody IFN-120574 level and lymphocyteproliferation response could be induced in mice immunizedwith DNA vaccine co-expressing the modified GP5 andPoIFN-1205821 more than those which received DNA vaccine onlyexpressing the modified GP5 The results demonstrate thatPoIFN-1205821 could significantly enhance the humoral and cellu-lar immune responses andmay provide protection which wasinduced by pcDNA31-PoIFN-1205821-SynORF5 against PRRSVchallenge in piglets

To our knowledge this study is the first demonstrationthat porcine IFN-1205821 fused the modified GP5 of PRRSV couldmarkedly enhance the immune responses PoIFN-1205821 mightbe a useful molecular adjuvant in improving PRRSV immuneresponse andmaybe it will be further used in PRRSV vaccine

Conflict of Interests

The authors declare no potential conflict of interests withrespect to the research authorship andor publication of thispaper

Authorsrsquo Contribution

Luping Du and Bin Li contributed equally to this work

Acknowledgments

This work was supported by the National Natural SciencesFoundation of China (31001066 and 31225027) the PriorityAcademic Program Development of Jiangsu Higher Educa-tion Institutions and the Jiangsu province Natural SciencesFoundation (BK 20131330)

References

[1] D D PolsonW EMarsh and G D Dial ldquoFinancial evaluationand decision making in the swine breeding herdrdquo The Veteri-nary Clinics of North America Food Animal Practice vol 8 no3 pp 725ndash747 1992

[2] S A Dee H S Joo D D Polson andW E Marsh ldquoEvaluationof the effects of nursery depopulation on the profitability of 34pig farmsrdquoVeterinary Record vol 140 no 19 pp 498ndash500 1997

[3] J G Cho and S A Dee ldquoPorcine reproductive and respiratorysyndrome virusrdquo Theriogenology vol 66 no 3 pp 655ndash6622006

[4] R Bilodeau S Dea G P Martineau and R A SauvageauldquoPorcine reproductive and respiratory syndrome in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[5] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Practitioners Newsletter vol 1no 2 pp 1ndash10 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swinedisease in the Netherlands the isolation of Lelystad virusrdquoVeterinary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] J J M Meulenberg M M Hulst E J de Meijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[9] J J MMeulenberg A P Besten E P de Kluyver R JMMoor-mannWMM Schaaper andGWensvoort ldquoCharacterizationof proteins encoded by ORFs 2 to 7 of Lelystad virusrdquo Virologyvol 206 no 1 pp 155ndash163 1995

[10] M P Murtaugh M R Elam and L T Kakach ldquoComparisonof the structural protein coding sequences of the VR-2332 andLelystad virus strains of the PRRS virusrdquo Archives of Virologyvol 140 no 8 pp 1451ndash1460 1995

[11] Q Zheng D Chen P Li et al ldquoCo-expressing GP5 and Mproteins under different promoters in recombinant modifiedvaccinia virus ankara (rMVA)-based vaccine vector enhancedthe humoral and cellular immune responses of porcine repro-ductive and respiratory syndrome virus (PRRSV)rdquoVirus Genesvol 35 no 3 pp 585ndash595 2007

[12] B Pirzadeh and S Dea ldquoImmune response in pigs vaccinatedwith plasmidDNA encodingORF5 of porcine reproductive andrespiratory syndrome virusrdquo Journal of General Virology vol 79no 5 pp 989ndash999 1998


[13] AM BarfoedM Blixenkrone-MoslashllerMH Jensen A Boslashtnerand S Kamstrup ldquoDNA vaccination of pigs with open readingframe 1ndash7 of PRRS virusrdquo Vaccine vol 22 no 27-28 pp 3628ndash3641 2004

[14] A Kheyar A Jabrane C Zhu et al ldquoAlternative codon usageof PRRS virus ORF5 gene increases eucaryotic expression ofGP5 glycoprotein and improves immune response in challengedpigsrdquo Vaccine vol 23 no 31 pp 4016ndash4022 2005

