本文選題：豬偽狂犬病病毒 + 單克隆抗體　； 參考：《南京農業(yè)大學》2015年碩士論文

【摘要】：偽狂犬病(Pseudorabies,PR)是一種由偽狂犬病病毒(Pseudorabies virus,PRV)引起的豬、牛、羊等多種哺乳動物的重要傳染病,給全球養(yǎng)豬業(yè)帶來巨大的經濟損失。目前,疫苗免疫成為防控偽狂犬病的主要手段。但是,2011年以來,我國許多規(guī)�；i場再次出現PRV大流行,給我國養(yǎng)豬業(yè)帶來嚴重經濟損失。本研究從我國發(fā)病豬場分離得到1株PRV強毒株,采用大腸桿菌表達系統表達獲得的PRV超強毒株ZJ01 gB和gE重組蛋白,制備獲得4株抗PRV單克隆抗體,闡明了 gB蛋白一個抗原表位,采用重組gB蛋白和辣根過氧化物酶標記的gB蛋白單抗,成功建立了檢測PRV抗體的阻斷ELISA方法,為該病防控奠定了重要基礎。具體研究內容包括:1.江蘇省豬偽狂犬病病毒強毒株分離與致病性研究從臨床發(fā)病豬群分離獲得一株PRVNJ毒株,gC和gD基因推導的氨基酸序列與先前報道的毒株相比分別有7個與2個氨基酸的插入,基因進化樹結果顯示,該分離株位于相對獨立的分支,與亞洲分離株親緣關系較近。100日齡健康豬攻毒比較結果顯示,PRVNJ株滴鼻接種組豬出現明顯臨床癥狀,攻毒后7d內全部死亡,組織病理變化明顯,而傳統毒株LA接種組僅表現體溫變化與輕微呼吸道癥狀,組織病理變化輕微,表明PRV NJ分離毒株毒力明顯增強。2.豬偽狂犬病病毒gB和gE蛋白單克隆抗體的制備與鑒定本研究采用大腸桿菌表達系統表達獲得PRV超強毒株ZJ01 gB和gE重組蛋白,免疫BALB/c小鼠,通過細胞融合、克隆和間接ELISA方法篩選,制備獲得4株能穩(wěn)定分泌抗PRV單克隆抗體的雜交瘤細胞,細胞培養(yǎng)上清ELISA抗體效價為1:1600～6400,腹水抗體效價達1:4×105;連續(xù)培養(yǎng)20代,抗體效價基本一致�？筭B單抗B1B6和B3D7屬于IgG2b,抗gE單抗E1B11和E5C10屬于IgG1,輕鏈均為1κ型。Western blot和IFA結果顯示,4株單抗均能與PRV流行毒株ZJ01、HZ、SD、NJ和LA發(fā)生特異性反應,B1B6和B3D7還能與PRV gE基因缺失疫苗毒株Bartha-K61發(fā)生反應,但E1B11和E5C10b不能與Bartha-K61反應,為PRV功能蛋白和抗體鑒別診斷試劑盒研制奠定了重要基礎。3.豬偽狂犬病毒ZJ01毒株gB蛋白單克隆抗體的B}0胞表位鑒定為了對已獲得的2株抗PRV gB蛋白的單克隆抗體針對的抗原表位進行定位,本研究利用原核表達系統截斷表達了 7個重組gB蛋白。通過SDS-PAGE和Western blot鑒定目的蛋白成功表達后,再通過Western blot對2株抗PRV gB蛋白的單克隆抗體所識別的抗原表位進行定位。結果顯示,抗gB單抗B1B6和B3D7識別相同的抗原表位E(104)YGDLDART(112),與不同來源的PRV毒株進行序列比對分析,結果發(fā)現該表位在所參考毒株中高度保守。本研究為gB蛋白功能分析研究和PRV診斷方法的建立奠定了基礎。4.豬偽狂犬病病毒阻斷ELISA抗體r檢測方法的建立與應用本研究采用豬偽狂犬病毒(PRV)ZJ01毒株重組gB蛋白和HRP標記單克隆抗體,建立檢測PRV gB抗體的阻斷ELISA方法,其優(yōu)化的反應條件為:抗原包被濃度0.5μg·mL-1,待檢血清1:1稀釋,作用時間37℃1h,酶標單抗稀釋度為1:15000,作用時間為37℃ 0.5h.血清樣品阻斷率PI≥28.67%時判為陽性,PI≤18.27%時判為陰性,18.27%PI28.67%時判為可疑。該方法與PRRSV、PCV2、CSFV和FMDV抗體陽性血清無交叉反應性。批間和批內變異系數均小于10%。敏感性、特異性分別為80.9%和96.4%。與IDEXX PRV gB抗體檢測試劑盒符合率為85.1%。535份臨床豬血清樣品檢測結果顯示,PRV抗體陽性率達61.87%。
[Abstract]:Pseudorabies (PR) is an important infectious disease of pigs, cattle, sheep and other mammals caused by Pseudorabies virus (PRV). It has brought huge economic losses to the global pig industry. At present, vaccine immunity has become the main means to prevent and control pseudorabies. But since 2011, many large-scale pig farms in our country have been re established. The PRV pandemic has brought serious economic loss to the pig industry in China. 1 PRV strains were isolated from the swine farm of our country. The superstrong PRV strain ZJ01 gB and gE recombinant protein expressed in the Escherichia coli expression system was used to prepare 4 monoclonal anti PRV monoclonal antibodies, and the epitope of gB protein was clarified, and the recombinant gB was used as a recombinant gB. The gB protein monoclonal antibody labeled with protein and horseradish peroxidase successfully established the blocking ELISA method for detecting PRV antibody and laid an important foundation for the prevention and control of the disease. 1. the specific research contents include: isolation and pathogenicity of the virulent strain of porcine pseudorabies virus in Jiangsu Province, a PRVNJ strain was isolated from the clinical swine, gC and gD genes were pushed. 