采用非洲地区广泛应用的绵羊痘弱毒株构建表达小反刍兽疫病毒H蛋白的重组疫苗

摘要

为构建表达小反刍兽疫病毒(PPRV)H基因的重组绵羊痘病毒(SPV),本研究利用eGFP报告基因和gpt筛选基因筛选纯化了PPRV H基因的重组SPV (rSPV-PPRV-H).通过PCR、序列测定和间接免疫荧光的方法对rSPV-PPRV-H进行鉴定.结果显示PPRV的H基因重组至rSPV-PRRV-H中并表达PPRV H蛋白;同时病毒的生长曲线也显示外源H基因的插入并未影响亲本病毒的复制.将该重组病毒经皮内注射途径免疫绵羊,免疫剂量为105.5 TCID50,间隔4周,免疫两次,中和试验结果显示rSPV-PPRV-H免疫绵羊后能够诱导其产生高滴度的抗PPRV的特异性中和抗体,抗体转阳率为100％(6/6).本研究为rSPV-PPRV-H作为PPRV疫苗提供了实验依据.%To construct sheep pox virus expressing H protein of peste des petits ruminants virus (PPRV),in this study,we used eGFP reporter gene and gpt selective gene to screen the recombinant sheep poxvirus (rSPV-PPRV-H).The rSPV-PPRV-H was identified by PCR,sequencing and indirect immunofluorescence.The results showed that PPRV H gene was inserted into rSVP-PPRV-H and PPRV H protein could be expressed in vitro.At the same time,the growth curve of the virus also showed that the insertion of the exogenous H gene did not affect virus replication compared with that of the parent virus.Six goats were immunized with 105.5 TCID50 rSPV-PPRV-H through intradermal injection,and were immunized for the second time at 28 days with the same dose.Neutralization test results showed that rSPV-PPRV-H immunized sheep were able to induce high titers of anti-PPRV-specific neutralizing antibodies,and 100％ sheep had seroconverted (6/6).This study shows that rSVP-PPRV-H can be used as a candidate vaccine against PPRV,which has good application prospect.