摘要
本研究利用对应于ＡＴＣＣＶＲ－２３３２及ＬＶＯＲＦ７基因保守序列的１对均为２８个碱基的引物１００１１ＰＣＳ、１０１０ＰＣＲ对ＰＲＲＳＶ中国分离株Ｂ１３进行ＲＴ－ＰＣＲ，结果扩增出了１条包括完整ＯＲＦ７基因的５１０ｂｐ的ＤＮＡ片段。纯化此扩增产物，并对其进行ＥｃｏＲＩ／ＰｓｔＩ双酶切，与用ＥｃｏＲＩ、ＰｓｔＩ双酶切及碱性磷酸酶处理的ＰＵＣ１８载体连接。转化大肠杆菌。结果得到了１个Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒ＰＵＣ１８Ｂ１３ＯＲＦ７１０。通过ＥｃｏＲＩ／ＰｓｔＩ及ＰＣＲ证明此重组质粒即为Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒。从而为ＯＲＦ７基因及所表达的核衣壳蛋白进一步研究奠定了基础。
The viral genome RNA of China isolate B 13 of PRRSV was amplified by RT PCR using the primer pair 1011PCS and 1010PCR which are both 28bp in lenth designed according to the conserved sequence of ORF 7 gene of ATCCVR 2332 and LV.A DNA fragment was amplified which contains complete ORF 5 gene and the size was about 510bp.The amplified product was purified and digested with EcoRI/PstI,and finally ligated into PUC18 plasmid vector which had been digested with EcoRI/PstI and treated with CIAP.Transferring it into E.coli DH 5α , a recombinant plasimid of B 13 ORF 7 gene and PUC18 vector were obtained.Using the methods of digestion with EcoRI/PstI and PCR,the recombinant plasmid was identified that it is recombined with B 13 ORF 7 gene and PUC18 vector.This research will lay a foundation for further study on ORF 7 gene and capsid protein expressed by ORF 7 gene.
出处
《中国兽医杂志》
CAS
北大核心
1998年第10期3-5,共3页
Chinese Journal of Veterinary Medicine
