人乳头瘤病毒16 E7基因的原核表达及表达条件的优化

目的:构建pET-28a(+)-HPV16 E7原核表达质粒,并对其表达条件进行优化,为进一步研究HPV16E7致病机制及HPV疫苗提供相应的技术平台。方法:应用基因重组技术,构建pET-28a(+)与HPV16 E7(标准株和湖北株)的原核表达重组质粒,用PCR、限制性内切酶酶切和核酸序列检测对重组质粒DNA进行鉴定,将其转入宿主菌E.coliBL21(DE 3)进行诱导以表达蛋白;SDS-PAGE及Western blot检测鉴定表达蛋白的分子量及特异性。优化诱导表达的条件,如温度、IPTG浓度、适用的培养基,探讨不同温度下蛋白的表达形式;纯化回收HPV16 E7蛋白。结果:PCR、限制性内切酶酶切和核酸序列检测证实重组质粒中插入的目的基因,其DNA大小、方向、插入位点和阅读框架均正确;HPV16 E7(标准株)蛋白得到高效原核表达,最佳表达条件:表达HPV16 E7的工程菌BL21(DE 3)的最佳诱导温度是37℃,诱导剂IPTG的浓度为0.3-0.4 mmol/L,最佳培养基为1.5倍LB。结论:pET-28a(+)-HPV16 E7标准株和湖北株重组载体构建成功,人乳头瘤病毒16 E7标准株获得高效原核表达。

Prokaryotic Expression of HPV16 E7 and Optimization of the Expression Conditions

Objective: To express the protein of HPV16 E7 based on pET-28a(+) at high levels and study the optimization of expression.Methods: The HPV16 E7(wild type and Hubei type) gene were amplified by PCR and cloned into pET-28a(+).The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG.SDS-PAGE and Western blot analysis were used to detect the confusion protein.Finally,the optimization of expression conditions,such as temperature,concentration of IPTG and media,were studied.Results: The recombinant plasmids(wild type and Hubei type) were identified and confirmed with enzyme digestion and sequencing.SDS-PAGE and Western blot showed that a confusion protein(wild type) of(20 000) in molecular weight was expressed.The optimum conditions of expression were 37 ℃,(0.3)-0.4 mmol/L IPTG and 1.5×LB.Conclusion: Prokaryotic expression vector pET-28a(+)-HPV16 E7(wild type and Hubei type) were successfully constructed.High-level expression of HPV 16 E7(wild type) was achieved in E.coli BL21(DE 3).