SSA抗原真核表达载体的构建  

Construction of Eukaryotic Expression Vector of SSA Antigen

作　　者：郑宗富 兰小鹏[2] 杨湘越[2] 苏东辉[3] 

机构地区：[1]南京军区福州总院四七六临床部检验科,350002 [2]南京军区福州总医院全军检验医学研究所 [3]福建医科大学

出　　处：《中国误诊学杂志》2008年第17期4040-4042,共3页Chinese Journal of Misdiagnostics

基　　金：福建省青年人才科技创新基金资助项目(编号:2002J060)

摘　　要：目的:通过基因克隆构建表达人干燥综合征抗原A(SSA)的巴斯德毕赤酵母分泌型表达载体。方法:从人白血病淋巴细胞HL-60株中提取RNA,反转录出cDNA,然后以cDNA为模板,通过PCR法扩增目的基因SSA,经纯化、双酶切、DNA浓缩回收,插入到酵母分泌型表达载体pPIC9k,用热冲击法转化感受态细胞DH5α,筛选阳性克隆提取质粒,双酶切鉴定并测序。结果:PCR产物大小符合要求,重组质粒pPIC9k-SSA双酶切鉴定与预期一致,测序结果正确。结论:成功构建SSA毕赤酵母分泌型表达载体,为今后获取高纯度、高产量的SSA抗原,研制新型抗SSA诊断试剂盒作前期准备。Objective：To construct and express the Pichia pastoris secreted vector of human SSA antigen from gene cloning. Method：With reverse transcription method, cDNA was prepared from RNA that was extracted from human lymphocyte leukemia （HL-60）. It was then used as template to prepare the target gene SSA through PCR amplified method. The obtained SSA mixture was purified,double digested,and DNA was recovered. SSA was then inserted into pastoris secreted vector pPlC9k,and transferred to competent cells DH5α using thermal shock method. The plasmid was extracted after screening the positivecells and cloning, and then detected and sequenced by double digest. Results：The size of PCR product was in the required range. The double digest results of recombinant plasmid of pPIC9k-SSA were in consistent with that expected. The sequenced results were also correct. Conclusion ：The Pichia pastoris secreted vector of SSA is successfully constructed. This will provide clues and early preparation for future product of high purified and highly productive SSA antigen,as well as development of novel anti-SSA diagnosis reagent kit.

关 键 词：干燥综合征/免疫学 核糖核蛋白类/遗传学/生物合成 转染 毕赤酵母/遗传学 遗传载体 

分 类 号：R442.8[医药卫生—诊断学]