[15] W Jiang P Jiang Y Li J Tang XWWang and SMa ldquoRecom-binant adenovirus expressing GP5 and M fusion proteins ofporcine reproductive and respiratory syndrome virus induceboth humoral and cell-mediated immune responses in micerdquoVeterinary Immunology and Immunopathology vol 113 no 1-2pp 169ndash180 2006

[16] H J Qiu Z J Tian G Z Tong et al ldquoProtective immunityinduced by a recombinant pseudorabies virus expressing theGP5 of porcine reproductive and respiratory syndrome virus inpigletsrdquoVeterinary Immunology and Immunopathology vol 106no 3-4 pp 309ndash319 2005

[17] Y B Jiang L R Fang S B Xiao et al ldquoImmunogenicity andprotective efficacy of recombinant pseudorabies virus express-ing the two major membrane-associated proteins of porcinereproductive and respiratory syndrome virusrdquo Vaccine vol 25no 3 pp 547ndash560 2007

[18] R G Bastos O A Dellagostin R G Barletta et al ldquoImmuneresponse of pigs inoculated with Mycobacterium bovis BCGexpressing a truncated form of GP5 and M protein of porcinereproductive and respiratory syndrome virusrdquo Vaccine vol 22no 3-4 pp 467ndash474 2004

[19] Y H Hou J Chen G Z Tong et al ldquoA recombinant plasmidco-expressing swine ubiquitin and the GP5 encoding-gene ofporcine reproductive and respiratory syndrome virus inducesprotective immunity in pigletsrdquoVaccine vol 26 no 11 pp 1438ndash1449 2008

[20] G Shen N Jin M Ma et al ldquoImmune responses of pigsinoculated with a recombinant fowlpox virus coexpressingGP5GP3 of porcine reproductive and respiratory syndromevirus and swine IL-18rdquo Vaccine vol 25 no 21 pp 4193ndash42022007

[21] B Li S B Xiao Y W Wang et al ldquoImmunogenicity ofthe highly pathogenic porcine reproductive and respiratorysyndrome virus GP5 protein encoded by a synthetic ORF5generdquo Vaccine vol 27 no 13 pp 1957ndash1963 2009

[22] E Arico and F Belardelli ldquoInterferon-120572 as antiviral and anti-tumor vaccine adjuvants mechanisms of action and responsesignaturerdquoThe Journal of Interferon and Cytokine Research vol32 no 6 pp 235ndash247 2012

[23] S Hong J Qian H Li et al ldquoCpG or IFN-120572 are morepotent adjuvants than GM-CSF to promote anti-tumor immu-nity following idiotype vaccine in multiple myelomardquo CancerImmunology Immunotherapy vol 61 no 4 pp 561ndash571 2012

[24] P Rizza I Capone F Moretti E Proietti and F BelardellildquoIFN-120572 as a vaccine adjuvant recent insights into the mech-anisms and perspectives for its clinical userdquo Expert Review ofVaccines vol 10 no 4 pp 487ndash498 2011

[25] S Xiao H Chen L Fang et al ldquoComparison of immuneresponses and protective efficacy of suicidal DNA vaccineand conventional DNA vaccine encoding glycoprotein C ofpseudorabies virus in micerdquo Vaccine vol 22 no 3-4 pp 345ndash351 2004

[26] L R Fang Y B Jiang S B Xiao C S Niu H Zhang andH C Chen ldquoEnhanced immunogenicity of the modified GP5

of porcine reproductive and respiratory syndrome virusrdquo VirusGenes vol 32 no 1 pp 5ndash11 2006

[27] M Ostrowski J A Galeota A M Jar K B Platt F A Osorioand O J Lopez ldquoIdentification of neutralizing and nonneu-tralizing epitopes in the porcine reproductive and respiratorysyndrome virus GP5 ectodomainrdquo Journal of Virology vol 76no 9 pp 4241ndash4250 2002