7 and 2 amino acids were inserted in the amino acid sequence compared with the previously reported strains. The results of gene evolution tree showed that the isolate was located in a relatively independent branch. Compared with the Asian isolates, the results showed that the swine inoculation group of PRVNJ strains had obvious clinical symptoms and 7 after attack of.100 day old pigs. 7 D all died and the pathological changes were obvious, while the traditional LA inoculation group showed only the temperature change and the mild respiratory symptoms, and the histopathological changes were slight. It showed that the virulence of the PRV NJ isolates significantly enhanced the preparation and identification of the monoclonal antibodies of the.2. porcine pseudorabies virus gB and gE protein. PRV superstrong strain ZJ01 gB and gE recombinant protein were obtained, and BALB/c mice were immunized by cell fusion, cloning and indirect ELISA screening. 4 hybridoma cells capable of stable secretion of anti PRV monoclonal antibodies were prepared. The titer of the cell culture supernatant ELISA antibody was 1:1600 to 6400 and the antibody titer of ascites reached 1:4 * 105; the antibody titer was continuously cultured for 20 generations. The anti gB monoclonal antibody B1B6 and B3D7 belong to IgG2b, and the anti gE monoclonal antibody E1B11 and E5C10 belong to IgG1, and the light chains are all 1 kappa.Western blot and IFA results. It can react with Bartha-K61 and establish an important basis for the development of the PRV functional protein and antibody differential diagnostic kit. The B}0 cell epitopes of the gB protein monoclonal antibody of the.3. porcine pseudorabies virus ZJ01 strain are identified in order to locate the antigen epitopes against the obtained 2 monoclonal antibodies against PRV gB protein. This study uses the prokaryotic expression system. 7 recombinant gB proteins were truncated. After the target protein was successfully expressed by SDS-PAGE and Western blot, the antigen epitopes identified by 2 monoclonal antibodies against PRV gB protein were identified by Western blot. The results showed that the antigen epitope E (104) of the anti gB McAb B1B6 and B3D7 identified (104), 112, and different sources. Sequence alignment analysis showed that the epitope was highly conserved in the reference strain. This study laid the foundation for the establishment and application of the gB protein function analysis and the establishment of the PRV diagnosis method for the.4. porcine pseudorabies virus blocking the ELISA antibody r detection method. The study adopted the recombinant gB eggs of the porcine pseudorabies virus (PRV) ZJ01 strain. White and HRP labeled monoclonal antibodies were used to establish a blocking ELISA method for detecting PRV gB antibodies. The optimal reaction conditions were: the antigen inclusion concentration was 0.5 mu g. ML-1, the serum 1:1 was diluted, the action time was 37 1H, the dilution of the enzyme labeled monoclonal antibody was 1:15000, the action time was 37 C 0.5H. serum sample blocking rate PI > 28.67% and PI was less than 18.27% 18.27%PI28.67% was found to be doubtful. The method had no cross reactivity with PRRSV, PCV2, CSFV and FMDV antibody positive serum. The coefficient of variation between batch and batch was less than 10%. sensitivity, the specificity was 80.9% and the coincidence rate of 96.4%. and IDEXX PRV gB antibody detection kit was shown in 85.1%.535 portion of clinical pig serum samples. The positive rate of antibody is 61.87%.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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