表达H9N2亚型禽流感病毒HA2蛋白的延迟裂解型沙门菌DNA疫苗载体的构建

Construction of regulated delayed lysis Salmonella delivering DNA vaccine expressing HA2 protein of H9N2 subtype AIV

为了构建表达禽流感病毒H9N2亚型血凝素HA2蛋白的延迟裂解型沙门氏菌载体,本试验首先将绿色荧光片段EGFP插入到真核表达载体p YA4545中,构建重组质粒p YA4545-EGFP,并转染巨噬细胞系RAW264.7,共聚焦显微镜下观察其表达情况;再通过PCR技术扩增HA2基因,将其克隆到沙门菌载体p YA4545中,构建重组质粒p YA4545-HA2,继而转染HEK-293T细胞,收集细胞总蛋白,通过Western-blot检测HA2蛋白的表达情况;最后将重组质粒电转到沙门菌宿主χ11218中,检测重组菌对阿拉伯糖的依赖性。结果,p YA4545真核表达体系能够有效表达;重组质粒p YA4545-HA2的HA2蛋白成功表达;重组菌对阿拉伯糖具有明显依赖性。本试验成功获得了以延迟裂解型沙门氏菌为载体表达禽流感病毒HA2蛋白的菌株,为后续开发以HA2为目标抗原的新型口服疫苗奠定了基础。

To construct recombinant Salmonella vector expressing HA2 protein of H9N2 subtype influenza virus,the EGFP gene was firstly inserted into pYA4545,yielding pYA4545-EGFP,which was transfected to RAW264.7 cells and the fluorescence was observed under confocal microscope.The HA2 gene was then amplified by PCR and inserted into pYA4545 to construct pYA4545-HA2.After transfection into HEK293 T cells,the total protein was collected and subjected to Western blotting;The pYA4545-HA2 was then transformed into regulated delayed lysis Salmonella χ 11218 and the dependence on arabinose was determined.The results demonstrated that EGFP and HA2 proteins were successfully expressed,and the recombinant strain was obviously dependent on arabinose for growth,indicating its possible application in oral vaccine production.