[28] S Wang L Fang H Fan et al ldquoConstruction and immuno-genicity of pseudotype baculovirus expressing GP5 and Mprotein of porcine reproductive and respiratory syndromevirusrdquo Vaccine vol 25 no 49 pp 8220ndash8227 2007

[29] W Jiang P Jiang X W Wang Y F Li Y Du and X WangldquoEnhanced immune responses of mice inoculated recombinantadenoviruses expressing GP5 by fusion with GP3 andor GP4of PRRS virusrdquoVirus Research vol 136 no 1-2 pp 50ndash57 2008

[30] R G Bastos O A Dellagostin R G Barletta A R Doster ENelson and F A Osorio ldquoConstruction and immunogenicityof recombinantMycobacterium bovis BCG expressing GP5 andM protein of porcine reproductive respiratory syndrome virusrdquoVaccine vol 21 no 1-2 pp 21ndash29 2002

[31] J Li P Jiang Y F Li et al ldquoHSP70 fused with GP3 and GP5 ofporcine reproductive and respiratory syndrome virus enhancedthe immune responses and protective efficacy against virulentPRRSV challenge in pigsrdquo Vaccine vol 27 no 6 pp 825ndash8322009

[32] X Wang J Li P Jiang et al ldquoGM-CSF fused with GP3 andGP5 of porcine reproductive and respiratory syndrome virusincreased the immune responses and protective efficacy againstvirulent PRRSV challengerdquo Virus Research vol 143 no 1 pp24ndash32 2009

[33] D Q Zhang Q X Xia J Q Wu D Liu X Wang and Z NiuldquoConstruction and immunogenicity of DNA vaccines encodingfusion protein ofmurine complement C3d-p28 andGP5 gene ofporcine reproductive and respiratory syndrome virusrdquo Vaccinevol 29 no 4 pp 629ndash635 2011

[34] Y J Du J Qi Y Lu et al ldquoEvaluation of a DNA vaccinecandidate co-expressing GP3 and GP5 of porcine reproductiveand respiratory syndrome virus (PRRSV) with interferon 120572120574in immediate and long-lasting protection against HP-PRRSVchallengerdquo Virus Genes vol 45 no 3 pp 474ndash487 2012

[35] B Pirzadeh and S Dea ldquoMonoclonal antibodies to the ORF5product of porcine reproductive and respiratory syndromevirus define linear neutralizing determinantsrdquo Journal of Gen-eral Virology vol 78 no 8 pp 1867ndash1873 1997

[36] E Weiland M Wieczorek-Krohmer D Kohl K K Conzel-mann and F Weiland ldquoMonoclonal antibodies to the GP5 ofporcine reproductive and respiratory syndrome virus are moreeffective in virus neutralization than monoclonal antibodies tothe GP4rdquo Veterinary Microbiology vol 66 no 3 pp 171ndash1861999

[37] P Gonin B Pirzadeh C A Gagnon and S Dea ldquoSeroneutral-ization of porcine reproductive and respiratory syndrome viruscorrelates with antibody response to the GP5 major envelopeglycoproteinrdquo Journal of VeterinaryDiagnostic Investigation vol11 no 1 pp 20ndash26 1999

[38] L Yang M L Frey K J Yoon J J Zimmerman and K B PlattldquoCategorization of North American porcine reproductive andrespiratory syndrome viruses epitopic profiles of theNM GP5and GP3 proteins and susceptibility to neutralizationrdquo Archivesof Virology vol 145 no 8 pp 1599ndash1619 2000

[39] D Wang L Fang F Zhao R Luo H Chen and S XiaoldquoMolecular cloning expression and antiviral activity of porcine


interleukin-29 (poIL-29)rdquo Developmental and ComparativeImmunology vol 35 no 3 pp 378ndash384 2011

