猪戊型肝炎病毒结构蛋白片段的表达及其在ELISA中的初步应用

Expression of Swine Hepatitis E Virus ORF2 and Development of ELISA Based on the Expressed Protein

为建立一种快速的猪戊型肝炎病毒抗体检测方法,根据已克隆的戊型肝炎病毒株DQ1结构蛋白基因(ORF2)的抗原性及水溶性分析,在其序列上设计一对引物,上游引物和下游引物分别带有BamHⅠ、HindⅢ酶切位点,用PCR方法扩增,获得369 bp大小的相应片段,位于ORF2 128-497 bp处。将扩增片段克隆到pET-32a构建了原核表达载体pET32a-DQ01,经测序证明该片段正确插入。将pET32a-DQ01转化BL21并诱导表达,SDS-PAGE结果显示表达蛋白大小为33 ku,Western-blot结果表明表达的蛋白质具有生物活性,将表达蛋白作为诊断抗原,对反应条件进行优化,初步建立了ELISA诊断方法。

To develope a method to detect the antibody of swine hepatitis E virus in pigs, a pair of primers were designed according to the ORF2 of swine hepatitis E virus DQ1 strain, and the ORF2 about 369 bp was amplified by PCR. Then the fragment was inserted into pET-32a to establish a expression vector which was named pET32a-DQ1. The sequence analysis showed that the sequence was correct. Then the pET32a-DQ1 was transformed into E. coli BL21, the segment was expressed. The result of SDS-PAGE showed that the expressed protein was about 33ku. Western-blot results indicated that the protein could reacted with swine HEY antiserum. With the protein, a ELISA method for detection of HEV was developed and the primary test revealed the feasibility for clinical use.