摘要
通过RT-PCR技术从细胞毒液中扩增N蛋白cDNA,克隆至原核表达载体pET-28a(+)中,所获得的重组质粒pET-28a-N经酶切鉴定正确后,将其转化入表达菌E.coliBL21plysS诱导表达,用SDS-PAGE与Western blotting对表达产物进行鉴定。结果表明,通过RT-PCR扩增获得长度为372 bp的N蛋白基因,诱导表达重组质粒pET-28a-N,经SDS-PAGE检测,IPTG终浓度为1 mmol/L时,诱导4 h蛋白表达量最高,出现分子质量约为18 ku的目的蛋白带,与N蛋白的理论值相符。经Western blotting检测,该表达产物可与PRRSV阳性血清发生特异性反应。获得的PRRSV N蛋白为建立针对该病毒抗体的间接ELISA检测方法,以及为进一步研发PRRS抗体检测试剂盒奠定了基础。
N gene fragment was amplified from PRRSV of cell RNA by RT-PCR and expressed it by prokaryotic expression vector pET-28a（＋）.A recombinant plasmid pET-28a-N was constructed and identified by restriction endonuclease digestion,then transformed into the E.coli BL21（DE3） plysS.The PRRSV N gene was expressed as a recombinant fusion protein and detected by SDS-PAGE and Western blotting.N gene fragment of 372 bp was amplified by RT-PCR,the recombinant bacteria was induced with IPTG and the expression protein was analyzed by SDS-PAGE.The results showed that bacteria contained the positive plasmid was induced to express with 1 mmol/L IPTG and 4 hours.A unique band was detected with the molecular mass of approximately 18 ku by SDS-PAGE.By analysis of Western blotting,the expressed production was reactive with the positive swine serum of PRRSV.PRRSV N gene was amplified and recombinant prokaryotic expression plasmid was constructed successfully.This showed that the high efficient expression of N protein was obtained which system of prokaryotic and eukaryotic expression.Using target protein after purified as coating antigen,an indirect ELISA was developed for detecting the anti-N antibody in the PRRSV serum by exploring the concentration of coating antigen and dilution degree of serum.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第1期61-64,共4页
China Animal Husbandry & Veterinary Medicine
关键词
猪繁殖与呼吸综合征病毒
核蛋白
原核表达
porcine reproductive and respiratory syndrome virus
nucleoprotein
prokaryotic expression
