表达H1N1亚型猪流感病毒HA蛋白的重组伪狂犬病病毒rPRV-optiHA的构建及生物学特性

Construction of a recombinant pseudorabies virus expressing the HA protein of the H1N1 subtype swine influenza virus

为研发针对猪流感病毒流行毒株和猪伪狂犬病病毒变异毒株更为有效的基因工程疫苗,本研究以变异伪狂犬病病毒基因缺失毒株(rPRVΔTKΔgI/gE)为载体,采用同源重组的方法,成功构建了表达经密码子优化的H1N1亚型猪流感病毒HA基因的重组病毒rPRVΔTKΔgI/gE-optiHA。对获得的重组病毒在多种细胞上的增殖特性、一步生长曲线、遗传稳定性以及对小鼠的安全性等生物学特性进行了研究。结果表明,构建的rPRVΔTKΔgI/gE-optiHA重组病毒能够稳定表达HA蛋白,并且经过优化后,目的基因的表达量有明显提高。该重组病毒在ST、PK-15、Vero和BHK-21细胞上的增殖滴度均高于10-7.0/0.1mL,对PK-15细胞的敏感性最高。一步生长曲线结果表明,重组病毒的增殖特性未受影响。连续传代20代后,该重组病毒依然具有较好的遗传稳定性,并且对小鼠安全性良好。本研究为重组病毒rPRV-optiHA的进一步研究与应用奠定了基础。

To develop an effective genetic engineering vaccine candidate for both swine influenza virus and swine pseudorabies virus(PRV)variant,a recombinant pseudorabies virus(rPRVΔTKΔgI/ gE-optiHA)which is able to express the HA protein of the H1N1subtype swine influenza virus(SIV)was constructed by using rPRVΔTKΔgI/gE mutant as the vector.A series of further tests including the growth properties and virus titers on multiple cells,the genetic stability and the preliminary safety experiment on mice model were performed to understand the basic phenotypical characteristics of the virus.The results showed that the rPRVΔTKΔgI/gE-optiHA recombinant virus was able to express the SIV-optiHA protein after 20generations of continuous passage.The titers of the virus on ST,PK-15,Vero and BHK-21celllines were higher than 10-7.0/0.1mL,and it was more sensitive to PK-15than to other cell lines.One-step growth assay showed that the recombinant virus displayed a similar growth property to rPRVΔTKΔgI/gE,suggesting the insertion of the SIV-HA gene did not have any effect on its proliferation.Mice experiment showed that rPRVΔTKΔgI/gE-optiHAwas safe to mice.It lays the foundation for the further development and utilization of the recombinant virus rPRV-optiHA.