Dissertation > Medicine, health > Basic Medical > Medical Immunology > Immunobiology

Cloning and Expression of Latent and Lytic-Aassociated Genes of Kaposi’s Sarcoma-Associated Herpesviru in E.coli and Immunogenic Analysis of Its Expressed Protein

Author HuangLi
Tutor LuChun
School Nanjing Medical University
Course Microbiology
Keywords Kaposi’s sarcoma-associated herpesvirus ORF73C prokaryotic expression Western blot affinity chromatography ELISA
CLC R392.1
Type Master's thesis
Year 2004
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Kaposi’ s sarcoma-associated herpesvirus is a newly identified γ 2-herpesvirus in lesion tissue of KS, and becomes the most common neoplasm in patients infected with HIV-1. In this study, six latent and lytic-associated genes were cloned and expressed in E.coli and polyclonal antibodies against recombinant proteins were further prepared and identified.1. Cloning and sequencing of six genesBased on KSHV gene sequence, six pairs of PCR primers were designed, in which restriction enzyme cut sites were engineered at the two 5’ends of primers, respectively. ORF73C, K8.1, ORF26, ORF73N, ORF59, and ORF65 were amplified by PCR taking the BCBL-1 total DNA as templates. Amplified PCR fragments were subcloned into prokaryotic expression vector to construct six recombinant expression plasmids. Nucleotide sequences analysis and blasting showed that the isolated and cloned genes encoding ORF26, K8.1, ORF65, ORF59, ORF73N, and ORF73C have 98%, 100%, 100%, 99%, 98%, and 99% similarity to the corresponding genes which have been registered in GenBank, respectively.2. Expression of relative genes in E.coli and preparation of polyclonal antibodies against the expressed proteinsFive recombinant plasmids were transformed to E.coli strain BL21(DE3) and BL21 (RP), respectively. The expression of fusion proteins were induced by isopropyl- [3 -D-thiogalactoside (IPTG) and finally, three fusion proteins encoded by ORF73C, ORF65, and ORF26 were exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After correlative disposal, the proteins with adjuvants were used to immunize mice to produce polyclonal antibodies. Western blot analysis was carried out to confirm the activity of produced antibody. The results showed that only antibodies againstORF73C and ORF26 could react to recombinant ORF73C and ORF26 fusionprotein.3. Establishment of preliminary ELISA assay for detection of KSHV latentinfectionAfter optimising the expressing condition, most of the ORF73C recombinant protein can be soluble in the supernatant of cells lysate. The protein was purified by Glutathione Sepharose 4B using glutathione S-transferase (GST) of pGEX series. Then the purified protein was used to immunize mice to obtain immune sera. Western blot analysis was performed to examine the reactivity of the immune sera to LANA protein of KSHV. The results demonstrated that the immune sera could recognize LANA protein. Finally, a preliminary ELISA assay for detection of KSHV latent infection was established by using above-mentioned immune sera as candidate antibody to detect concentrated KSHV particles.

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