传染性支气管炎病毒NP基因在大肠杆菌中的可溶性表达

目的]生产具有生物活性的、可用于临床检测的NP融合蛋白抗原,建立可用于临床检测IB抗体诊断的试剂盒。方法]利用RT-PCR技术扩增传染性支气管炎病毒M41株的NP基因,将之克隆并重组于原核表达载体pET-28a,经PCR、酶切鉴定及序列分析构建重组表达载体pET-NP,研究NP基因在大肠杆菌中的可溶性表达。结果]将重组载体pET-NP转化表达菌BL21,获得阳性重组菌pET-NP-BL。阳性重组子经IPTG诱导后,目的基因NP获得了高效表达。表达产物经过SDS-PAGE与Western blot检测表明,NP分子量约为49 kD,且以可溶性蛋白形式存在;表达产物可与特异性传染性支气管炎病毒抗血清发生免疫反应。表达产物可以用作检测IB抗体的抗原。结论]该融合蛋白可用于生产检测IB的诊断试剂,生物安全性高,生产方便。但尚需建立适当的检测方法,进一步研究其实际诊断价值。同时也可以研制新型IB重组亚单位疫苗,进一步研究其刺激动物机体产生的细胞与体液免疫,深入研究新功能,筛选新的抗原表位,为生产新型IB基因工程疫苗奠定基础。

Soluble Expression of NP gene of Infectious Bronchitis Virus in E.coliBL21

The objective of this study is to produce NP fusion protein antigen that is biologically active and can be used in clinic detection and establish reagent kits used in clinic detection of IB antibody diagnosis.A pair of primers was designed by employing DNAStar software and consulting the NP nucleotide sequences of M41 strain of IBV.Then the NP gene with flanking restriction sites was successfully PCR-amplified.The NP gene was excised with BamH Ⅰand Xho Ⅰand inserted into the expression plasmid pET-28a to obtain the recombinant expression vector pET-NP,which was used to transform E.coli BL21 competent cells for gaining recombinant bacteria pET-NP-BL.And then the recombinant pET-NP-BL was induced by IPTG.SDS-PAGE and Western blotting detection confirmed that the gene had been expressed solubly in recombinant pET-NP-BL cells,the size of the NP being approximately 49 kD.The expression product could specially react to the anti-sera of IBV.The product could be used as antigen for detection of antibody to IBV clinically.This study has laid a solid foundation for production of new type IB gene engineering vaccines.