摘要
为构建能稳定表达猪繁殖与呼吸综合征病毒(PRRSV)ORF5-ORF6融合基因的重组减毒猪霍乱沙门菌,以p MD19-T-ORF5-7为模板,PCR分别扩增出ORF5和ORF6,将2个基因先后插入表达载体PYA3493,构建了重组质粒PYA3493-ORF5-ORF6。PCR、双酶切及测序结果表明,成功构建了重组质粒p YA3493-ORF5-ORF6。将该重组质粒电转至减毒猪霍乱沙门菌ΔcrpΔcyaΔasd C78-1基因缺失株,获得PRRSVORF5-ORF6融合基因重组减毒猪霍乱沙门菌ΔcrpΔcyaΔasd C78-1(p YA3493-ORF5-ORF6),并对该重组减毒猪霍乱沙门菌的生长特性、遗传稳定性和表达特性进行了测定。进一步对重组减毒猪霍乱沙门菌ΔcrpΔcyaΔasd C78-1(p YA3493-ORF5-ORF6)研究发现,其生长速度比强毒株C78-1明显减慢,与ΔcrpΔcyaΔasd C78-1缺失株及ΔcrpΔcyaΔasd C78-1(p YA3493)互补菌株基本一致。在体外连续传代能够稳定遗传ORF5-ORF6融合基因。重组菌培养上清经SDS-PAGE能检测到ORF5-ORF6蛋白条带,且经Western-blot分析证实,重组菌共表达的GP5和M蛋白能与PRRSV阳性血清特异性结合。以上结果表明,能稳定携带ORF5-ORF6融合基因的重组减毒猪霍乱沙门菌ΔcrpΔcyaΔasd C78-1(p YA3493-ORF5-ORF6)构建成功,为开发猪繁殖与呼吸综合征和仔猪副伤寒基因工程二联疫苗奠定了良好基础。
This study aimed to construct a recombinant attenuated Salmonella choleraesuis serovar Typhimurium strain for stable delivery of PRRSV ORF5-ORF6 fusion gene. Firstly,the ORF5 geneand ORF6 gene were amplifiedfrom pMD19-T-ORF5-7 by PCR,and inserted into the vector pYA3493. The results of PCR, dual-enzyme digestion and sequencing showed that recombinant plasmid pYA349 3-ORF5-ORF6 was successfully constructed. The recombinant plasmid pYA3493-ORF5-ORF6 was electro-transformed into the attenuated choleraesuis serovar Typhimurium A crp k cya A asdC78-1 to obtain the recombinant attenuated S. choleraesuis serovar Typhimurium A crp k cya a asd C78-1 （pYA3493-0RF5-ORF6） strain. Then the growth charact-eristics, genetic stability and expression property of the recombinant attenuated S. choleraesuis serovar Typhimurium crp a cya A asd C78-1 （pYA3493-0RF5-0RF6） were anal yzeck The growth rate of recombinant attenuated S. cho 1 eraesui s serovar Typhimurium z~ crp z~ cya ~ asd C78-1 （pYA3493-0RF5-ORF6） strain was more slowly than the C78-1 strain, but almost identical to A crpk cyaa asdC78-1strain and k crpzl cyaA asdC78-1 （pYk3493） strain. The ORF5-0RF6 fusion gene was inherited stably and ORF5-ORF6 protein was detected in the cultured superna tant of a crp a cya A asd C78-1 （pVA3493-OPF5-ORF6）strain. The expression product was verified with better reactinogenicity by Westernblot. These results showed that the recombinant attenuated S. choleraesuis serovar Typhimurium crp A cya asd C78-1 （pYA3493-ORF5-ORF6） strain was successfully constructed,which laid a good founda- tion for development of a bivalent vaccine against piglet paratyphoid and porcine reproductive and respiratory syndrome.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第1期47-53,共7页
Chinese Veterinary Science
基金
河南省科技攻关项目(122102110109)
