表达PRRSV GP5蛋白重组减毒猪霍乱沙门菌C78-1的构建及生物学特性

Construction and biological characteristics of recombinant attenuated Salmonella choleraesuis C78-1 strain expressing GP5 antigen of porcine reproductive and respiratory syndrome virus

为了构建表达猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白口服重组减毒猪霍乱沙门菌活载体疫苗株,通过PCR克隆去除信号肽序列的PRRSV ORF5基因,将其插入表达载体pYA3493中,构建了重组质粒pYA3493-dORF5。将该重组质粒电转入猪霍乱沙门菌基因缺失株(缺失crp,asd基因),获得PRRSV ORF5基因减毒猪霍乱沙门菌ΔcrpΔasdC78-1(pYA-dORF5)重组菌。对重组菌表型性状、生长特性、稳定性及安全性进行测定,并对重组菌表达的GP5蛋白进行SDS-PAGE和Western-blot分析。结果显示,该菌株保留了在DAP阴性环境中生存的能力;不能利用麦芽糖、蔗糖及果糖等碳源,但保留了利用葡萄糖的能力,其生长速度低于强毒株C78-1,而与ΔcrpΔasdC78-1相比变化不明显;在体外连续传代能够稳定遗传dORF5目的基因片段。Western-blot分析证实重组菌表达的GP5蛋白能与PRRSV阳性血清特异性结合;通过口服途径测得亲本强毒株C78-1和猪霍乱沙门菌弱毒疫苗株C500的毒力分别是重组菌ΔcrpΔasdC78-1(pYA-dORF5)的492倍和1.31倍。表明,成功构建了能稳定表达PRRSV GP5蛋白的口服重组减毒猪霍乱沙门菌,为研究PRRSV口服基因工程疫苗奠定了良好的基础。

In order to construct a recombinant attenuated Salmonella choleraesuis oral live vector strain expressing GP5 antigen of porcine reproductive and respiratory syndrome virus （PRRSV）, the PRRSV ORF5 gene with deletion of the N-terminal signal peptide sequence was cloned and inserted into the pYA3493 vector. The recombinant plasmid pYA-dORF5 was finally electro-transformed into attenuated S. choleraesuis AcrpAasdC78-1. The recombinant strain AcrpAasdC78-1 （pYA-dORFS） was identified by GP5 protein expression,phenotypic traits, stability, growth curve and safety in vitro. The growth ability of recombinant strain AcrpAasdC78-1（pYA-dORFS） in a circumstance without DAP was persisted. The abili- ty of fermentation for maltose, sucrose, fructose was lost, but the recombinant can ferment glucose. Com- pared with a C78-1 strain, the growth of recombinant strain AcrpAasdC78-1 （pYA-dORFS） was reduced significantly, but the growth was almost the same as that of AcrpAasdC78-1. The gene of dORF5 was be transmit- ted stably in the liquid nutrient medium of Luria Bertani after 60 generations. Furthermore, the successfulexpression of dORF5 gene was confirmed by SDS-PAGE. The expression product with better reactinogeni- city was verified by Western-blot. The KM mouse lethal experiment showed that the virulence of C78-1 and C500 was 492 times and 1.31 times for the recombinant strain AcrpAasdC78-1（pYA-dORF5） through oral infection, respectively. These results showed that Acrl）AasdC78-1 （pYA-dORF5） can be used to carry foreign gene stably as a live carrier of vaccines.