抗登革病毒E蛋白单克隆抗体的制备及鉴定
摘要
目的:登革病毒(Dengue virus,DV)感染致死率高,登革热在世界范围内发病率不断升高,而目前尚无有效疫苗和特效药物,因此登革病毒感染的快速诊断有重要意义。登革病毒包膜蛋白E蛋白是重要的抗原蛋白,与免疫保护密切相关。本研究通过原核表达纯化E蛋白,利用杂交瘤技术制备DV-E特异性单克隆抗体,为进一步制备胶体金试纸奠定基础。
方法:
1.采用PCR法扩增E基因片段,原核表达载体pET 32-a及PCR产物经双酶切后,用T4DNA连接酶将两者连接并转化到大肠杆菌JM109构建并筛选重组质粒。随后将重组质粒转入表达宿主BL21感受态细胞,经IPGT诱导表达,对表达蛋白进行SDS-PAGE电泳分析和Western-blot检测。以Ni2+-NTA树脂亲和层析纯化重组蛋白。
2.用重组E蛋白作为抗原免疫Balb/c小鼠,采用杂交瘤技术,制备抗DV-E的单克隆抗体,用ELISA方法筛选分泌抗DV-E单克隆抗体的杂交瘤细胞株,采用间接ELISA方法和Western-blot进行McAb特异性鉴定;同时采用间接ELISA方法鉴定McAb的Ig亚类,检测McAb的效价。
结果:
1.采用PCR方法扩增得到与预期大小一致的目的基因片段。重组质粒经PCR鉴定及测序分析证实重组质粒克隆成功,命名为pET 32-a-E。转化表达宿主菌后成功诱导出分子量为70kD的重组E蛋白,与预期分子量相符。Western-blot检测表明诱导表达的融合蛋白能与抗DV的血清发生特异性结合。采用Ni2+-NTA树脂亲和层析纯化得到重组蛋白。
2.成功筛选出一株可分泌特异性McAb的杂交瘤细胞7C7,其抗体亚类为IgG1;将生长良好的杂交瘤细胞接种到预处理的Balb/c小鼠腹腔中制备腹水型DV-E的单克隆抗体,酶联免疫吸附试验(ELISA)证明腹水效价可达10-6; Western-blot鉴定结果提示该单克隆抗体可以特异结合DV-E。
结论:综上所述,我们运用重组DNA技术与原核表达方法获得了重组E蛋白,利用该蛋白成功制备了抗DV-E单克隆抗体,所得抗体具有生物学活性,其抗体亚类为IgG1。为进一步制备快速检测登革病毒的胶体金试纸条奠定了坚实的基础。
Objectives: Dengue(DV) disease is a life-threatening disease. And nowadays the inciDVce of Dengue fever(DF) is increasing in the world. But we have had no utility vaccinum and specific medicine yet. So rapid diagnosis of the Dengue viral infection is important and emergent.The E protein , envelope protein of DV, is an important antigen protein and intimate to immunoprotection of Dengue infection. In this study, we constructed a prokaryotic expression vector to express DV-E in E.coli and obtained purified E fusion protein. Then, the specific monoclonal antibodies (McAbs) against DV-E were prepared by hybridoma cell techniques, which served as a basement of the gold colloid test paper.
Methods:
PCR was deployed to amplify E gene segment. After double enzyme digestion, prokaryotic expression vector pET32-a and PCR products were conjuncted by T4DNA ligase and converted into E.coli JM109 to construct and biopan the recombinant plasmid which was transformated into E.coli -BL21 later. After be induted by IPGT, the fusion protein was migration in SDS-PAGE gel electrophoresis analysis and detectived by Western-blot. Then the fusion protein was purified by Ni2+-NTA-chelated agarose affinity chromatography method.
Balb/c mice were immunized with E fusion protein and monoclonal antibodies against DV-E were prepared with hybridoma method. The positive hybridomas secreting anti-E McAb were screened by ELISA. The antibody activity and specificity were analyzed by indirect ELISA assay. The McAb immunoglobulin (Ig) subtype and the titer of the selected E McAb were determined. The specificity of monoclonal antibody was tested by Western blot analysis.
Results:
The target gene segment, just the same size as expected, was amplified by PCR. The recombinant plasmid was successfully constructed and named pET32-a-E. And through be successfully transformating and inducting, we got the fusion protein, which molecularweight is 70kD, just as expected. Western-blot result indicated that the inductive expressed fusion protein could specificly bind with anti-DV serum. Through the Ni2+-NTA-chelated agarose affinity chromatography, we obtained the fusion protein with a purity of 90%.
We screening out one hybridoma, named 7C7, the Ig subtype of 7C7 McAb is IgG1 subclass. Balb/c mice predisposaled were inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-DV-E monoclonal antibody and their titer reached about 10~(-6); Western blot result suggested that McAb could bind specially to the antigen.
Conclusion:
The E fusion protein was obtained by recombinant DNA techniques and prokaryotic expression methods, and the specific monoclonal antibodies against DV-E were prepared. The obtaind monoclonal antibodies with high biological activity belong to the subtypes IgG1.
