羊口疮病毒陕西株B2L基因的克隆表达及间接ELISA检测方法的建立

发布时间：2018-06-13 17:07

本文选题：ORFV + B2L　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：羊口疮(Orf)是由羊口疮病毒(Orf virus,ORFV)引起的传染性很强的传染病,呈全球性分布,羔羊对本病较为敏感,常造成重大的经济损失。本试验应用聚合酶链反应(PCR)技术扩增了ORFV Shaanxi分离株的B2L基因,并对其进行了序列分析及原核表达,对表达的重组蛋白进行纯化,并以该重组蛋白为包被抗原,通过优化各种条件,初步建立了检测其抗体水平间接ELISA方法。获得如下研究结果:1.扩增了ORFV Shaanxi分离株的B2L基因,目的基因片段长度为1137 bp,测序后,与GenBank中已收录的其他11株病毒毒株的B2L蛋白进行了序列分析,核苷酸序列和氨基酸序列的相似性在95.9%~97.4%和97.8%~99%之间。将目的基因成功连接到原核表达载体pET-28a,构建了重组质粒pET-28a-B2L。在大肠埃希菌原核表达系统中对重组质粒pET-28a-B2L进行诱导表达,获得了42 kD的B2L重组蛋白。2.以纯化的B2L重组蛋白为包被抗原,通过优化各种条件,建立了检测ORFV抗体水平的间接ELISA方法。优化的各条件是:抗原包被量为600 ng,血清和酶标二抗的稀释浓度1∶200和1∶5000;含5%脱脂奶粉的PBST封闭时间是1.5 h;血清和酶标二抗孵育1 h和0.5 h;TMB显色10 min。该方法特异性强、敏感性和重复性良好。对344份临床血清样品进行了检测,阳性检出率为96.8%。
[Abstract]:Orfis is an infectious disease caused by Orf virus (Orf virus), which is distributed globally. Lambs are sensitive to the disease and often cause great economic losses. In this experiment, the B2L gene of ORFV Shaanxi isolate was amplified by polymerase chain reaction (PCR) technique. The B2L gene was sequenced and expressed in prokaryotic cells. The recombinant protein was purified, and the recombinant protein was used as the coating antigen. By optimizing various conditions, an indirect Elisa method was established to detect its antibody level. The following results are obtained: 1: 1. The B2L gene of ORFV Shaanxi isolate was amplified. The length of the target gene was 1137 BP. After sequencing, the B2L protein was sequenced with the other 11 virus strains in GenBank. The similarity between nucleotide sequence and amino acid sequence was 95.9% and 97.89%, respectively. The target gene was successfully ligated to the prokaryotic expression vector pET-28a and the recombinant plasmid pET-28a-B2L was constructed. The recombinant plasmid pET-28a-B2L was induced and expressed in Escherichia coli prokaryotic expression system, and 42 KD B2L recombinant protein was obtained. Using purified B2L recombinant protein as coating antigen, an indirect Elisa method for detection of ORFV antibody was established by optimizing various conditions. The optimized conditions were as follows: the amount of antigen coating was 600 ng, the dilution concentration of serum and enzyme labeled second antibody was 1: 200 and 1: 5 000; the blocking time of PBST containing 5% skim milk powder was 1.5 h; the second antibody of serum and enzyme was incubated for 1 h and the second antibody of enzyme was incubated for 10 min. The method is specific, sensitive and reproducible. 344 clinical serum samples were tested and the positive rate was 96. 8%.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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