人乳头瘤病毒16型E4蛋白表达、纯化及抗血清制备

目的 表达人乳头瘤病毒16型(HPV16) E4蛋白,并制备小鼠抗HPV16 E4血清.方法 将HPV16 E4基因克隆入pQE30,重组质粒pQE30-HPV 16E4经鉴定后转化大肠埃希菌M15(PREP4),诱导表达并鉴定表达产物.因纯化、变性和复性方法,制备可溶性HPV16 E4蛋白.免疫Bal B/C小鼠制备抗血清,检测小鼠IFN-γ水平、CD4/CD8比值和抗血清滴度变化.结果 酶切和测序结果表明pQE30-HPV16F4构建成功.表达分子相对分子量(Mr)为10 000,Western blot法证明具有较高特异性.小鼠抗血清效价升高,CD4/CD8无升高,小鼠γ干扰素(IFN-γ)无升高.结论 成功制备可溶性HPV16 E4蛋白和小鼠抗HPV16 E4高效价的抗血清.

Expression and purification of human papillomavirus 16 E4 protein and preparation of its antiserum

Objective To express and purify human papillomavirus type 16 (HPV16) F4 protein in prokaryotic bacteria and prepare the antiserum of HPV16 E4 protein.Methods HPV16 E4,amplified by PCR was inserted into pQE30 plasmid.The recombinant pQE30-HPV16E4 vector was transferred into M15 (pREP4).Expression product was identified after induction.Following with purification,denaturation and renaturation,soluble protein was obtained.We immunized Balb/c mice with HPV16 E4 protein.Mouse IFN-γ,CD4/CD8 ratio and antiserum titer were examined.Results Restriction digestion and DNA sequencing showed pQE30-HPV16E4 was constructed successfully.Relative molecular mass (Mr) of HPV16 E4 was 10 000 in SDS-PAGE and specificity of the protein was confirmed with Western blotting.The antiserum could specifically bind with HPV16 E4 protein.In the immunized Balb/c mice,high antiserum titer was detected,while IFN-γand CD4/CD8 ratio did not change obviously.Conclusions Soluble HPV16 E4 protein was obtained successfully.The antiserum against HPV16 E4 was prepared in mice.