人类睾丸凋亡相关基因蛋白(TSARG2)在大肠杆菌中的表达和纯化  

Expression and Purification of Human Testis Spermatocyte Apoptosis-related Gene,Tsarg2 Protein in E.coli

作　　者：邓云[1,2] 聂东宋[1,2] 卢光琇[1,2] 

机构地区：[1]人类干细胞国家工程研究中心 [2]中南大学生殖与干细胞研究所,湖南长沙410078

出　　处：《怀化学院学报》2006年第8期70-73,共4页Journal of Huaihua University

基　　金：国家重点基础研究发展规划项目973课题:人类精子发生相关新基因的筛选及克隆(G1999055901).

摘　　要：目的获得重组TsARG2基因蛋白,为进一步研究其功能和制备特异抗体奠定基础.方法根据已报道的TsARG2基因序列,采用RT-PCR的方法获得TsARG2基因.将所得的PCR产物插入原核表达载体pQE30中,得重组质粒(pQE/TSARG2)并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有六个组氨酸的融合蛋白,采用镍固定金属亲和层析法纯化.结果序列分析表明TSARG2基因成熟肽编码区含有918bp,编码305个氨基酸;与GenBank(AY040204)中已报道的TSARG2分离物的核苷酸序列有100%同源性.经IPTG诱导表达,SDS-PAGE电泳分析显示,表达的融合蛋白占菌体蛋白总量的66%,分子质量约为35kDa.经Ni2+-NTAagarose纯化获得SDS-PAGE电泳下单一条带.结论在大肠杆菌中获得了TsARG2基因蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础.Objective To obtain recombinant TSARG2 protein by prokaryotic expression for its functioned study. Methods The encoding sequence for mature peptide of human TSARG2 was amplified with RT- PCR and inserted into pQE30 vector to establish the prokaryotic expressing system coupled with 6 × His. The competent cells of host strain of M15 were transformed by the recombinant plasmid （pQE30/TSARG2）. Expression of the target protein was induced with IPTG and assayed by SDS - PAGE after sequenclng. The recombinant products were purified by Ni^2＋ - NTA agarose column. Results： The cloned fragment of human TSARG2 was 100% consistent with that in Genbank （AY040204）, which was deduced to express 305 Aa mature peptide of human TSARG2 correctly. The expressed fusion protein was 35 kD in SDS- PAGE as expected and 66% in the total quatiy of germ proteins. Conclusion ： The homologous recombinant human TSARG2 was obtained after Ni - affinity chromatograph, which could be used for its functioned study and preparation of specific antibodies.

关 键 词：TSARG2 表达与纯化 大肠杆菌 

分 类 号：Q781[生物学—分子生物学]