george asanga ndeta · cdna synthesis primer (10µm) 1.0 µl (mix content & spin) then incubate...
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Molecular Basis of Arrested Liver Stage Development of the Gamma-irradiated Plasmodium yoelii exo-erythrocytic form.
by
George Asanga Ndeta
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INTRODUCTIONMalaria is one of the deadliest of human parasitic diseases that results in the dead of about 3 million individuals annually in the tropical and sub-tropical parts of the world.
Efforts to check the spread of malaria has recently been complicated by the problem of rise in resistance to chloroquine by the malaria parasite as well as rise in resistance to insecticides by the mosquito vector.
A possible solution to these problems of resistance is to explore alternate forms of therapies; the gamma-irradiated sporozoites as a vaccine candidate need further studies to understand the molecular events that results in its developmental arrest in the liver so as to improve on its efficacy.
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-Gamma-irradiated and non-irradiated Plasmodium yoelii (17XNL) sporozoites were isolated from Anopheles stephensi and purifiedon a renografin-60 gradient.-Sporozoites were axenically cultured at 37oC for 24 hours.-Total RNA was isolated with chloroform/isopropanol.-cDNA (1) library representing the gamma-irradiated EEF andcDNA (2) library representing the non-irradiated EEF were synthesized using the PCR-Select cDNA Subtraction Kit.-cDNA were digested with Rsa I restriction enzyme followedby adaptor ligation. -The two tester cDNA libraries were subjected to two roundsof hybridization followed by1ery and 2ndry PCR amplification.-Cloning
Experimental design For SSH
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Synthesis Of First cDNA Strand
� Poly A+ RNA (2µg) 2-3 µL
� cDNA Synthesis Primer (10µM) 1.0 µL � (mix content & spin)
� Then incubate at 70oC for 2 min in a thermal cycler
� Cool on ice for 2 min and briefly centrifuge
� Add the following reagents to each reaction:
� 5X First-Strand Buffer 2.0 µL
� dNTP Mix (10mM each) 1.0 µL
� Sterile H20 1.0 µL
� AMV Reverse Transcriptase 1.0 µL
� 420C x 1.5Hrs
� Stop Rxn on ice
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2nd Strand cDNA Synthesis
� Sterile H20 48.4 µL
� 5x Second-Strand buffer 16.0 µL
� dNTP Mix (10 mM) 1.6 µL
� 20x Second-Strand Enzyme Cocktail 4.0 µL
� 16oC x 2.0Hrs
� T4 DNA Polymerase 2.0 µL
� 16oC x 30min
� Stop Rxn – 4.0 µL of 20x EDTA/Glycogen
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Recover Synthesize cDNA
� 100 µL of Phenol:Chloroform:Isoamyl Alc
� 100 µL of Chloroform:Isoamyl Alcohol 2X
� 40 µL NH4OAc & 300 µl of 95% ETOH
� 500 µL of 80% ETOH
� Air Dry cDNA x 10 min.
� Dilute with 50.0 µL of H2O
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Rsa I Digestion
� ds cDNA 43.5 µL
� 10 x Rsa I Restriction Buffer 5.0 µL
� Rsa I Restriction Enzyme 1.5 µL
� Mix & Incubate @ 37oC x 1.5 Hrs
� Stop Rxn ĉ 2.5 µL of 20x EDTA/Glycogen
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Recover Blunt End cDNA
� 50 µL Phenol:Chloroform:Isoamyl alcohol
� 50 µL Chloroform:Isoamyl alcohol 2x
� 25 µL NH4OAc & 187.5 ul Etoh
� 200 µL 80% Etoh
� Air dry x 10 min
� Dissolve ĉ 5.5 µL of H2O
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Ligation
� Make 1:5 dilution of blunt end cDNA
� Ligation mix:
� Sterile H20 3.0 µL
� 5x Ligation Buffer 2.0 µL
� T4 DNA Ligase 1.0 µL
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Adaptor ligation
� Dilute cDNA………
� Adaptor 1………….
� Adaptor 2………….
� Master mix………..
� Final Volume……..
� 16oC overnight
� Stop Rxn ĉ 1ul of EDTA/Glycogen
� Heat at 72oC to inactivate Ligase
2.0uL
2.0uL
6.0uL 6.0uL
2.0uL
2.0uL
10.0uL10.0uL
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1-1 2-2
+cDNA2NI
+cDNA1I
2-11-2
+cDNA2NI
+cDNA1I
Experimental (tester) cDNA-1Irradiated cDNA
Experimental (tester) cDNA-2Non-Irradiated-cDNA
Mix withAdaptor 1
Mix withAdaptor 2 Mix with
Adaptor 1Mix withAdaptor 2
First HybridizationDriver with no Adaptors
Second HybridizationDriver with no Adaptors
+cDNA2NI
+cDNA1I
1o and 2o (PCR) 1o and 2o (PCR)
POOLPOOL
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-Amplified cDNA was cloned to TOPO vector (plasmid).-Cloned products were used to transform TOP 10 OneShot chemically competent cells.
-Transformed bacteria were incubated at 37oC for 24 hrson plates with x-gal and ampicillin.
