猪瘟病毒SM株单克隆抗体的制备、初步鉴定及应用研究
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摘要
本研究采用单克隆抗体技术，用纯化的猪瘟病毒石门毒株(CSFV-SM)免疫BALB／C小鼠，取免疫鼠脾细胞与骨髓瘤细胞SP2／0进行细胞融合，经筛选克隆后，获得了10株能够稳定分泌抗CSFV的单克隆抗体(Monoclonal antibody，McAb)的杂交瘤细胞株，分别命名为2F_4、2F_7、2E_(10)、2B_2、4G_5H_2、5F_(11)、5C_9B_1、4A_1C_9、3C_4、5H_(11)，间接ELISA测定杂交瘤细胞培养上清和接种小鼠腹腔制备的腹水McAb效价，分别在1：160～1：640之间和1：800～1：6400之间；在-80℃超低温下保存3个月后复苏的各杂交瘤细胞仍然能稳定分泌抗CSFV的McAb。
免疫荧光(IF)试验对McAb的特异性检测结果表明，2F_7、2E_(10)、2B_2、4G_5H_2、5F_(11)、5C_9B_1、4A_1C_9与接种CSFV-SM毒的细胞能产生特异荧光，尤以4G_5H_2株最强，效价高达1：1000；McAb与CSFV E2蛋白反应结果表明，2E_(10)、2B_2、4G_5H_2、2F_7是抗E2蛋白的McAb。McAb与SDS处理前后的CSFV-SM抗原均有反应，推测McAb针对的抗原表位可能是线性表位。
利用4G_5H_2 McAb对接种CSFV-SM株、梧州株、博白株、北海株和疫苗株病毒的PK_(15)细胞进行免疫酶试验，结果均呈阳性反应。表明该McAb能识别CSFV强毒、野毒和弱毒等病毒株抗原；用4G_5H_2 McAb包被酶标板建立了抗原捕获ELISA(ACI-ELISA)方法，对22份来自不同地区的组织病料进行了检测，结果15份为阳性，与PCR检测的阳性结果(13份)基本一致。这表明ACI-ELISA以其操作简便、能多个样品同时检测、特异性较高等优点在临床上有较大的应用前景。
In this study ten monoclonal antibodies designated as 2F4,2F7,2E10,2B2, 4G5H2, 5F11, 5C9B1,, 4A1C9, 3C4, 5H11 against classical swine fever virus Shimen strain(CSFV-SM) were generated by immunization of BALB/C mice with purified CSFV-SM strain, using monoclonal antibody technique. The antibody of the supernatant and ascitic fluid from seven hybridomas were detected by indirect ELISA.The titers respectively ranges 1:160 to 1:640 and 1:800 to 1:6400. They can still stably excreted McAb after being stored at -80C.for three months. 2F7, 2E10, 2B2, 4G5H2, 5F11, 5C9B1, and 4A1C9 can reacte with CSFV-SM strain prepared in PK15 cell in 96 microplates using indirect immunofluorescent assay(IFA), especially 4G5H2 has strong fluorescent reaction , it has a titer of 1:1000. The reaction of McAb With CSFV E2 protein by indirect ELISA indicated that 2F7, 2E10, 2B2, 4G5H2 are McAb against CSFV E2 protion. 2F7, 2E10, 2B2, 4G5H2, 5F11, 5C9B, and 4A1C9 could still react with CSFV-SM strsin treated by SDS, indicating the anti
gen epitope of McAbs are linear epitope.
The 4G5H2 could react with CSFV-SM strain, wuzhou strain , BoBai strain, BeiHai strain and vaccine strain prepared in PK15 cell in 96 microplate by immunoenzymatic essay, demonstrating 4G5H2 can recognize all these CSFV strain. 4G5H2 McAb was boated in the 96 well microplates. 22 samples from different areas were detected by the antigen capture assay. 15 clinic samples were positive of CSFV. The result is in agreement with that of PCR based technique.
免疫荧光(IF)试验对McAb的特异性检测结果表明，2F_7、2E_(10)、2B_2、4G_5H_2、5F_(11)、5C_9B_1、4A_1C_9与接种CSFV-SM毒的细胞能产生特异荧光，尤以4G_5H_2株最强，效价高达1：1000；McAb与CSFV E2蛋白反应结果表明，2E_(10)、2B_2、4G_5H_2、2F_7是抗E2蛋白的McAb。McAb与SDS处理前后的CSFV-SM抗原均有反应，推测McAb针对的抗原表位可能是线性表位。
利用4G_5H_2 McAb对接种CSFV-SM株、梧州株、博白株、北海株和疫苗株病毒的PK_(15)细胞进行免疫酶试验，结果均呈阳性反应。表明该McAb能识别CSFV强毒、野毒和弱毒等病毒株抗原；用4G_5H_2 McAb包被酶标板建立了抗原捕获ELISA(ACI-ELISA)方法，对22份来自不同地区的组织病料进行了检测，结果15份为阳性，与PCR检测的阳性结果(13份)基本一致。这表明ACI-ELISA以其操作简便、能多个样品同时检测、特异性较高等优点在临床上有较大的应用前景。
In this study ten monoclonal antibodies designated as 2F4,2F7,2E10,2B2, 4G5H2, 5F11, 5C9B1,, 4A1C9, 3C4, 5H11 against classical swine fever virus Shimen strain(CSFV-SM) were generated by immunization of BALB/C mice with purified CSFV-SM strain, using monoclonal antibody technique. The antibody of the supernatant and ascitic fluid from seven hybridomas were detected by indirect ELISA.The titers respectively ranges 1:160 to 1:640 and 1:800 to 1:6400. They can still stably excreted McAb after being stored at -80C.for three months. 2F7, 2E10, 2B2, 4G5H2, 5F11, 5C9B1, and 4A1C9 can reacte with CSFV-SM strain prepared in PK15 cell in 96 microplates using indirect immunofluorescent assay(IFA), especially 4G5H2 has strong fluorescent reaction , it has a titer of 1:1000. The reaction of McAb With CSFV E2 protein by indirect ELISA indicated that 2F7, 2E10, 2B2, 4G5H2 are McAb against CSFV E2 protion. 2F7, 2E10, 2B2, 4G5H2, 5F11, 5C9B, and 4A1C9 could still react with CSFV-SM strsin treated by SDS, indicating the anti
gen epitope of McAbs are linear epitope.
