猪流感病毒核蛋白基因的原核表达及其在诊断中的初步应用

根据GenBank中A/Swine/Hong Kong/126/82(H3N2)株猪流感病毒(SIV)NP基因核苷酸序列设计合成一对引物,经RT-PCR扩增了我国SIV分离株A/Swine/Heilongjiang/3/2000(H3N2)的核蛋白(NP)基因.将扩增所得NP基因克隆于pMD18-T载体,酶切鉴定后对其序列分析表明NP基因与香港分离株同源性高达93.8%,然后定向亚克隆NP基因于原核表达载体pET30(a)的多克隆位点中,经酶切鉴定后,将含有NP基因的重组质粒命名为pET-NP.用pET-NP转化受体菌BL21,用诱导剂IPTG以不同浓度进行诱导,并在不同诱导时间收集样品.经SDS-PAGE电泳分析证实NP基因获得了表达,并在终浓度为1mM的IPTG诱导下,6 h其表达量达到高峰,经Western blot分析证实表达的重组核蛋白具有反应活性,大小约为60kD.用表达产物作琼脂扩散试验,结果表明表达产物与SIV抗血清可形成清晰的反应沉淀线,而与其它9种猪病原阳性血清不发生反应.正常菌体蛋白与SIV阳性血清也不发生反应.通过对40份送检血清样品的检测结果显示其与HI试验的相符率高达95%以上.试验证明利用原核表达系统制备的重组核蛋白作为诊断用抗原,不仅制备过程简单而且所建立的琼扩诊断方法特异、快速、简便,其灵敏度也能满足现地的使用要求.目前正在进行现地的中试试验.

Primary application of swine influenza virus nucleporitein expressed in prokaryotic espression system

According to the sequence of Nucleoprotein(NP) gene of swine influenza virus(SIV) A/Swine/HongKong/126/82(H3N2), cDNA encoding for NP of a SIV isolate, A/Heilongjiang/3/2000(H3N2), was amplified and cloned into pMD18-T plasmid. Sequencing analysis indicated the homology between HongKong strain and Heilongjiang strain is 93.8 %. The NP gene then was subcloned into prokaryotic expression plasmid pET30(a). The recombinant plasmid carrying NP gene was designated as pET-NP. After transformation of BL21 with pET-NP, an expressed fusion protein was identified by SDS-PAGE in E.coli. BL21 after induced by IPTG. The recombinant NP protein is about 60 kD in size. The immune reactivities of the recombinantprotein were confirmed by western-blot and AGIP test with polyclonal antibodies.In the AGIP test recombinant NP as antigen, no reaction was shown to sera from other nine swine infectious pathogens. The total of 40 serum samples were randomly collected from field and evaluated by AGIP with recombinant NP and HI test, the coincidental rate between the two tests is about 95 %. The results above showed that recombinant NP based AGIP is specific, economic, and easy to perform for serologically diagnosis of SIV infection in field condition.