人乳头瘤病毒16L1-减毒志贺氏杆菌为载体疫苗的构建及免疫效应初步鉴定

为预防高危型人乳头瘤病毒16型(HPV16)诱发宫颈癌，制备以减毒志贺氏杆菌为载体的HPV16预防疫苗，以期载体可介导机体产生粘膜免疫反应，达到预防HPVl6感染的目的。为此构建了以HPV16L1为免疫原的减毒志贺氏杆菌苗，并初步鉴定候选疫苗的减毒特性和免疫效果。利用基于志贺氏杆菌virG／icsA基因的表达载体(pHS3199)，将HPV16L1基因插入后构成pHS3199-hpv16L1质粒，电穿孔法将其转入减毒志贺氏杆菌sh42，经筛选获得重组减毒sh42-HPV16L1工程菌。用免疫印迹法检测HPV16L1蛋白表达，连续传代法确定其传代和目的蛋白表达的稳定性；豚鼠角膜巩膜炎症试验检测细菌的毒力和菌苗的免疫效果；小鼠红细胞凝集抑制试验检测免疫血清对病毒样颗粒(VLP)的中和活性。免疫印迹检测证实，重组菌株sh42-HPV16L1可稳定表达HPV16L1；豚鼠角膜巩膜炎症试验证实，该候选菌苗无致病性。减毒sh42-HPV16L1经结膜囊途径免疫豚鼠，可以产生特异性体液免疫应答，免疫动物体内的血清、肠道、阴道分泌物中抗HPV16L1 VLPIgG、IgA含量显著高于对照组，并且sh42-HPV16L1免疫动物血清可明显抑制HPV16L1 VLP引起的小鼠红细胞凝集。因而sh42-HPV16L1将是一种潜在的HPV16候选预防疫苗。

Construction, Characterization and Animal Testing of Attenuated Shigella Flexneri 5a sh42 Recombinant for Human Papillomavirus Type 16 L1 Protein

In the present study,we used recombinant attenuated Shigella flexneri strain carrying the HPV16 major capsid protein coding sequence(HPV16-L1)as candidate vaccine to induce effective mucosal immunity through mucosal route.HPV16-L1 gene was inserted into virG/icsA gene based plasmid pHS3199,and then electroporated into attenuated strains of shigella sh42,Western blot technique was used to detect the expression of HPV16L1 protein,the genetic stability of recombinant strain sh42-HPV16L1 was monitored in consecutively passaged cultures.The candidate vaccines virulence and immunogenicity were evaluated with guinea pigs keratoconjuntivitis model(Sereny test).Neutralizing activity of the antiserum was assayed by inhibition of mouse erythrocyte agglutination induced by HPV16-L1 VLP(hemagglutination inhibition test HAI).Western-blot showed that recombinant sh42-HPV16L1 could express HPV16L1 protein stably.Sereny test was negative and specific anti-HPV16L1 IgG,IgA were demonstrated in immunized animals sera,intestinal and vaginal lavages by ELISA.HAI also demonstrated that the antiserum could inhibit HPV VLP-induced mouse erythrocyte agglutination.The results above indicated that HPV16-L1 protein was consecutively expressed in sh42-HPV16L1,and the recombinant vaccine was safe and could stimulate efficient immune response in guinea pigs mucosal site through mucosal route.It may be a potent strategy for developing HPV prophylactic vaccine.