CAJ | 학술논문

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pET28a-rHN,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/mL,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。
위건립검측신성역병독( NDV)항체적간접ELISA방법,구건료신성역( ND) xx08주HN기인항원표위집중구기인편단적중조표체질립pET28a-rHN,가입IPTG진행유도표체,경SDS-PAGE감정표명,표체적중조단백대소약위28 ku。 Western blot분석표명,해중조단백구유량호적반응성。이순화적HN단백위포피항원건립검측NDV항체적간접ELISA방법,우화시험조건후,항원최괄포피농도위5μg/mL,혈청최괄희석비례위1∶80,여HI시험결과부합솔교고,차여계전염성법씨낭병독(IBDV)、계전염성지기관염병(IBV)、계금류감병독( AIV)화계마립극씨병독( MDV)등표준양성혈청무교차반응。표명건립적간접ELISA방법가이용우림상신성역항체적검측。
In order to obtain the protein of HN gene epitope concentration region of Newcastle disease vi-rus xx08 strain,a method of detecting antibody of Xinxiang NDV was established. The gene was cloned into prokaryotic expressing pET-28a vector to obtain the recombinant expression plasmid pET28a-rHN. The recombinant expression plasmid was induced by IPTG. The expressed protein was separated by SDS-PAGE 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 anti-serum. 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 microti-eration plate was 5 μg/mL 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 be-tween the antibodies against other virus IBD,IBV,AIV and MDV. This study established a rapid and ac-curate detection method,provided rapid diagnosis on Newcastle disease.