JC病毒t抗原的融合蛋白表达及抗体制备  

Expression of t antigen fusion protein of JC virus and preparation of its polyclonal antibody

作　　者：郑铁龙[1] 王殿丽[2] 李兴旺[1] 毛羽[1] 洪源[1] 王琦[1] 成军[1] 

机构地区：[1]北京地坛医院传染病研究所,100011 [2]大连市第二人民医院药剂科

出　　处：《中华传染病杂志》2009年第7期403-407,共5页Chinese Journal of Infectious Diseases

基　　金：北京市科委资助项目（D0906003040291）

摘　　要：目的构建JC病毒t抗原的原核融合蛋白表达载体，表达并纯化该融合蛋白。方法采用PCR方法从患者脑脊液中扩增JC病毒t抗原，测序正确后再克隆入pET32a（＋）质粒，构建pET32a（＋）-t表达重组体，并诱导表达t抗原融合蛋白。大量制备该融合蛋白并以镍柱亲和层析纯化。然后以纯化蛋白免疫小鼠制备多克隆抗体。结果pET32a（＋）-t表达出相对分子质量为41000左右的重组蛋白。十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SD-PAGE）分析显示，异丙基-β-D硫代半乳糖苷诱导后3．5～20．0h融合蛋白均高水平表达。Western印迹证实具有良好的抗原性，免疫BALB／c小鼠后，成功制备了鼠多克隆抗体。结论成功构建原核表达载体pET32a（＋）-t，表达纯化t抗原融合蛋白，成功制备了JC病毒小包膜蛋白t的抗体，为进一步流行病学调查及该基因的功能研究奠定了基础。Objective To construct prokaryotic expression vector carrying JC virus （JCV） t-antigen gene, express and purify this fusion protein. Methods The JCV t-antigen gene from a cerebrospinal fluid sample was amplified using polymerase chain reaction （PCR） method. After sequencing, the gene was cloned into plasmid pET32a （＋） to construct recombinant prokaryotic expression vector pET32a（＋）-t. The tantigen fusion protein was expressed by isopropy-β-D-thiogalactoside （IPTG） induction and prepared in large scale, then purified by Ni＋ affinity column chromatography. The polyclonal antibody was obtained from the BALB/c mouse immunity by the purified protein. Results The relative molecular mass of recombinant protein expressed by pET32a （＋）t was about 41 000. Sodium dodecylsulfate-polyacrylamide gel electrophoresis （SDS-PAGE） showed that the fusion protein was highly expressed after 3. 5- 20. 0 h of IPTG induction. The antigenicity of the purified protein was well confirmed by Western blot. The anti-mouse polyclonal antibody was obtained successfully from immunized BALB/c mice. Conclusions The prokaryotic expression vector pET32a（＋）-t is successful constructed and the fusion protein is expressed and purified. Furthermore, the antibody of JCV small envelop protein t is successfully prepared. This work provides valuable information for further study on epidemiology and biological function of t antigen.

关 键 词：JC病毒 抗原 病毒 肿瘤 抗体生成 基因表达 重组融合蛋白质类 

分 类 号：R686[医药卫生—骨科学]