人乳头瘤病毒18型E2基因的原核表达载体构建与表达

Prokaryotic Expression of HPV18 E2

目的将人乳头瘤病毒18型(Human papillomavirus 18,HPV18)E2基因插入到原核表达载体pET28a(+)T7启动子下游,构建原核表达载体pET28a(+)-HPV18E2,原核表达并纯化HPV18E2蛋白,为研究HPV18 E2蛋白相关疫苗奠定实验基础。方法1)以HPV18型基因组DNA为模板,运用聚合酶链反应扩增E2基因,将其插入到原核表达载体pET28a(+)中,构建重组表达质粒pET28a(+)-HPV18E2;2)PCR、限制性内切酶酶切分析以及核酸序列测定重组质粒pET28a(+)-HPV18E2的正确性;3)将重组表达质粒pET28a(+)-HPV18E2转化大肠杆菌BL21(DE3),IPTG诱导表达,经镍螯合亲和层析胶体纯化HPV18E2蛋白;4)十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定HPV18E2蛋白的正确表达。结果1)PCR、限制性内切酶酶切分析和核酸序列分析鉴定证实了重组表达质粒pET28a(+)-HPV18E2的成功构建;2)SDS-PAGE电泳分析,HPV18E2蛋白在大肠杆菌BL21中表达量占菌体总蛋白的21%,纯化后的蛋白纯度大于90%;经Western blot鉴定,显示HPV18E2蛋白与标签蛋白(6xHis)形成相对分子质量约42KD的蛋白,与理论预计值相符。结论1)成功构建了原核表达载体pET28a(+)-HPV18E2;2)原核表达获得了HPV18E2蛋白。

Objective Construction of Prokaryotic Expression vector,pET28a（＋）-HPV18E2 expressing human papillomavirus（HPV） 18 E2 gene.Prokaryotic expression and purification of HPV18 E2 protein,for future study of HPV18 E2 vaccine against HPV related cervical cancer.Methods 1） Amplification of HPV18 E2 gene cDNA by PCR from HPV 18 genomic DNA template,insert of E2 cDNA fragment into prokaryotic expression vector pET28a（＋）,to construct recombinant prokaryotic expression vector pET28a（＋）-HPV18E2;2） Confirmation of the recombinant prokaryotic expression vector by PCR,restrictional enzymes digestion and DNA sequencing;3） Transformation of BL21 E.coli with pET28a（＋）-HPV18E2.Expression of HPV18 E2 protein by IPTG induction and protein purification with Ni-NTA gel chromatography column;4） E2 protein expression was verified by SDS-PAGE and Western blotting.Results 1） The construction of recombinant plasmid was confirmed by PCR,double enzymes digestion and DNA sequencing.2） Expression of HPV E2 protein was purified and analyzed by SDS-PAGE and Western blotting assay and it was shown that the protein prokaryotic expressed was corrected and purified.Conclusions HPV18 E2 protein was prokaryotic expressed and purified,and would be used for future study of vaccine against HPV related cervical cancer.