舟山眼镜蛇毒细胞毒素的克隆及原核表达  

Cloning,Expression of Cytotoxin(CTX) from Chinase Cobra(Naja Naja Atra)

作　　者：陈瑜[1] 王玥[2] 黄燕愉[3] 许云禄[3] 

机构地区：[1]福建医科大学基础医学院生化与分子生物学系,福州350004 [2]天津师范大学化学与生命科学学院,天津300387 [3]福建医科大学药学院药理系蛇毒研究所,福州350004

出　　处：《海峡药学》2010年第5期228-232,共5页Strait Pharmaceutical Journal

基　　金：福建省发改委高技术产业开发项目080886(200701)

摘　　要：目的构建舟山眼镜蛇毒细胞毒素(Cytotoxin,CTX)原核表达载体(pET42α(+)-CTX),并在大肠杆菌中表达CTX重组蛋白。方法从舟山眼镜蛇毒中分离纯化细胞毒素(CTX),测定N-末端氨基酸序列并据此设计引物,以提取的舟山眼镜蛇毒腺总RNA为模板,通过RT-PCR方法扩增细胞毒素基因序列(CTXcDNA)。将CTXcDNA定向克隆到原核表达载体pET42α(+)中,双酶切图谱、PCR和DNA测序鉴定,转化宿主菌E.coliBL21(DE3),异丙基硫代-β-D半乳糖苷(IPTG)诱导,SDS-PAGE凝胶电泳分析表达产物,GSTrap FF亲和层析纯化融合蛋白CTX;Western-blotting鉴定。结果成功合成CTXcDNA,并构建原核表达质粒pET42α(+)/GST-CTX,在大肠杆菌E.coliBL21(DE3)中表达融合蛋白CTX,SDS-PAGE凝胶浓度扫描显示表达量占菌体总蛋白的28.9%,Western-blotting证实纯化蛋白为重组CTX融合蛋白(rCTX)。结论成功构建CTX基因的原核表达载体,并在大肠杆菌中表达,获得rCTX具有眼镜蛇毒CTX的抗原性。OBJECTIVE The CTX gene was cloned into Prokaryotic expression vector pET42α（＋） and the CTX recombinant protein was expressed in E.Coli.METHODS The CTX of Naja naja atra venom Isolate and the N-terminal amino acid was determined,the primers were designed according to the sequences,one primer contain KpnI site and another primer contain SacI site.the CTX cDNA was synthesed using the mRNA from the gland of Naja naja atra by RT,the CTX gene was amplified by PCR and cloned into pET42α（＋） vector.The positive clones were screened by digestion and were confirmed by gene sequencing.We found that the gene sequence are same as the CTX gene sequence reported.The positive recombinant plasmids were transformed into BL21（DE3）,the CTX fusion protein was induced by IPTG,the over-expressed protein was checked by SDS-PAGE,the fusion protein was purified by GSTrap FF column and the protein was also identified by Western-blotting using anti-CTX antibody.RESULTS The amplified CTX cDNA by RT-PCR was cloned into pET42α（＋）vector and the cloned gene was over expressed successfully in BL21（DE3）,the SDS-PAGE results indicated that the fusion protein are soluble and the over-expressed recombinant protein are 28.9% of the total proteins of the E.Coli.The purified CTX has characters of CTX antigen confirmed by Western-blotting.CONCLUSIONS The amplified CTX cDNA by RT-PCR was cloned into pET42α（＋）vector and the cloned gene was over expressed successfully in E.Coli,the purified recombinant protein has characters of CTX antigen.

关 键 词：眼镜蛇蛇毒 细胞毒素 克隆 表达 

分 类 号：R914[医药卫生—药物化学] R915[医药卫生—药学]