【作者】 张凯；

【导师】 彭军； 王刚；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2022， 硕士

【摘要】 非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）引起的猪的一种高度致死性病毒性传染病,是世界动物卫生组织法定报告的动物疫病,在我国属于一类动物疫病。该病在2018年8月传入我国,由于其很高的死亡率,使得存栏母猪量迅速下降,导致猪肉价格上涨,给养猪业的发展和社会经济稳定带来了严峻的挑战。目前ASF没有有效的治疗方法和效果确实的疫苗能够用于防控,防控手段主要依靠生物安全和扑杀。然而,随着该病的逐渐流行和发生,部分动物会表现出亚急性感染并存活下来。ASF感染后的康复动物具有较高的抗体水平,血液中通常检测不到病毒,因此抗体检测对ASF的筛查以及净化具有重要的意义。本研究通过原核表达方式制备ASFV p22蛋白,以此建立适用于ASFV抗体检测的ELISA方法。本研究根据NCBI公布的ASFV中p22基因序列（Gen Bank:MK128995.1）,通过DNASTAR软件分析其氨基酸序列,筛选出亲水性和抗原性较好的第24-145位氨基酸序列,对其进行密码子优化、在片段下游添加6×His标签基因,合成上述融合蛋白表达的基因片段并连接至原核表达载体p ET-32a质粒中。将重组质粒p ET32a-p22转化感受态大肠杆菌BL21（DE3）表达宿主菌中,经IPTG诱导表达后表达目的蛋白,再经Ni柱亲和层析纯化,用BCA法测定重组蛋白浓度,其蛋白浓度为2.5μg/μL。Western Blot试验结果表明,本研究所表达的p22蛋白能够与灭活的ASFV阳性血清发生特异性反应,而不能和PRRSV、PCV2、CSFV、PRV等其他常见病原的阳性血清以及健康猪的血清发生反应。用纯化后的His-p22重组蛋白作为抗原进行包被ELISA反应板,建立并优化猪血清中ASFV抗体检测的间接ELISA方法,标准的ASFV灭活阳性血清和阴性血清均由中国动物卫生与流行病学中心惠赠。经过对检测条件的摸索和优化,最终确定最佳的抗原包被浓度为0.125μg/m L,被检血清稀释倍数为1:800,4℃过夜包被抗原,封闭液选择5%脱脂奶粉（每孔200μL）,封闭时间为60 min,血清在37℃条件下作用时间为60 min,二抗以1:10,000稀释（100μL/孔）,37℃作用90 min,TMB显色液（100μL/孔）最佳显色时间为15 min。应用优化完的ELISA方法检测60份无ASFV感染的猪阴性血清,根据统计学原理计算该ELISA方法的临界值。当血清样品的OD₄₅₀值≥0.384时为抗体阳性;OD₄₅₀值<0.384时为抗体阴性。使用本检测方法检测PCV2、PRRSV、PRV、CSFV的阳性血清,结果均为阴性,说明该检测方法的特异性比较好。重复性试验结果显示,同一批血清样本在同一批试验和不同批次的试验中数值别分为4.5%～7.5%和1.7%～9.08%,均小于10%,表明该间接ELISA方法具有良好的重复性。使用本试验建立的间接ELISA方法和动物卫生与流行病学中心的非洲猪瘟病毒抗体检测试剂盒同时检测263份临床样品,结果显示本实验室建立的间接ELISA方法和商品化试剂盒符合率为97.7%,无显著差异;表明本文建立的p22抗体间接ELISA检测方法可用于ASFV p22抗体的检测。综上所述,本研究基于原核表达系统表达ASFV p22蛋白,以此建立了适用于抗体检测的间接ELISA方法,该方法具有良好的灵敏度、重复性和特异性,为我国ASFV的防控提供了技术支撑。更多还原

【Abstract】 African swine fever(ASF)is a highly lethal viral infectious disease of pigs caused by African swine fever virus(ASFV),which is a legally reported animal disease by the World Organization for Animal Health(OIE)and belongs to a category of animal diseases in China.The disease was introduced into China in August 2018,and due to its very high mortality rate,it has caused a rapid decline in the stock of sows,leading to an increase in pork prices and posing a serious challenge to the development of the pig industry and socioeconomic stability.At present,there is no effective treatment for ASF and no effective vaccine that can be used for prevention and control,and the means of prevention and control mainly rely on biosecurity and culling.However,with the gradual prevalence and occurrence of the disease,some animals exhibit subacute infection and survive.recovered animals with ASF infection have high antibody levels and the virus is usually undetectable in the blood,so antibody detection is important for ASF screening and decontamination.In this study,we prepared ASFV p22 protein by prokaryotic expression and used it to establish an ELISA method suitable for ASFV antibody detection.In this study,the amino acid sequence of p22 gene in ASFV was analyzed by DNASTAR software based on the sequence of p22 gene published by NCBI(Gen Bank:MK128995.1),and the amino acid sequence of positions 24-145 with good hydrophilicity and antigenicity was screened,codon optimized,and 6×His tag gene was added downstream of the fragment to synthesize the fusion protein expressed by the above-mentioned fusion protein.The gene fragment of the above fusion protein was synthesized and ligated into the prokaryotic expression vector p ET-32 a plasmid.The recombinant plasmid p ET32a-p22 was transformed into the receptor E.coli BL21(DE3)expression host,and the target protein was expressed after IPTG induction,then purified by Ni column affinity chromatography,and the concentration of recombinant protein was determined by BCA at 2.5 μg/μL.The results of Western Blot assay showed that the p22 protein expressed in this study was able to reacted specifically with inactivated ASFV-positive sera,but not with positive sera of other common pathogens such as PRRSV,PCV2,CSFV,PRV,and sera of healthy pigs.The purified His-p22 recombinant protein was used as the antigen for encapsulating ELISA reaction plates to establish and optimize an indirect ELISA method for the detection of ASFV antibodies in pig sera,and standard ASFV inactivated positive and negative sera were kindly provided by the China Animal Health and Epidemiology Center.After the exploration and optimization of the assay conditions,the optimal antigen coating concentration was finally determined to be 0.125 μg/m L,the dilution of the examined sera was 1:800,the antigen was coated overnight at 4℃,the closure solution was selected from5% skim milk powder(200 μL per well),the closure time was 60 min,the serum was acted at37℃for 60 min,the secondary antibody was diluted at 1:10,000(The optimized ELISA method was applied to detect 60 negative sera from pigs without ASFV infection,and the critical values of the ELISA method were calculated based on statistical principles.When the OD450 value of the serum samples was ≥ 0.384,they were positive;when the OD450 value was < 0.384,they were negative.The positive sera of PCV2,PRRSV,PRV and CSFV were tested using this assay and the results were negative,indicating that the specificity of the assay is relatively good.The results of the repeatability test showed that the values of the same serum samples in the same batch of tests and different batches of tests were 4.5%～7.5%and 1.7%～9.08%,respectively,which were less than 10%,indicating that the indirect ELISA method has good reproducibility.The results showed that the indirect ELISA method established in our laboratory and the commercialized kits met 97.7% with no significant difference;it indicated that the indirect ELISA method established in this paper for the detection of p22 antibody can be used for the detection of ASFV p22 antibody detection.In conclusion,this experiment was based on the expression of p22 protein in the prokaryotic expression system,and an indirect ELISA method for the detection of ASFV antibodies based on ASFV p22 protein was established,which has good reproducibility and specificity.It laid the foundation for the development of ASFV vaccine in China.更多还原