The 4G5H2 could react with CSFV-SM strain, wuzhou strain , BoBai strain, BeiHai strain and vaccine strain prepared in PK15 cell in 96 microplate by immunoenzymatic essay, demonstrating 4G5H2 can recognize all these CSFV strain. 4G5H2 McAb was boated in the 96 well microplates. 22 samples from different areas were detected by the antigen capture assay. 15 clinic samples were positive of CSFV. The result is in agreement with that of PCR based technique.
引文
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[3]. Susa M, Konig M, Saalmuller A, et al, [J] Virol, 1992,66:1171～1175.
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[6]．BE斯特劳，SD阿莱尔，WL蒙加林等．猪病学(第八版)[M]．，北京：中国农业出版社，2000，157～171．
[7]．周泰冲．CSFV与防制猪瘟的研究进展[J]，中国兽医杂志，1980，16(2)：23～39．
[8]. Nishimori Y. Identification of hog cholera virus using monoclonal antibodies [J]. Vet Med Sci, 1996,581:707～712
[9]. Meyers,Grumenapf,T..Thiel,H ,J., Virology,1989,171,556～567.
[10]. Moormanna, R.J.M.,Warmerdam,P,A,M Vam der Meer, B.,Virology, 1990,177,184～198.
[11]. Isbikawa K,.Nagai,H.,Kalayama K.,et al.,ARCH. Virol.,1995,140,1385～1391.
[12]. Moormanna, R..J.M., Warmerdam . VanGennip,H. G.P.,Mledema,G.K.W.,et al.,J.Virol., 1996,70:763～770.
[13]. Moormanna, R., J. M. et al.,Nucleotide sequence of hog cholera virus RNA:properties of the polyproten encoded by the open reading frame spanning the viralgenomic RNA [J].Vet Microbiol. 1990,23:185～191.
[14]. Ighidawa, K. Comparison of the entire nucleotide and deduced amine acid sequence of the attenuated hog cholera vaccine strain GPE-and the wild-type patental strain ALD [J].Arch Virol,1995,140:1385～1391.
[15]. Lowing, J., P. Paton, D. J. Sands. Classixal swine ferver: genetic detection and analysis of differenxes between vires isolates [J]. Gen Virol, 1994,75:3461～3468.
[16]. Wensvoort,G;et al:Vet. Microbiol, 1990,177,184～198.
[17]. Colett,M. S.,Gomp.Immun.Mocrobiol.,Infect,Dis., 1992,15,145～154.
[18]. Pauly, T. Elbers.,Konig,M.,et al.,J.Gen. Virol., 1995,76,3039～3049.
[19]．金扩世，涂长春，殷震等．CSFV保护性囊膜糖白E1(gp55)基因研究进展．病毒学
报，1996，12(3)：287～289
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[21]. Van Rijn P A, Miedema G K W , Wensoort G ,et al. Antigenic structure of envelop glycoprotein in E1 (gp55) of hog cholera vires. [J.] Virol,1994,68:3934～3942
[22]. Wensvoort G et al. Immunoaffinity purification and characterization of envelope protein E1(gp55) of hog cholera virus. [J.] Gen Virol,1990,71:531～540
[23]. Huslt M M ,.Glycoprotein E1 of hog cholera vires expressed in insect cell protects swine from hog cholera. [J]. Virol,1993,71:5435～5442
[24]. Wensvoort,G.;Terpstea, C.;De K luojver, E.P.; Kragten, C.; and Warnaar, J.C., Antigen differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus. Vet Microbiol, 1989,21:9～20.
[25]. Edwards ,S.,Moenning,V.;and wensvoort, G..,The development of an international reference panal of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses.Vet Microbiol, 1991,29:101～108.
[26]. Paton,D .J.,Peativirus diversity. [J] Comp Pathol [Ⅰ], 1995, 2:215～236.
[27]．蔡宝祥．家畜传染病学(第四版)[M]．北京：中国农业出版社，．2001：201～206
[28]. Vanthemsche P.,Classical swine fever 1993-1994 Belgium [J].Pig Journal,1996,37:43～53.
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