CAJ | 학술논문

本研究旨在以复制缺陷型人5型腺病毒为载体,构建一株同时表达猪繁殖与呼吸综合征病毒(PRRSV)变异株GP5蛋白和猪瘟病毒(CSFV)E2蛋白的重组腺病毒疫苗.首先利用重叠PCR将GP5和E2基因通过口蹄疲病毒(FMDV)2A序列连接,形成一个完整的ORF,并将其克隆到腺病毒穿梭载体中,通过细茵内同源重组构建共表达PRRSV GP5蛋白和CSFV E2蛋白的重组腺病毒(rAdV-GP52AE2).间接免疫荧光试验和western blot检测证实2个外源基因均获得表达.在小鼠上进行的免疫效力评价结果显示,rAdV-GP52AE2免疫组针对CSFV的中和抗体滴度可达1:128,针对PRRSV的中和抗体滴度为1:16;在淋巴细胞增殖试验中,免疫组与阴性对照组在增殖指数上有显著差异,表明该重组腺病毒可以同时诱导抗PRRSV和CSFV的特异性体液免疫和细胞免疫.这些结果显示,利用具有自动剪切功能的FMDV 2A多肽构建的重组腺病毒有望开发成一种同时预防PRRS和CSF的新型载体疫苗.
본연구지재이복제결함형인5형선병독위재체,구건일주동시표체저번식여호흡종합정병독(PRRSV)변이주GP5단백화저온병독(CSFV)E2단백적중조선병독역묘.수선이용중첩PCR장GP5화E2기인통과구제피병독(FMDV)2A서렬련접,형성일개완정적ORF,병장기극륭도선병독천사재체중,통과세인내동원중조구건공표체PRRSV GP5단백화CSFV E2단백적중조선병독(rAdV-GP52AE2).간접면역형광시험화western blot검측증실2개외원기인균획득표체.재소서상진행적면역효력평개결과현시,rAdV-GP52AE2면역조침대CSFV적중화항체적도가체1:128,침대PRRSV적중화항체적도위1:16;재림파세포증식시험중,면역조여음성대조조재증식지수상유현저차이,표명해중조선병독가이동시유도항PRRSV화CSFV적특이성체액면역화세포면역.저사결과현시,이용구유자동전절공능적FMDV 2A다태구건적중조선병독유망개발성일충동시예방PRRS화CSF적신형재체역묘.
Classical swine fever (CSF) and porcine reproduction and respiratory syndrome (PRRS) are both economically important,highly contagious diseases worldwide.To develop an effective vaccine to control the two diseases,we constructed a recombinant adenovirus (rAdV-GP52AE2) using a replication-defective human adenovirus serotype 5 (HAd5) as a delivery vector,to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus (PRRSV) and the E2 protein of classical swine fever virus (csrv).The 2A peptide of Foot-and-mouth disease virus (FMDV) was used as a linker between GP5 and E2 proteins to allow automatic self-cleavage of the expressed fusion protein.Expression of the GP5 and E2 genes were detected by immunofluorescenee assay (IFA) and western blot.The results showed that rAdV-GP52AE2 could induce both effective humoral and cellular immune responses in mice.Our study provided supporting evidence for the use of FMDV 2A in the development of a novel multi-gene engineering vaccine.