猪细小病毒1型VP2基因的原核表达、多克隆抗体制备及间接ELISA方法的建立

发布时间：2018-05-06 00:00

本文选题：猪细小病毒1型(Porcine + parvovirus　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：猪细小病毒(Porcine parvovirus,PPV)属于细小病毒科细小病毒属,是一种自主复制型病毒,是造成母猪繁殖障碍性疫病的主要病原体之一;母猪感染该病毒后可导致流产、木乃伊胎及死胎等,其给国内外养猪业造成了严重的经济损失。Mary与Mahnel在1966年首次发现了该病毒,而后发展到世界多个国家和地区;在20世纪80年代,我国的多个地方都分离发现猪细小病毒病。经典的猪细小病毒病是由猪细小病毒1型(Porcine parvovirus type 1,PPV1)感染引起的,自此预防、控制并最终根除该病成了我国当前面临的一项艰巨任务。因此,如何准确、及时地检测该病原的抗体情况在该病的防控中起着重要作用。本课题开展了针对PPV1 VP2蛋白的多克隆抗体的制备和PPV1间接ELISA检测方法的建立,为后续检测试剂盒的研发做好铺垫。本试验主要以NADL-2经典毒株的VP2基因为参考序列,以猪细小病毒AV30毒株的DNA为模板,设计了编码VP2蛋白第155-439位氨基酸的表达引物,成功克隆出VP2基因,并将其成功连接到表达载体pET-30a(+)上,转化到BL21(DE3)后进行诱导表达。可溶性分析表明原核表达的融合蛋白是以包涵体的形式存在,复性后蛋白进行Western bolt检测表明其具有较好的抗原性;利用Ni-NTA树脂亲和层析纯化重组VP2蛋白,并作为免疫原,经四次免疫新西兰大白兔,制备PPV1 VP2蛋白的多克隆抗体,Western bolt和间接ELISA检测证明抗血清具有较好的特异性,抗体效价为1:25 600;免疫兔血清经硫酸铵沉淀、Protein A树脂亲和层析纯化后,得到较高纯度的多克隆抗体;通过优化反应条件,建立了PPV1间接ELISA检测方法,该方法不与NA-PRRSV、PCV2、PRV和CSFV抗体发生交叉反应,表明该方法具有很好的特异性;通过与国、内外检测PPV1抗体的同类检测试剂盒的比较,结果显示,该间接ELISA方法对参考阳性血清的检出限可以达到1:800倍稀释度,临床敏感性为94.3%(132/140),略高于武汉科前公司的试剂盒的敏感性,低于西班牙INGEZIM PPV间接ELISA试剂盒97.1%(136/140)的敏感性;该方法临床特异性为96%(96/100),高于武汉科前试剂盒94%(94/100),略低于西班牙INGEZIM PPV间接ELISA试剂盒的特异性97%(97/100)。与国内外试剂盒的比较结果表明,本研究建立的PPV1间接ELISA方法具有很好的临床特异性和敏感性,具有较大的开发利用价值。
[Abstract]:Porcine parvovirus (PPVV) belongs to the genus Parvovirus of the family Parvoviridae. It is an autonomous replicative virus and is one of the main pathogens causing reproductive disorders in sows. Infection of the virus in sows can lead to abortion, mummies and stillbirths. This virus was first discovered by Mary and Mahnel in 1966, and then developed to many countries and regions in the world. In 1980s, porcine parvovirus was isolated in many parts of China. The classical porcine parvovirus (PPV1) infection caused by porcine parvovirus type 1 (PPV1) has become a difficult task to prevent, control and eventually eradicate the disease in China. Therefore, how to accurately and timely detect the antibodies plays an important role in the prevention and control of the disease. In this paper, the preparation of polyclonal antibody against PPV1 VP2 protein and the establishment of PPV1 indirect ELISA detection method were carried out, which paved the way for the research and development of subsequent detection kit. Using the VP2 gene of classical NADL-2 strain as reference sequence and the DNA of porcine parvovirus AV30 strain as template, the expression primers encoding the 155-439 amino acid of VP2 protein were designed and the VP2 gene was cloned successfully. It was successfully ligated to the expression vector pET-30a () and transformed into BL21 (DE3) to induce the expression. Soluble analysis showed that the fusion protein expressed in prokaryotic cells existed in the form of inclusion body, and the refolding protein had good antigenicity by Western bolt detection, and the recombinant VP2 protein was purified by Ni-NTA resin affinity chromatography and used as immunogen. New Zealand white rabbits were immunized for four times. The polyclonal antibody of PPV1 VP2 protein was prepared by Western bolt and indirect ELISA. The antibody titer was 1:25 600, and the serum of immunized rabbits was purified by ammonium sulfate precipitation protein A resin affinity chromatography. The method of PPV1 indirect ELISA detection was established by optimizing the reaction conditions. The method did not cross react with NA-PRRSVG PCV2PRV and CSFV antibodies, which indicated that the method had good specificity. The results showed that the detection limit of the indirect ELISA assay for the reference positive serum could reach 1: 800 times dilution, and the clinical sensitivity was 94.33 / 132 / 140, which was slightly higher than the sensitivity of the kit of Wuhan Qianjian Company. The sensitivity of this method was lower than that of the indirect ELISA kit of Spanish INGEZIM PPV 97.6 / 140. The clinical specificity of this method was 96 / 96 / 100, which was higher than that of the pre-Wuhan kit 94 / 94 / 100, and slightly lower than the specificity of 97 / 97 / 100 of the Spanish INGEZIM PPV indirect ELISA kit. The results of comparison with domestic and foreign kits show that the indirect ELISA method developed in this study has good clinical specificity and sensitivity, and has great value of development and utilization.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

【相似文献】