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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。
To construct recombinant live attenuated Salmonella choleraesuis vaccine strain expressing GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV), the PRRSV ORF5 gene with signal peptide sequence was cloned by PCR and inserted into the expression vector pYA3493 to construct Recombinant plasmid pYA3493-dORF5. The recombinant plasmid was electroporated into S. choleraesuis gene deletion strain (deletion of crp, asd gene) to obtain recombinant attenuated Salmonella choleraesuis ΔcrpΔasdC78-1 (pYA-dORF5) of PRRSV ORF5 gene. The phenotypic traits, growth characteristics, stability and safety of the recombinant bacteria were determined. The GP5 protein expressed by recombinant bacteria was analyzed by SDS-PAGE and Western-blot. The results showed that this strain retained the ability to survive in a DAP negative environment; it could not utilize carbon sources such as maltose, sucrose and fructose, but retained the ability to utilize glucose, and its growth rate was lower than that of the virulent strain C78-1 but not with ΔcrpΔasdC78 -1 was not significantly different; continuous passage in vitro can stably inherit dORF5 gene fragment. Western-blot analysis confirmed that the GP5 protein expressed by the recombinant bacteria could specifically bind to the PRRSV positive sera. The virulence of the virulent strain C78-1 and the attenuated Salmonella choleraesuis strain C500 by oral route were respectively determined by the recombinant strains ΔcrpΔasdC78- 1 (pYA-dORF5) 492-fold and 1.31-fold. The results showed that oral recombinant attenuated Salmonella choleraesuis strain which can stably express PRRSV GP5 protein was successfully constructed, which laid a good foundation for the research of PRRSV oral genetically engineered vaccine.