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。

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-16L1E7CTA2