CTLA-4在大肠杆菌中的克隆和表达

Gene Cloning and Expression of CTLA-4 in E． Coli

目的 在原核表达系统大肠杆菌中表达具有生物活性的人毒性 T淋巴细胞相关蛋白-4可溶性部分 (human soluble CTLA-4)。方法 PCR扩增获得 CTLA-4编码基因，利用表达载体 pGEX-2T构建重组质粒 2TC，在 BL21(DE3)宿主菌中表达。观察不同 IPTG浓度及诱导时间对蛋白表达的影响并测定其免疫活性。结果 PCR扩增所获 DNA片段与文献报道 CTLA-4序列一致。在重组大肠杆菌中 0.10 mmol/L IPTG诱导 4 h可大量生成相对分子质量 40 000的 GST-CTLA4融合蛋白。 Western blot分析表明原核表达产物 CTLA-4具抗原抗体结合活性。结论 hsCTLA-4可在原核表达系统中表达并具有免疫活性。

Objective To express hsCTLA-4 in E.coli. Methods The hsCTLA-4 gene was obtained by PCR amplification from pE plasmid which contains CTLA-4 gene and was inserted into the expression vector pGEX-2T． The recombination strain was induced by IPTG with different concentrations and time． Results The sequence of PCR amplified DNA fragments was identical with the reported CTLA-4 gene． SDS-PAGE and Western blot showed that 0.10 mmol/L IPTG can induce higher production of the fusion protein with mo1ecular weight 40 000 after addition of IPTG 4 hours and GST-CTLA4 had immuno1ogical activity． Conclusions hsCTLA-4 can be expressed in soluble form high efficiency．