CAJ | 학술논문

目的：构建HPV18E7基因重组质粒，并探索其在大肠杆菌中的最佳表达条件。方法以提取的 HeLa细胞株中的DNA为模板PCR扩增 HPV18E7基因；将HPV18E7基因与载体pET-32a（＋）连接为重组质粒pET-32a（＋）-HPV18E7；将该重组质粒转入大肠杆菌BL21-DE3-pLysS细胞中，探索优化表达的条件，以获得大量HPV18E7致癌蛋白。结果 PCR扩增的目标基因大小序列与HeLa细胞中的HPV18E7基因的大小序列一致。用LB培养基，IPTG和乳糖诱导表达显示不同浓度、不同温度、不同诱导起始量等表达量均不高，尝试用ZYM-5052自动诱导培养基诱导，HPV18E7融合蛋白的表达量远远高于用异丙基-β-D-硫代吡唃半乳糖苷（IPTG）和乳糖诱导表达的表达量。结论测序正确的 HPV18E7重组质粒在自动诱导培养基ZYM-5052中获得远远高于用异丙基-β-D-硫代吡唃半乳糖苷（IPTG）和乳糖诱导表达的HPV18E7融合蛋白。
목적：구건HPV18E7기인중조질립，병탐색기재대장간균중적최가표체조건。방법이제취적 HeLa세포주중적DNA위모판PCR확증 HPV18E7기인；장HPV18E7기인여재체pET-32a（＋）련접위중조질립pET-32a（＋）-HPV18E7；장해중조질립전입대장간균BL21-DE3-pLysS세포중，탐색우화표체적조건，이획득대량HPV18E7치암단백。결과 PCR확증적목표기인대소서렬여HeLa세포중적HPV18E7기인적대소서렬일치。용LB배양기，IPTG화유당유도표체현시불동농도、불동온도、불동유도기시량등표체량균불고，상시용ZYM-5052자동유도배양기유도，HPV18E7융합단백적표체량원원고우용이병기-β-D-류대필곡반유당감（IPTG）화유당유도표체적표체량。결론측서정학적 HPV18E7중조질립재자동유도배양기ZYM-5052중획득원원고우용이병기-β-D-류대필곡반유당감（IPTG）화유당유도표체적HPV18E7융합단백。
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .