本文選題：ORFV + B2L　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：羊口瘡(Orf)是由羊口瘡病毒(Orf virus,ORFV)引起的傳染性很強(qiáng)的傳染病,呈全球性分布,羔羊?qū)Ρ静≥^為敏感,常造成重大的經(jīng)濟(jì)損失。本試驗應(yīng)用聚合酶鏈反應(yīng)(PCR)技術(shù)擴(kuò)增了ORFV Shaanxi分離株的B2L基因,并對其進(jìn)行了序列分析及原核表達(dá),對表達(dá)的重組蛋白進(jìn)行純化,并以該重組蛋白為包被抗原,通過優(yōu)化各種條件,初步建立了檢測其抗體水平間接ELISA方法。獲得如下研究結(jié)果:1.擴(kuò)增了ORFV Shaanxi分離株的B2L基因,目的基因片段長度為1137 bp,測序后,與GenBank中已收錄的其他11株病毒毒株的B2L蛋白進(jìn)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%之間。將目的基因成功連接到原核表達(dá)載體pET-28a,構(gòu)建了重組質(zhì)粒pET-28a-B2L。在大腸埃希菌原核表達(dá)系統(tǒng)中對重組質(zhì)粒pET-28a-B2L進(jìn)行誘導(dǎo)表達(dá),獲得了42 kD的B2L重組蛋白。2.以純化的B2L重組蛋白為包被抗原,通過優(yōu)化各種條件,建立了檢測ORFV抗體水平的間接ELISA方法。優(yōu)化的各條件是:抗原包被量為600 ng,血清和酶標(biāo)二抗的稀釋濃度1∶200和1∶5000;含5%脫脂奶粉的PBST封閉時間是1.5 h;血清和酶標(biāo)二抗孵育1 h和0.5 h;TMB顯色10 min。該方法特異性強(qiáng)、敏感性和重復(fù)性良好。對344份臨床血清樣品進(jìn)行了檢測,陽性檢出率為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%.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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