A组轮状病毒VP7基因表达及ELISA检测的方法研究

Study on the expression of GroupA rotavirus VP7 gene and the ELISA Detection Method

目的纯化原核系统中表达的A组轮状病毒VP7基因,建立间接ELISA方法,为进一步大量表达VP7基因,构建基因工程疫苗和建立快速临床检测奠定基础。方法反转录并扩增VP7基因,连接到载体中,转换乳酸杆菌,诱导并纯化重组蛋白。将纯化蛋白免疫家兔制备抗血清,并建立间接ELISA方法。结果成功获得VP7基因的扩增片段,筛选阳性克隆,得到大小为28 kD的大量表达蛋白。确定了间接ELISA法的最佳反应条件和工作浓度。结论构建的重组乳酸杆菌能够大量表达VP7蛋白,纯化的VP7蛋白有很好的免疫原性,制备的抗血清用于ELISA方法能够得到准确的检测结果。

Objective To purify the cloned Group A rotavirus VP＇/gene expressed in prokaryotic system and construct a double antibody sandwich ELISA, so as to further highly express the VP＆7 protein and lay a foundation for the construction of genetic engineering vaccine and clinical quick detection. Methods The VP7 gene fragment was reverse transeripted and amplified, then combined with the vector and transformed into lacotobacillus. The recombination protein was induced and purified. The purified protein was used to immune rabbit to produce antiserum and construct the double antibody sandwich ELISA. l^esarlts The VP7 gene was successfully amplified, DNA （ ＋ ） recombinant was selected, and a protein of about 28 kD was found after the induction. The optimal reaction con- dition and working concentration of ELISA was confirmed. Conclusion Restructuring laeotobacillus could highly express VP7 protein and the purified protein has good i mmunogenicity. The ELISA detection method could achieve precise results with the antiserum produced by the purified recombination pretein.