新城疫病毒xx08株HN蛋白主要抗原区的原核表达及其在抗体检测中的应用

Prokaryotic Expression of HN Protein Main Antigen Region of NDV xx08 Strain and Application in Detecting Antibody of NDV

为建立检测新城疫病毒(NDV)抗体的间接ELISA方法,构建了新城疫(ND)xx08株HN基因抗原表位集中区基因片段的重组表达质粒p ET28a-r HN,加入IPTG进行诱导表达,经SDS-PAGE鉴定表明,表达的重组蛋白大小约为28 ku。Western blot分析表明,该重组蛋白具有良好的反应性。以纯化的HN蛋白为包被抗原建立检测NDV抗体的间接ELISA方法,优化试验条件后,抗原最适包被浓度为5μg/m L,血清最适稀释比例为1∶80,与HI试验结果符合率较高,且与鸡传染性法氏囊病毒(IBDV)、鸡传染性支气管炎病(IBV)、鸡禽流感病毒(AIV)和鸡马立克氏病毒(MDV)等标准阳性血清无交叉反应。表明建立的间接ELISA方法可以用于临床新城疫抗体的检测。

In order to obtain the protein of HN gene epitope concentration region of Newcastle disease virus xx08 strain,a method of detecting antibody of Xinxiang NDV was established.The gene was cloned into prokaryotic expressing p ET-28 a vector to obtain the recombinant expression plasmid p ET28a-r HN.The recombinant expression plasmid was induced by IPTG.The expressed protein was separated by SDSPAGE and identified by Western blot assay.The results showed that the HN protein was overly expressed and the molecular weight was about 28 ku.The expressed protein specifically reacted with anti-NDV antiserum.Using recombinant HN protein as antigen,an ELISA for the detection of antibodies against NDV was developed.The result showed that optimum amount of HN protein coated onto the polystyrene microtieration plate was 5 μg / m L and appropriate dilution of the serum samples was 1∶ 80,and the coincidence of established ELISA method with HI test was high.It was confirmed that there was no cross reaction between the antibodies against other virus IBD,IBV,AIV and MDV.This study established a rapid and accurate detection method,provided rapid diagnosis on Newcastle disease.