【摘要】 将人乳头瘤病毒(Human papillomavirus,HPV)16 L1E7基因插入携带霍乱毒素基因的pET CTA2B载体,获得HPV16L1E7与CTA2B融合表达质粒pET L1E7CTA2B。在此基础上,通过PCR基因克隆技术,将pET载体的T7启动子与pTETnir15载体的大肠杆菌硝酸盐还原酶基因B启动子nirB进行分割重组,将nirB启动子的厌氧调控元件和启动子基本元件FNR TATA盒与T7 启动子的核糖体结合位点(SD)序列融合,形成nirB T7 杂合启动子。最后获得HPV16重组减毒沙门氏细菌疫苗表达载体pNir 16L1E7CTA2B,并进一步构建对照质粒pNir 16L1E7。Western blot结果证实以上质粒nirB T7杂合启动子能够在沙门氏细菌中表达L1E7 融合蛋白。口服免疫接种小鼠可成功诱导小鼠生殖道产生HPV16 特异性粘膜免疫,且霍乱毒素CTA2B可以增强粘膜免疫诱导能力,为开发廉价有效的HPV16预防性疫苗打下了基础。更多还原

【Abstract】 Vaginal tract mucosae were the main place of Papillomaviruses replication and sexually transmission. More than 70% of women suffering from cervical intraepithelular neoplasia do not have HPV-specific antibodies in the genital secretion. Therefore, it is very important to develop vaccines, which mainly induce the mucosal immunity against Papillomavirus infection. An attenuated salmonella-based Papillomavirus vaccine was developed. pET-16 L1E7CTA2B plasmids were constructed by inserting the L1E7 DNA fragment upstream of CTA2B in pET-20b under the control of IPTG-inducible promoter T7. The pNir-16L1E7CTA2B contained the hybrid NirB promoter and were developed by digestion and reassemble of both the T7 promoter of pET-20b and the nirB of pTETnir15, so hybrid nirB-T7 promoter contained the FNR and the basic element (TATA box) form nirB and ribosome-binding site form T7 promoter, could drive the transcription and expression the target genes. The BALB/c mice were inoculated with the pNir-16L1E7- and pNir-16L1E7CTA2B- transformed S.SL3261, it could induce the HPV16 specific mucosal immunity in the vaginal tract, and the CTA2B adjuvant was able to enhance the mucosal immune reaction. This is fundamental work for further developing the cost-effective Papillomavirus vaccine in the developing countries.更多还原