[40] F A Osorio J A Galeota E Nelson et al ldquoPassive transfer ofvirus-specific antibodies confers protection against reproduc-tive failure induced by a virulent strain of porcine reproductiveand respiratory syndrome virus and establishes sterilizingimmunityrdquo Virology vol 302 no 1 pp 9ndash20 2002

[41] O J Lopez M F Oliveira E A Garcia B J Kwon A Dosterand F A Osorio ldquoProtection against porcine reproductive andrespiratory syndrome virus (PRRSV) infection through passivetransfer of PRRSV-neutralizing antibodies is dose dependentrdquoClinical and Vaccine Immunology vol 14 no 3 pp 269ndash2752007

[42] O J Lopez and F A Osorio ldquoRole of neutralizing antibodiesin PRRSV protective immunityrdquo Veterinary Immunology andImmunopathology vol 102 no 3 pp 155ndash163 2004

[43] E Mateu and I Diaz ldquoThe challenge of PRRS immunologyrdquoVeterinary Journal vol 177 no 3 pp 345ndash351 2008

[44] M P Murtaugh Z Xiao and F Zuckermann ldquoImmunologicalresponses of swine to porcine reproductive and respiratorysyndrome virus infectionrdquo Viral Immunology vol 15 no 4 pp533ndash547 2002

[45] J F Lowe R Husmann L D Firkins F A Zuckermannand T L Goldberg ldquoCorrelation of cell-mediated immunityagainst porcine reproductive and respiratory syndrome viruswith protection against reproductive failure in sows duringoutbreaks of porcine reproductive and respiratory syndrome incommercial herdsrdquo Journal of the American Veterinary MedicalAssociation vol 226 no 10 pp 1707ndash1711 2005

[46] F A Zuckermann E AGarcia I D Luque et al ldquoAssessment ofthe efficacy of commercial porcine reproductive and respiratorysyndrome virus (PRRSV) vaccines based on measurementof serologic response frequency of gamma-IFN-producingcells and virological parameters of protection upon challengerdquoVeterinary Microbiology vol 123 no 1-3 pp 69ndash85 2007

[47] W Jiang P Jiang X W Wang Y F Li X Wang and Y DuldquoInfluence of porcine reproductive and respiratory syndromevirus GP5 glycoprotein N-linked glycans on immune responsesin micerdquo Virus Genes vol 35 no 3 pp 663ndash671 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


used three methods to modify the PRRSV GP5 exhibitedsignificantly enhanced immunogenicity particularly in theability to induce neutralizing antibody responses and cel-lular immune responses compared to the native GP5 [21]Consequently this modified GP5 may be useful to facilitatethe development of the new generation of vaccines such asDNA vaccines live attenuated chimeric virus vaccines andlive virus-vectored vaccines against the highly pathogenicPRRSV in the future

Type III interferon a new interferon family was firstlyreported in 2003 and different from the types I and IIinterferon including IFN-1205821 IFN-1205822 and IFN-1205823 IFN-120572and IFN-120573 belonging to type I interferon were confirmedto be adjuvants to improve the vaccinesrsquo immune responses[22ndash24] In addition previous studies have shown that typeIII interferon has almost the same biological activity ofother interferons such as anti-viral antitumor and immuneregulation but when compared with type I interferon itsside effects are obviously little Thus the research on type IIIinterferon will play a role in promoting the control of animaldiseases and medical treatment of human disease

In view of the above information in this study weconstructed the DNA construct units encoding pcDNA31-PoIFN-1205821-SynORF5 and find that pcDNA31-PoIFN-1205821-SynORF5 could induce stronger cellular and humoralimmune responses than the responses induced by pcDNA31-SynORF5 Therefore PoIFN-1205821 might be a promising candi-date molecular adjuvant to develop more effective vaccines

2 Material and Methods

21 Plasmids and Cells pcDNA31-SynORF5 which wasbased on the native ORF5 gene of highly pathogenic PRRSVstrain (constructed and kept in our lab) pcDNA31Hela cellsand Marc-145 cells were kept in our lab