Template plasmid, spin column purified with a SV miniprep kit; Sequencing; Dye terminator Method.
Insert check was done on positive clones to ascertain the success of cloning.
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Insert check of cloned cDNA from gamma-irradiated exo-erythrocytic form (EEF)
mak
er
500 bp-
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Irradiated clones Contig Protein Identified
I 1-4 Malpy000471 Hypothetical proteinDI 2-2 Malpy01825 Hypothetical proteinDI 2-11 Malpy00500 Putative yir1 proteinDI 2-5 Malpy00137 Putative yir4 proteinDI 1-12 Malpy00664 Tubulin-tyrosine ligase familyDI 2-2 Malpy01557 Putative yir4 proteinI 1-6 Malpy01953 Hypothetical proteinDI 1-15 Malpy01115 Uncharacterized protein familyDI 2-15 Malpy00137 Putative yir3 proteinDI 2-9 Malpy00296 hypothetical proteinDI 2-13 Malpy00664 CCAAT-box DNA binding protein subunit
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Non-irradiated clones Contig Identified Protein
NI 1-5 Malpy02019 CCAAT-box DNA binding proteinNI 1-11 Malpy01430 235 kDa rhoptry proteinNI 1-12 Malpy00905 Putative bir1NI 2-14 Malpy02672 Ribosomal protein S4NI 2-10 Malpy00013 Uncharacterized protein familyNI 2-12 Malpy00271 Erythrocyte membrane proteinNI 2-2 Malpy00014 Putative uncharacterized protein familyNI 2-19 Malpy00954 Putative yir4 proteinNI 1-1 Malpy02241 Hypothetical proteinDNI 2-3 Malpy01423 Heat shock protein 60DNI 2-7 Malpy00271 Uncharacterized protein familyNI 1-5 Malpy02019 Hypothetical protein
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Screening with un-subtracted cDNA probes
Un-subtracted tester probe Un-subtracted driver probe
Screening with subtracted cDNA probes
Forward subtracted probe Reverse subtracted probe
++ +++
+
++
++++
- --- --
--
- ---
-
--
-
+
SD IN
SDIN
--
1
141 1
1
14
14 1415
15
15
15 26
2626
26
27
27
27
27
40
40
40
40
41
41
41
41
5252
52 52
5353
535366
6666
66
67
67
67
67
78
78
78
78
79
7979
79 92
92
92
92
93
93
93
93
104
104
104
104
Gamma-irradiated P.yoelii EEF subtracted cDNA library dot blots
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Dot blots screened with un-subtracted cDNA probes
Dot blots screened with subtracted cDNA probes
Un-subtracted tester probe Un-subtracted driver probe
Forward subtracted probe Reverse subtracted probe
Non-irradiated P. yoelii EEF subtracted cDNA library dot blots
1
1 1
1
14 14
14 14
15 15
15 15
26 26
2626
27 27
2727
40 40
4040
41 41
4141
52 52
52 52
53 53
5353
66 66
66 66
67 67
67 67
78 78
7878
79 79
79 79
92 92
9292
9393
9393
104 104
104104
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Erythrocyte membrane protein
Control
p235kDa rhoptry protein
Tubulin tyrosine ligase
yir
NI I
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Liver Stage Parasites Blood Stage Parasites
Hep-17
500bp
200bp
Marker 1 2 3 4 5 6
7 LC
28SrRNA
MSP-1
Marker
500bp
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CONCLUSION
-SSH technique enabled us to generate two cDNA libraries from the IEEF and the NIEEF.
-Semi-quantitative RT-PCR analysis of selected clones showed the effect of radiation on sporozoites DNA when exposed to 12,000 rads of radiation from a 137Cesium source to be up regulation, down regulation or abrogation of some liver stage genes.
-The expression of the EMP gene in the NIEEF confirms a progressive transformation that will transition the sporozoite into the erythrocytic stage while abrogation of the EMP gene in the IEEF confirms the development arrest of sporozoites at the trophozoite stages.
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Acknowledgment
Dr. KellyDr. Sedegha
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SSH Primers
The following oligonucleotides are used at a concentration of 10 µM. Whenever possible, oligonucleotides should be HPLC- or gel-purified. 1. cDNA synthesis primers: 5′-TTTTGTACAAGCT(T)30-3′ 2. Adapters: (Ad1) –5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGG CAGGT-3′ 3′-GGCCCGTCCA-5′ (Ad2) – 5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGG GCGGT-3′ 3′-GCCTCCCGCCA-5′ (Ad2R) – 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGA GGT-3′ 3′-GCCGGCTCCA-5′ 3. PCR primers: (P1) – 5′-CTAATACGACTCACTATAGGGC-3′ (P2) – 5′-TGTAGCGTGAAGACGACAGAA-3′ Nested primer 1 (NP1) – 5′-TCGAGCGGCCGCCCGGGCAGGT-3′ Nested primer 2 (NP2) – 5′-AGGGCGTGGTGCGGAGGGCGGT-3′ Nested primer 2R (NP2R) – 5′-AGCGTGGTCGCGGCCGAGG