HP-PRRSV重组新城疫病毒载体疫苗构建及小鼠免疫评价

Construction and immunogenicity evaluation of recombinant NDV vaccine for HPPRRSV in mice

高致病性猪繁殖与呼吸综合征(highly pathogenic porcine reproductive and respiratory syndrome,HP-PRRS)在中国已流行多年,国内疫苗主要以减毒活疫苗为主,能有效保护猪不受病毒侵害,但易与野外毒株重组,导致新的变异株出现,而灭活苗安全性好但是疫苗效价低,因此本研究旨在研制一种不会重组且安全有效的疫苗。本研究利用反向遗传技术将HP-PRRSV具有抗原活性的GP5蛋白序列插入到新城疫病毒(Newcastle disease virus,NDV)载体,拯救稳定表达GP5的重组病毒。通过PCR、酶切、Western blot、间接免疫荧光(IFA)和毒力检测试验对重组病毒进行鉴定,结果表明,重组NDV rL-GP5_((LQ))拯救成功,能够稳定表达GP5蛋白。小鼠的疫苗免疫效果评价结果表明,重组NDV rL-GP5_((LQ))二免后14 d特异性抗体酶标检测450 nm吸光度值为0.47(P<0.01),中和抗体为1∶8(P<0.01),在小鼠体内能诱导有效的抗体表达。脾淋巴细胞增殖试验中rL-GP5_((LQ))组显著升高(P<0.01),说明rL-GP5_((LQ))能在体内诱导免疫应答,为防控HP-PRRS提供了新的疫苗候选和思路。

Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)has been raging in China for a long time,domestic vaccines are mainly live attenuated vaccines,which can effectively protect pigs from viruses,but are easy to recombine with wild strains,resulting in the emergence of new variants.Inactivated vaccines are safe but low vaccine titer,so this study is to develop a safe and effective vaccine that will not recombinant.Reverse genetic technology was used to insert HP-PRRSV antigenically active GP5 gene sequence into Newcastle disease virus vector.Recombinant viruses that stably express GP5 was rescued,identified by PCR,enzyme digestion,Western blot,indirect immunofluorescence(IFA)and virulence detection experiments.The results showed that the D value of specific antibody at 35 d was 0.47(P<0.01),and neutralizing antibody reached 1:8(P<0.01),which could induce efficient antibody expression in mice.The proliferation of splenic lymphocytes in rL-GP5_((LQ))group was significantly increased(P<0.01),indicating that rL-GP5_((LQ)) could induce the immune response in vivo,providing new vaccine candidates and ideas for the prevention and control of HP-PRRS.