摘要目的 建立人乳头瘤病毒(human papillomavirus,HPV)16型E7蛋白的原核表达系统,表达HPV16 E7蛋白,制备小鼠抗HPV16 E7血清.方法 采用PCR方法扩增HPV16 E7基因,构建入pET21b质粒,重组表达载体经鉴定后转化大肠埃希菌BL21(DE3).诱导表达后经十二烷基硫酸钠(so-dium dodecyl sulphate,SDS)-聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和Western blot鉴定表达产物.提取包涵体进行变性复性处理后纯化,纯化后的可溶性蛋白免疫Balb/C小鼠,检测小鼠体内 γ-干扰素(interferon-γ,IFN-γ)水平、CD4/CD8比值和抗血清滴度变化.结果 PCR扩增片段为0.3 Kb,酶切和序列测定证实重组质粒构建正确.SDS-PAGE在11 KD处出现蛋白条带.蛋白表达以包涵体为主.免疫后小鼠抗体效价升高,CD4/CD8比值无升高,IFN-γ无反应.结论 成功制备可溶性HPV16E7蛋白和小鼠抗HPV16E7高效价抗血清.

abstractsObjective To express and purify human papillomavirus type 16(HPV16)E7 protein from bacteria and to prepare the antiserum of HPV16 E7 protein. Methods HPV16 E7 amplified by PCR was in-serted into pET21b. The recombinant pET21b-HPV 16E7 vector was transferred into BL21(DE3). The expres-sion product was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting. BALB/ c mice were immunized with HPV16E7 soluble protein which was obtained by purifica-tion,denaturation and renaturation of the inclusion bodies. IFN-γ,CD4 / CD8 ratio,and antiserum titer were ex-amined. Results PCR fragment of HPV16 E7 was 0. 3Kb. Restriction digestion and DNA sequencing showed that pET21b-HPV 16E7 was constructed successfully. Relative molecular mass(Mr)of HPV16 E7 was 11000 in SDS-PAGE. In the immunized Balb/ c mice,high antiserum titers were detected,while IFN-γ and CD4 / CD8 rati-o did not change obviously. Conclusion Soluble HPV16 E7 protein and the antiserum against HPV16 E7 were obtained successfully.