【作者】 黄丽；

【导师】 卢春；

【作者基本信息】 南京医科大学 ， 微生物学， 2004， 硕士

【摘要】 卡波济肉瘤相关疱疹病毒（Kaposi’s sarcoma-associated herpesvirus,KSHV）是近年来在卡波济肉瘤（Kaposi’s sarcoma,KS）组织中发现的一种γ2型疱疹病毒，并且已成为HIV-1感染阳性患者较常出现的一种肿瘤。本研究的目的是对KSHV的六个潜伏与增殖相关基因进行克隆表达和表达产物的多克隆抗体制备与鉴定。 1．相关基因的克隆及测序鉴定 根据KSHV基因序列组成，设计六对引物并引入酶切位点，用PCR方法扩增六个目的基因，分别是ORF73C、K8.1、ORF65、ORF26、ORF73N和ORF59。将目的基因克隆至原核表达质粒，构建成六个重组质粒。重组质粒的核酸序列分析结果显示，克隆的目的基因ORF73C、K8.1、ORF65、ORF26、ORF73N和ORF59与GenBank中登记的基因序列同源性分别是99％、100％、100％、98％、98％和99％。 2．目的基因的原核表达与表达产物的多抗制备 将其中五个重组质粒分别转化大肠杆菌BL₂₁（DE₃）和BL₂₁（RP），经异丙基硫代-β-D-半乳糖苷（IPTG）诱导，SDS-聚丙烯酰胺凝胶电泳（PAGE）显示有三个重组质粒表达目的蛋白，即ORF73C、ORF26和ORF65蛋白。目的蛋白经相关处理后免疫小鼠，制备抗目的蛋白多抗。运用Western blot检测，证实两个多抗有效，即抗ORF73C和抗ORF26多抗。 3．KSHV潜伏感染ELISA检测方法的初步建立 优化ORF73C重组蛋白的表达条件，使蛋白主要以可溶状态出现在诱导表达菌上清中。借助于pGEX系统融合蛋白谷胱甘肽S-转移酶（GST），运用Glutathione Sepharose 4B亲和层析柱纯化ORF73C重组蛋白，获得纯化的目的蛋白ORF73C，并进一步免疫小鼠获得抗ORF73C抗体。采用ECL Western blot方法检测抗ORF73C抗体与BCBL-1细胞中KSHV LANA蛋白的反应特性。结果显示，该抗体与LANA反应呈阳性。进一步以浓缩的KSHV病毒颗粒为被检抗原，以ORF73C抗体为已知抗体，初步建立了KSHV潜伏感染的ELISA检测方法。更多还原

【Abstract】 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.更多还原