新城疫病毒HN蛋白在水稻中的表达及半定量快速检测抗体方法的建立

Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies

摘要:

本研究旨在利用水稻胚乳生物反应器表达新城疫病毒（Newcastle disease virus，NDV） HN蛋白，建立一种NDV抗体半定量、快速免疫层析检测方法。通过MlyⅠ和XhoⅠ双酶切重组质粒pUC57-HN，NaeⅠ和XhoⅠ双酶切包含有启动子、信号肽和终止子的pMP3中间载体，分别回收HN和pMP3片段并进行连接，构成重组质粒pMP3-HN1。然后利用EcoRⅠ和Hind Ⅲ双酶切pMP3-HN1和植物载体pCAMBIA1300，将HN1基因成功连接到pCAMBIA1300上，采用电转化方法将重组质粒pCAMBIA1300-HN1导入根癌农杆菌EHA105。通过农杆菌介导法将pCAMBIA1300-HN1转入水稻愈伤组织。经过暗培养、愈伤筛选、分化、生根和移栽，4个月后获得转基因水稻种子。经PCR鉴定，HN基因已插入到水稻基因组中。SDS-PAGE和Western blotting鉴定结果表明，水稻胚乳成功表达了HN蛋白，且该蛋白通过SP阳离子层析和凝胶过滤层析，其纯度达到90%以上。根据国家制定的新城疫血凝抑制（hemagglutination inhibition，HI）试验诊断技术标准（HI≥4log₂，检测的抗体为阳性），利用胶体金标记HN蛋白，通过双抗夹心法制备NDV抗体半定量快速检测试纸。结果显示：试纸与其他病毒的阳性血清无交叉反应，且试纸对新城疫标准阳性血清的敏感性可达到1：102 400。根据308份临床血清的检测结果，NDV抗体试纸与和HI试验的符合率为97.08%，Kappa值为0.942。综上所述，获得了高纯度水稻胚乳表达的重组HN蛋白，并研制了一种简单、快速、灵敏度高、特异性强的半定量免疫层析试纸。该试纸能够初步应用于新城疫疫苗的免疫评价。

Abstract:

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI ≥ 4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.