【作者】 张福良；

【导师】 张翊华；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2021， 博士

【摘要】 流行性日本乙型脑炎（Epidemic encephalitis B）,简称乙脑,是由日本乙型脑炎病毒（Japanese encephalitis virus,JEV）引起的一种人畜共患病,是威胁人类尤其是儿童健康的主要传染病之一。猪被认为是乙脑流行环节中的“放大宿主”,日本乙型脑炎病毒可在猪体内大量繁殖,从而引起明显的病毒血症。此外,日本乙型脑炎病毒还可以通过蚊子在猪群之间进一步传播,对中国养猪业造成重大经济损失。日本乙型脑炎的防控不仅需要疫苗的有效防疫,也需要简便、快捷、准确的诊断与检测。单克隆抗体（Monoclonal antibodies,Mc Ab）具有高度特异性的优点,基于单抗而建立的日本乙型脑炎抗原抗体诊断方法具有很大的应用前景。本研究进行了日本乙型脑炎病毒单克隆抗体的制备及其抗原抗体检测方法的建立与初步应用。本研究建立了一种较为温和地纯化日本乙型脑炎病毒的双重凝胶层析方法,与病毒原液相比,双重凝胶层析纯化后日本乙型脑炎病毒的回收率和总蛋白去除率分别为61.04%和99.71%,HCP和BSA去除率分别达到98.72%和99.72%,表明在经过膜包超滤浓缩及琼脂糖凝胶分子筛层析（TFF-SEC）后,进一步进行Capto ^TMCore700层析能够获得纯度更高、完整性更好,且具有良好免疫原性和生物学活性的病毒颗粒。以TFF-SEC-Capto ^TMCore700纯化的日本乙型脑炎病毒粒子作为抗原,采用IPMA、IFA和间接ELISA相结合的手段,建立了日本乙型脑炎病毒单抗体外（Vero细胞系）筛选的试验方法。研究共筛选到5株单克隆抗体:29E3、29F8、4B4、2F2和36A6,其杂交瘤细胞培养上清效价在1:800～1:1600之间;接种小鼠后,制备的腹水效价在1:5.12×10⁵～1:2.56×10⁶之间。类和亚型鉴定表明,4B4和36A6为Ig G1类,29E3、29F8和2F2为Ig G2α类,29E3、29F8和2F2轻链为κ链,4B4和36A6轻链为λ链。稳定性测定试验表明,五株杂交瘤细胞均能稳定、持续分泌特异性抗体。Western blot鉴定结果显示,29F8和2F2单克隆抗体可以特异性识别JEV的E蛋白。应用纯化的日本乙型脑炎病毒E蛋白单克隆抗体,制备了可用于检测日本乙型脑炎病毒的胶体金试纸。试纸条特异性强,检测极限约可达10³～10⁴TCID₅₀/100μL,重复性、稳定性良好,可广泛应用于生产、临床上多种样品检测。研究进一步优化了各种检测条件,并对临床检测日本乙型脑炎病毒阴性、阳性血清临界值进行了判定,建立了日本乙型脑炎病毒单抗阻断ELISA检测方法,最低可检测到1:1600倍稀释的阳性血清,具有较强的特异性;批内、批间重复性试验的变异系数均小于5%。综上所述,本研究成功建立了日本乙型脑炎病毒单抗体外筛选方法,并获得5株能稳定、持续分泌特异性抗体的杂交瘤细胞;成功建立了日本乙型脑炎病毒单抗阻断ELISA检测方法。更多还原

【Abstract】 Epidemic encephalitis B is caused by Japanese encephalitis virus（JEV）,which is a zoonotic natural epidemic disease transmitted by mosquitoes and is one of the major infectious diseases that threaten the health of humans,especially children.Pigs are considered to be the"amplifying host"in the epidemic of JE.Japanese encephalitis virus can multiply in pigs,causing significant viremia.In addition,the virus can be further transmitted between pigs through mosquitoes,The pig industry has caused significant economic losses.Prevention and control of Japanese encephalitis requires effective vaccine immunization on the one hand,and simple,fast,and accurate diagnostic methods on the other.Monoclonal antibodies（Mc Ab）have the advantages of high specificity,so the diagnostic method of Japanese encephalitis antigen based on monoclonal antibodies has great application prospects.In this study,the preparation of Japanese encephalitis virus monoclonal antibody and the establishment and preliminary application of its antigen-antibody detection method were carried out.In this study,a dual-chromatographic method for the gentle purification of JEV was established.Compared with the virus stock solution,the virus recovery and total protein removal rate of JEV after dual-gel chromatography purification were 61.04%and 99.71%,respectively.The removal rates of HCP and BSA reached 98.72%and 99.72%,respectively,indicating that Capto ^TMCore700 chromatography after ultrafiltration membrane package concentration and agarose gel molecular sieve chromatography（TFF-SEC）can obtain higher purity and integrity Well,virus particles with good immunogenicity and biological activity.BALB/c mice were immunized with JEV virons purified by TFF-SEC-Capto ^TMCore700.A total of 5 strains of monoclonal antibodies were screened in the study:29E3,29F8,4B4,2F2,and 36A6.The hybridoma cell culture supernatant titer was between 1:800and 1:1600.After inoculating the mice,the ascites titer was prepared.Between 1:5.12×10⁵and 1:2.56×10⁶.Class and subtype identification showed that 4B4 and 36A6 are Ig G1.29E3,29F8,and 2F2 are Ig G2α.29E3,29F8,and 2F2 light chains areκchains,and 4B4 and 36A6light chains areλchains.Stability testing showed that all five hybridoma cells were able to stably and continuously secrete specific antibodies.Western blot analysis showed that 29F8and 2F2 monoclonal antibodies could specifically recognize the E protein of JEV virus.The purified JEV E protein monoclonal antibody was used to optimize colloidal gold test paper for the detection of JEV virus.The test strip has strong specificity,and the detection limit can reach 10³～10⁴TCID₅₀/100μL,which has good repeatability and stability,and can be widely used in production and clinical detection of a variety of samples.The study further optimized various detection conditions,determined the thresholds of negative and positive serum for clinical tests,and established a JEV monoclonal antibody blocking ELISA detection method.The method has good specificity,and the lowest detectable sex serum is 1:1600 times.The coefficient of variation of the repeatability test within and between batches is less than 5%.The clinical sample tests show that JEV is still active in the pig farms and even the entire Henan Province for inspection,and vaccine immunization prevention must be strengthened.In conclusion,we have successfully established an in vitro screening method for Japanese encephalitis virus monoclonal antibody and obtained 5 hybridoma cells that can stably and continuously secrete specific antibodies;A blocking ELISA for the detection of Japanese encephalitis virus（JEV）was successfully established.更多还原