以沙门氏菌菌毛为载体的重组HIV-1 447-52D表位疫苗的构建和毒力检测

目的 构建菌毛上表达HIV-1 447-52D表位的减毒Salmonella typhimurium ST14028-3b aroA-44752D,并对其毒力进行检测.方法 利用同源重组技术,将447-52D表位整合至沙门氏菌细聚合菌毛上,Western blot检测其表达情况,通过刚果红结合实验分析外源表位对菌毛吸附能力的影响.感染动物后检测重组菌的毒力及其在小鼠体内的分布.结果 重组菌的嵌合基因片段测序结果和嵌合蛋白的表达分析表明,重组菌菌毛上成功表达外源447-52D表位.重组菌携带外源表位后对刚果红吸附的能力没有改变,不影响菌毛的正常生物学活性.动物感染实验结果显示,重组菌灌胃感染BALB/c小鼠的LD50为1.45×109 CFU,与野生型减毒株相似,且重组菌可以有效地分布于感染小鼠的派氏淋巴结和脾脏中.结论 成功构建菌毛携带HIV-1 447-52D表位的减毒Salmonella typhimurium ST14028-3b aroA-44752D.菌毛携带外源表位后不影响减毒菌的毒力和并在体内分布,有望作为为口服HIV-1疫苗的候选并对其他病原体表位疫苗的研究提供了新的思路.

Construction and infectivity analysis of live attenuated recombinant salmonella expressing HIV-1 447-52D epitope on fimbriae

Objective To construct recombinant attenuated Salmonella expressing the HIV-1 447-52D epitope on the thin aggregative fimbriae and to analyze the infectivity status of this recombinant strain.Methods The recombinant attenuated Salmonella ST-44752D was constructed by using pHSG415 gene replacement system to replace the wild-type agfA gene with the chimeric agfA:44752D gene and then verified by polymeruse chain reaction(PCR) and sequencing.The expression of the recombinant thin aggregative fimbriae was analyzed by Western blotting.Congo red binding test was performed to examine the adsorbability of fimbriae.50％ lethal dose (LD50) and organ distribution status of the 447-52D strain were determined in mice.Results The 447-52D expressing recombinant strain was successfully constructed and confirmed by DNA sequencing.Compared with wild-type ST,recombinant ST-44752D strain showed similar expression level of the thin aggregative fimbriae AgfA,and similar binding activity to Congo red reagent.The LD50 value of ST-44752D strain in mice was 1.45 × 109 CFU.After infection,ST-44752D strain could distribute effectively in spleen and lymph node,similar to wild-type ST.Conclusion The recombinant attenuated Salmonella typhimurium ST-44752D harboring HIV-1 447-52D epitope on fimbriae was successfully constructed.The 447-52D epitope within AgfA did not influence AgfA expression,fimbrial adsorbability,bacterial LDS0 value or organ distribution in mice.