弓形虫ZS株P22基因片段的克隆、表达与融合蛋白的纯化  

Cloning and Expression of the P22 Gene Fragment of Toxoplasma Gondii and Purification of the Fusion Protein in ZS Strain

作　　者：陈义忠[1] 章涛[1] 彭碧文[2] 黄清玲[1] 林建银[1] 

机构地区：[1]福建医科大学分子医学研究中心,福州350004 [2]武汉大学医学院生理学教研室,武汉430071

出　　处：《福建医科大学学报》2003年第2期133-136,共4页Journal of Fujian Medical University

摘　　要：目的　克隆弓形虫 ZS株表面抗原 P2 2编码基因 ,构建原核重组表达质粒 p Thio His A,B,C/ P2 2 ,并在大肠杆菌 (Top10 )中进行表达及纯化融合蛋白。　方法　 PCR扩增及限制性内切酶 Pst 和 Bgl 双酶切 4 96 bp的弓形虫表面抗原 P2 2编码基因目的片段 ,胶回收纯化 ,插入表达质粒载体 p Thio His A,B,C多克隆位点 ,构建重组体p Thio His A,B,C/ P2 2 ,并转化大肠杆菌 (E.coli) Top10 ,筛选阳性克隆 ,以限制性酶切分析及测序鉴定后 ,以异丙基硫代β- D-半乳糖苷 (IPTG)进行诱导 ,在 E.coli Top10中表达 ,用 SDS- PAGE与免疫印迹分析表达产物并纯化。　结果　 PCR扩增出约 4 96 bp的 P2 2编码基因目的片段 ,与预期片段大小相符 ;所构建的 p Thio His A,B,C/ P2 2重组体阳性克隆经双酶切与测序鉴定 ,与预期结果一致 ;SDS- PAGE与免疫印迹显示 ,表达融合蛋白产物约 35 .4 k D。　结论　成功构建弓形虫 ZS株表面抗原 P2 2编码基因 p Thio His A,B,C/ P2 2表达质粒 ,诱导表达弓形虫表面抗原 P2 2蛋白 。Objective To perform clone and express the P22 gene fragment and purificate the fusion protein of Toxoplasma gondii ZS strain. Methods The 496 bp P22 gene fragment was amplified by PCR, then digested with PstⅠ and BglⅡ. After purification, the fragment was inserted into the polylinker of the plasmid pThioHisA, B, C. And the recombination plasmid pThioHisA, B, C was transfered into Escherichia coli(E.coli) Top10. The positive clones Top10 containing recombination were idendified by the methods of restrictive digestion and the gene sequencing. The recombination E.coli was induced by IPTG to express the fusion protein. The expressed and purified product was analyzed by SDS\|PAGE and Western\|blot. Results The recombination plasmid pThioHisA, B, C/P22 was constructed and expressed in the E.coli Top10. The results of the SDS\|PAGE and Western\|blot showed that the expressed and purified P22 was about 35.4 kD. Conlusion A fragment of the gene encoding T. gondii P22 surface protein was successfully cloned into the plasmid pThioHis A, B, C and expressed in the E.coli Top10.

关 键 词：弓形虫 鼠 克隆 分子 基因表达 重组融合蛋白质类 抗原 原虫 质粒 

分 类 号：R392[医药卫生—免疫学]