22 Experimental Animals 6-week-old BALBc mice werepurchased from Yang Zhou University The mice were ran-domly divided into 3 groups and acclimated under controlledspecific pathogen-free (SPF) conditions for 1 week prior to thestart of the experiment

23 Cloning and Sequencing of PoIFN-1205821 Gene The primerswere designed for amplifying PoIFN-1205821 based on genesequence of porcine IFN-1205821 gene (GenBank accessionnumber FJ853390) PoIFN-1205821F 51015840-TTTGCTAGCGCCACC-ATGGCTACAGCTTGGATCGTGGTG-31015840 PoIFN-1205821RGAGGGTACCGCTACCACCACCACCGATGTGCAA-GTCTCCACTGGTAA-31015840 PCR reaction was performed inthe thermocycler with the following program denaturationat 95∘C for 5min 30 cycles were comprised of denaturationat 95∘C for 1min annealing at 60∘C for 1min and extensionat 72∘C for 1min and was ended with the final extension of10min at 72∘C PCR products obtained with primers PoIFN-1205821F and PoIFN-1205821R were inserted into vector pMD18-Tgenerating plasmids pMD18-T-PoIFN-1205821 cDNAs encodingPoIFN-1205821 were obtained subsequently by RT-PCR usingmRNAs from porcine peripheral blood mononuclear cells(PBMC) The sequence of the insert was confirmed bysequencing

24 Construction of pcDNA31-PoIFN-1205821-SynORF5 PlasmidsThe cloning product was inserted into pMD18-T vector andthen sequenced Based on the sequencing result the PCRproduction and pcDNA31-SynORF5 were digested with asimilar pair of restriction enzymes Nhe IKpn I then thecorresponding restriction fragments were linked using T4DNA ligase The standard molecular biological techniques toconstruct the pcDNA31-PoIFN-1205821-SynORF5 plasmid wereshown in Figure 1

25 Restriction Enzyme Digestion of the Plasmid DNA Therecombinant plasmids were purified by AxyPrep PlasmidMiniprep Kit (Axygen Biosciences Zhejiang China) Thenthe obtained plasmids were respectively digested with threepairs of restriction enzyme which included Nhe IKpn I NheIXho I and Kpn IXho I

26 Transfection andWestern Blotting Hela cells were seededat a concentration of 25 times 104 cellswell into 6-well tis-sue culture plate until the cells reached approximately 70ndash80 confluence Transfection was performed with Lipo-fectAMINE 2000 reagent (Invitrogen) as specified by themanufacturer The transfected cells were collected at 48 hafter transfection and lyzed in a buffer containing 10mMTris-HCl (pH 75) 150mMNaCl 1mM EDTA 1 NP-40 andprotease inhibitors cocktail (Roche) Protein quantificationwas carried out using a BCATM 223 protein assay kit(Pierce) Equal amounts of proteins were separated using10 SDS-PAGE and electroblotted onto a nitrocellulosemembrane The membrane was blocked with 5 nonfatmilk in phosphate-buffered saline (PBS) and incubated withGP5-specific monoclonal antibodies (kept in our lab) andsubsequently with HRP-conjugated goat anti-mouse IgG(Sigma) Signals were developed using SuperSignal West PioLuminol kit (Pierce)

27 Immunization of BALBcMice with Plasmid DNA Large-scale preparations of plasmid DNA including pcDNA31pcDNA31-SynORF5 and pcDNA-PoIFN-1205821-SynORF5 werepurified by EndoFree Maxi Plasmid Kit (TIANGEN BeijingON China) as instructed by themanufacturerThe plasmidsrespectively adjust to a final concentration of 1120583g120583L

