人乳头瘤病毒16型E7密码子优化后的原核表达及免疫原性研究  

Prokaryotic expression and immunogenicity of human papillomavirus type-16 E7 with codon optimization

作　　者：李菁华[1] 史红艳[1] 韩放[1] 逯茵茵[1] 徐国全 于丽 孙延波[1] 

机构地区：[1]吉林大学白求恩医学部基础医学院病原生物学系,吉林长春130021 [2]大庆市红岗区人民医院,黑龙江大庆163000

出　　处：《微生物学免疫学进展》2014年第2期7-11,共5页Progress In Microbiology and Immunology

基　　金：吉林省科技厅资助课题(吉林省科技发展计划项目:应用基础200705374)

摘　　要：目的构建人乳头瘤病毒16型(HPV16)E7基因密码子优化后的原核表达系统,通过特异性抗体制备评价E7融合蛋白的免疫原性。方法人工合成优化后的HPV16 E7基因,利用特异引物扩增HPV16 E7基因。将E7基因连接至原核表达载体构建重组表达质粒pMAl-c2X-E7,转化至感受态细胞DE3后,经IPTG诱导,SDS-PAGE分析表达产物E7融合蛋白。纯化后的E7融合蛋白免疫动物,采用ELISA检测动物血清效价。结果 E7基因PCR产物的测序结果与优化后的目的基因序列比对一致。表达的E7融合蛋白经SDS-PAGE分析表明:在相对分子质量50 000处有特异性表达带,与预期相符。以E7融合蛋白制备的多克隆抗体其血清效价可达1∶64 000。结论成功构建的重组表达质粒pMAl-c2X-E7可有效表达MBP-E7融合蛋白,E7融合蛋白具有良好的免疫原性。Objective To construct a prokaryotic expressing system that harbors human papillomavirus type-16 （HPV16） E7 gene with codon optimization and to evaluate the immunogenicity of fusion protein E7 through preparation of specific antibodies. Methods HPVl6 E7 gene with codon optimization was artificially synthesized and amplified by using specific primers. E7 gene was cloned into prokaryotic expressing vector to construct recombinant expressing plasmid pMAI-c2X-E7. After transfection of pMAI-c2X-E7 to DE3 competent cell with induction by IPTG, fusion protein E7 was analyzed by SDSPAGE. Animals were immunized with the fusion protein E7, and ELISA was employed to detect serum titer. Results The sequence of amplified E7 gene is identical to sequence with codon optimization. SDS-PAGE analysis showed that expressed fusion protein E7, with a relative molecular mass of about 50 000, could be found. The titer of specific polyclonal antibodies in immunized serum against fusion protein E7 reached to 1 ： 64 000. Conclusion Recombinant expressing plasmid pMAI-c2X-E7 could express fusion protein ET, effectively; and fusion protein E7 has a perfect immunogenicity.

关 键 词：人乳头瘤病毒16型 原核表达 多克隆抗体 

分 类 号：R392[医药卫生—免疫学] R737.33[医药卫生—基础医学]