人乳头瘤病毒16 E7基因的原核表达及表达条件的优化

Prokaryotic Expression of HPV16 E7 and Optimization of the Expression Conditions

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。

Objective： To express the protein of HPV16 E7 based on pET-28a（4-） at high levels and study the optimization of expression. Methods： The HPV16 ET（wild type and Hubei type） gene were amplified by PCR and cloned into pET-28a（4-）. 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（4-）-HPV16 E7 （wild type and Hubei type） were successfully constructed. High-level expression of HPV 16 ET（ wild type） was achieved in E. coli BL21（DE 3）.