Six-week-old BALBc mice were purchased from YangZhou University Twenty-one mice were randomly devidedinto three groups and mice were vaccinated intramus-cularly twice at 2-week intervals with pcDNA31-PoIFN-1205821-SynORF5 pcDNA31-SynORF5 and the empty vectorpcDNA31 (+) respectively Serum samples were collected 2and 4 weeks after primary inoculation for serological testsSix weeks after primary immunization mice were euthanizedand the sera were harvested for the detection of antibodiesagainst PRRSV and splenocytes were isolated as describedpreviously [25] for IFN-120574 assay and lymphocyte-proliferationassay

28 Serological Tests GP5-specific antibodies were deter-mined with an endpoint ELISA using the purified recom-binant GP5 as antigen as described previously [26] The


Ampicillin

BGH

pA

f1 or

i

SV40 oriNeomycin

SV40 pA

pUC ori


pcDNA31-SynORF5

pcDNA31-SynORF5




Nhe I

Kpn I

Xho I

PoIFN-1205821


PCM

V

pcDNA31 (+)5428bp











3 Results



(bp) 1 M (bp)

576

2000

1000750

500

250

100

(a)

2000

1000750

500

250

100

576

2692

(bp) (bp)1 M

(b)

2000

(bp) (bp)1 2 3 4 5 6 7 8 9 M1 M2

5428

1000

500250

300040005000

1239663576

(c)








1160

662450

350

250184144

(kDa)

1 2 3





0

500

1000

1500

2000

2500

3000


Endp

oint

dilu

tion

titer



02468

1012141618


Neu

tral

izin

g an

tibod

ies t

iters






0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5



4 Discussion


0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)


0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A











Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ampicillin

BGH

pA

f1 or

i

SV40 oriNeomycin

SV40 pA

pUC ori


pcDNA31-SynORF5

pcDNA31-SynORF5




Nhe I

Kpn I

Xho I

PoIFN-1205821


PCM

V

pcDNA31 (+)5428bp











3 Results



(bp) 1 M (bp)

576

2000

1000750

500

250

100

(a)

2000

1000750

500

250

100

576

2692

(bp) (bp)1 M

(b)

2000

(bp) (bp)1 2 3 4 5 6 7 8 9 M1 M2

5428

1000

500250

300040005000

1239663576

(c)








1160

662450

350

250184144

(kDa)

1 2 3





0

500

1000

1500

2000

2500

3000


Endp

oint

dilu

tion

titer



02468

1012141618


Neu

tral

izin

g an

tibod

ies t

iters






0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5



4 Discussion


0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)


0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A











Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(bp) 1 M (bp)

576

2000

1000750

500

250

100

(a)

2000

1000750

500

250

100

576

2692

(bp) (bp)1 M

(b)

2000

(bp) (bp)1 2 3 4 5 6 7 8 9 M1 M2

5428

1000

500250

300040005000

1239663576

(c)








1160

662450

350

250184144

(kDa)

1 2 3





0

500

1000

1500

2000

2500

3000


Endp

oint

dilu

tion

titer



02468

1012141618


Neu

tral

izin

g an

tibod

ies t

iters






0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5



4 Discussion


0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)


0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A











Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1160

662450

350

250184144

(kDa)

1 2 3





0

500

1000

1500

2000

2500

3000


Endp

oint

dilu

tion

titer



02468

1012141618


Neu

tral

izin

g an

tibod

ies t

iters






0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5



4 Discussion


0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)


0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A











Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

16

pcD

NA

31

pcD

NA

31-

SynO

RF5

Stim

ulat

ion

inde

x (S

I)

pcD

NA31

-PoI

FN-1205821-

SynO

RF5



4 Discussion


0

50

100

150

200

250

300

350

400

450

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

IFN

-120574pr

oduc

tion

(pg

mL)


0

05

1

15

2

25

3

35

4

pcD

NA

31

pcD

NA

31-

SynO

RF5

pcD

NA31

-PoI

FN-1205821

-Sy

nORF

5

Rela

tive q

uant

ity o

f IFN

-120574

mRN

A











Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article construction and immunogenicity of dna...

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