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以课题组保存的连接在pMD-18T载体上的香蕉条斑病毒河口分离物全基因组为模板,设计一对特异引物,经过PCR扩增,切胶回收,并通过与pET32(b)载体共同双酶切及T4连接酶连接,得到重组载体pET32-ORF2。将获得的重组质粒pET32-ORF2转化表达菌株E.coli BL21(DE3)感受态细胞,得到表达BSV的ORFⅡ的工程菌。经0.1mmol/L的IPTG诱导表达,超声波破碎后,获得的融合蛋白大多为包含体,但仍有少部分为可溶性蛋白可以进行后续的纯化步骤。将上述可溶性融合蛋白通过Ni2+-NTA亲和层析柱纯化,获得纯度较高的融合蛋白。以纯化蛋白作为抗原免疫家兔,得到特异性抗血清。间接ELISA以及western blotting表明,所得抗血清效价和特异性都较高,可以用于检测。为BSV的快速检测以及一些相关后续理论研究工作奠定了一定基础。
The whole genome of the banana streak virus echovirus isolated on the pMD-18T vector was used as a template to design a pair of specific primers. The PCR products were amplified by PCR, cut through the gel, and co-transfected with the pET32 (b) vector Digested by T4 ligase and ligated to obtain the recombinant vector pET32-ORF2. The obtained recombinant plasmid pET32-ORF2 was transformed into competent E. coli BL21 (DE3) competent cells to obtain an engineered bacterium expressing ORFII of BSV. After induced by 0.1 mmol / L IPTG, most of the fusion proteins were inclusion bodies after sonicated, but a small amount of soluble protein could be used for the subsequent purification steps. The soluble fusion protein was purified by Ni2 + -NTA affinity chromatography to obtain high purity fusion protein. Rabbit was immunized with purified protein as antigen to obtain specific antiserum. Indirect ELISA and western blotting showed that the titer and specificity of the obtained antisera were high and could be used for detection. It has laid a solid foundation for the rapid detection of BSV and some related theoretical researches.