烟草环斑病毒外壳蛋白基因片段原核表达载体的构建及表达

Construction of Prokaryotic Vector and Expression of the CP Fragment of Tobacco ringspot virus

根据烟草环斑病毒外壳蛋白(TRSV-CP)基因的保守序列设计引物,采用RT-PCR方法,获得烟草环斑病毒基因组中编码外壳蛋白部分基因片段,将其插入原核表达载体pET28a中,获得重组表达质粒pET-CP,转化大肠杆菌BL21(DE3)后,利用IPTG诱导蛋白大量表达。重组蛋白经过Ni+-NTA纯化后,SDS-PAGE和Western blotting分析结果表明,TRSV-CP基因在大肠杆菌中获得了高效表达,产生的融合蛋白分子质量约为34 ku,并具有免疫学活性。以纯化的表达蛋白免疫新西兰大白兔,多抗血清效价达1∶409 600,可以与重组蛋白发生抗原抗体反应。

The CP fragment of the Tobacco ringspot virus （TRSV） was amplified by RT-PCR, and inserted into the expression vector pET.28a. The recombinant plasmid pET-CP was transformed into Escherichia coli BL21（DE3）, the target protein was expressed with induction of IPTG and purified by Ni^＋-NTA metal chelation chromatography. The purified recombinant protein was analyzed by SDS- PAGE and Western blotting, the results showed that the CP fragment was highly expressed. The molecular weight of the recombinant protein was about 34 ku, which was immunological reactive. The product was used as antigen to immunize the rabbit,and the antiserum had a titer of 1 ： 409 600,it was reaction between antiserum and recombinant protein.