鸭肠炎病毒US10基因的原核表达及抗血清的制备

以鸭肠炎病毒 (DEV) C-KCE株基因组DNA为模板,PCR扩增获得US10基因。构建了其 原 核表达载体pET-32a-US10,经1.0 mmol／L IPTG诱导,目的蛋白在Rosetta(DE3) pLys宿主菌中获得了表达。SDS-PAGE电泳结果显示目的蛋白以包涵体形式表达,与预期大小相符。利用 亲和层析法纯化目的蛋白,以纯化的蛋白为免疫原制备其抗血清,琼脂扩散法测得抗体效价为 1∶4,间接ELISA法测定抗体效价为1∶102　400。间接免疫荧光试验证实制备的抗血清可特 异性识别鸭肠炎病毒的US10基因编码产物。

Prokaryotic Expression and the Antiserum Preparation of Duck Enteritis Virus US10 Gene

The US10 gene was expressed in pET-32a(+)/Rosetta(DE3) pLys  system usi ng the DNA of Duck enteritis virus (DEV) C KCE as temple, US10 gene ORF was amp l ified by polymerase chain reaction (PCR). Then the recombinant protein of US10 g ene induction with IPTG by SDS-PAGE analysis showed that the object protein is m ainly expressed as inclusion bodies. Using the Ni NTA Purification System, the  r ecombinant protein immunized to rabbits was purified to make the antiserum. The  titer of the antiserum tested by agar diffusion reaction was 1∶4. The titer of  t he antiserum tested by ELISA was 1∶102　400. The prepared antiserum has a h igh spe cificity with the western blot analysis, and the indirect immunofluorescence (I I F) test proved that the antiserum could react with DEV US10 encoded product, whi ch lays a foundation for further research on molecular structure and function.