HPV16 MuE6/E7嵌合蛋白的原核表达、纯化及其免疫效果

Prokaryotic Expression,Purification and Immune Effect of HPV16 MuE6/E7 Chimeric Protein

目的表达HPV16型MuE6/E7嵌合蛋白,并检测其免疫原性及抗肿瘤活性。方法将构建的重组MuE6/E7蛋白的表达载体转化大肠杆菌BL21(λDE3)进行诱导表达,表达的包涵体蛋白经分离、纯化、变性及复性后,通过离子交换和分子筛层析进行纯化。纯化蛋白经SDS-PAGE、Western blot、HPLC和质谱分析鉴定,并免疫C57BL/6小鼠,检测其免疫原性和抗肿瘤活性。结果重组MuE6/E7蛋白相对分子质量约为21 000,表达量约为23.06%,表达形式为包涵体,经复性、纯化后,纯度达97.17%。免疫3针后,小鼠可产生高滴度的HPV特异性抗体,并且与注射剂量呈正相关。免疫小鼠能抵抗TC-1肿瘤细胞的攻击,并可延长TC-1致瘤小鼠的存活期。结论已表达并纯化了重组MuE6/E7蛋白,其对TC-1致瘤小鼠具有一定的免疫治疗作用,为进一步研制HPV治疗性疫苗奠定了基础。

Objective To express HPV16 MuE6/E7 chimeric protein and study its immunogenicity and antitumor activity. Methods Transform the constructed recombinant expression vector for MuE6/E7 protein to E. coli BI21 （λDE3） for expression under induction of IFTG. The expressed product, in a form of inclusion body, was separated, purified, de-naturalized and re-naturalized, then further purified by ion exchange and molecular sieve chromatography, and identified by SDS-PAGE, Western blot, HPLC and mass spectrography. Immunize C57BL/6 mice with the purified protein to evaluate the immunogenieity and antitumor activity. Results The expressed product, with a relative molecular mass of about 21 000, contained about 23.06% of total somatic protein and reached a purity of 97.17 % after purification. High titer specific antibody against HPV was induced in the mice immtmized with 3 doses of purified MuE6/E7 protein. The antibody titer was positively related to the dosage of purified protein. The protein protected the immunized mice against challenge with TC-1 tumor cells and prolonged the survival time of mice with tumor caused by TC-1 cells. Conclusion Recombinant MuE6/E7 protein was expressed and purified and showed a certain curative effect on the mice with tumor caused by TC-1 cells, which laid a foundation of further development of therapeutic HPV vaccine.