方法:
1.采用PCR法扩增E基因片段,原核表达载体pET 32-a及PCR产物经双酶切后,用T4DNA连接酶将两者连接并转化到大肠杆菌JM109构建并筛选重组质粒。随后将重组质粒转入表达宿主BL21感受态细胞,经IPGT诱导表达,对表达蛋白进行SDS-PAGE电泳分析和Western-blot检测。以Ni2+-NTA树脂亲和层析纯化重组蛋白。
2.用重组E蛋白作为抗原免疫Balb/c小鼠,采用杂交瘤技术,制备抗DV-E的单克隆抗体,用ELISA方法筛选分泌抗DV-E单克隆抗体的杂交瘤细胞株,采用间接ELISA方法和Western-blot进行McAb特异性鉴定;同时采用间接ELISA方法鉴定McAb的Ig亚类,检测McAb的效价。
结果:
1.采用PCR方法扩增得到与预期大小一致的目的基因片段。重组质粒经PCR鉴定及测序分析证实重组质粒克隆成功,命名为pET 32-a-E。转化表达宿主菌后成功诱导出分子量为70kD的重组E蛋白,与预期分子量相符。Western-blot检测表明诱导表达的融合蛋白能与抗DV的血清发生特异性结合。采用Ni2+-NTA树脂亲和层析纯化得到重组蛋白。
2.成功筛选出一株可分泌特异性McAb的杂交瘤细胞7C7,其抗体亚类为IgG1;将生长良好的杂交瘤细胞接种到预处理的Balb/c小鼠腹腔中制备腹水型DV-E的单克隆抗体,酶联免疫吸附试验(ELISA)证明腹水效价可达10-6; Western-blot鉴定结果提示该单克隆抗体可以特异结合DV-E。
结论:综上所述,我们运用重组DNA技术与原核表达方法获得了重组E蛋白,利用该蛋白成功制备了抗DV-E单克隆抗体,所得抗体具有生物学活性,其抗体亚类为IgG1。为进一步制备快速检测登革病毒的胶体金试纸条奠定了坚实的基础。
Objectives: Dengue(DV) disease is a life-threatening disease. And nowadays the inciDVce of Dengue fever(DF) is increasing in the world. But we have had no utility vaccinum and specific medicine yet. So rapid diagnosis of the Dengue viral infection is important and emergent.The E protein , envelope protein of DV, is an important antigen protein and intimate to immunoprotection of Dengue infection. In this study, we constructed a prokaryotic expression vector to express DV-E in E.coli and obtained purified E fusion protein. Then, the specific monoclonal antibodies (McAbs) against DV-E were prepared by hybridoma cell techniques, which served as a basement of the gold colloid test paper.
Methods:
PCR was deployed to amplify E gene segment. After double enzyme digestion, prokaryotic expression vector pET32-a and PCR products were conjuncted by T4DNA ligase and converted into E.coli JM109 to construct and biopan the recombinant plasmid which was transformated into E.coli -BL21 later. After be induted by IPGT, the fusion protein was migration in SDS-PAGE gel electrophoresis analysis and detectived by Western-blot. Then the fusion protein was purified by Ni2+-NTA-chelated agarose affinity chromatography method.
Balb/c mice were immunized with E fusion protein and monoclonal antibodies against DV-E were prepared with hybridoma method. The positive hybridomas secreting anti-E McAb were screened by ELISA. The antibody activity and specificity were analyzed by indirect ELISA assay. The McAb immunoglobulin (Ig) subtype and the titer of the selected E McAb were determined. The specificity of monoclonal antibody was tested by Western blot analysis.
Results:
The target gene segment, just the same size as expected, was amplified by PCR. The recombinant plasmid was successfully constructed and named pET32-a-E. And through be successfully transformating and inducting, we got the fusion protein, which molecularweight is 70kD, just as expected. Western-blot result indicated that the inductive expressed fusion protein could specificly bind with anti-DV serum. Through the Ni2+-NTA-chelated agarose affinity chromatography, we obtained the fusion protein with a purity of 90%.
We screening out one hybridoma, named 7C7, the Ig subtype of 7C7 McAb is IgG1 subclass. Balb/c mice predisposaled were inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-DV-E monoclonal antibody and their titer reached about 10~(-6); Western blot result suggested that McAb could bind specially to the antigen.
Conclusion:
The E fusion protein was obtained by recombinant DNA techniques and prokaryotic expression methods, and the specific monoclonal antibodies against DV-E were prepared. The obtaind monoclonal antibodies with high biological activity belong to the subtypes IgG1.
引文
[1]. Castleberry JS,Mahon CR.Dengue fever in the Western Hemisphere.Clin Lab Sci,2003,16:34-38.
[2]. Malavige GN,Fernando S,Fernando DJ.Dengue viral infections[J].Postgrad Med J,2004,80(948);588—601.
[3]. Gibbons RV,Vaughn DW.Dengue:an escalating problem[J].BMJ,2002,324:1563—1566.
[4]. 王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].中山医科大学学报,2001,22(6):443.6.
[5]. World Health Organization.Dengue haemorrhagic fever:diagnosis,treatment,prevention and control[M],2nd ed.World Health Organization,Geneva,Switzerland.1997:205.
[6]. De Paula SO,Pires Neto Pd,Correa JA,et a1.The use of reverse Transcription-polymerase chain reaction (RT—PCR) for the rapid detection and iDVtification of Dengue virus in an endemic region;avalidation study.Trans R Soc Trop Med Hyg,2002,96:266-269.
[7]. 李志军,王树声。登革病毒快速检测鉴定技术研究进展。广西预防医学,l997,3:40_43.
[8]. Rey F A,Heinz F X,Mandel C,et a1.The envelope glycoprotein from tick-brone encephalitis at 2A resolution [J]. Nature,1995,375:291—298.
[9]. 闻玉梅,现代医学微生物学[M].上海:上海医科大学出版社.1999.1ll7.1142
[10]. Rochrig J T, Risi P A, Mathews J H .T-Helper cell epitopes on the E-glycoprotein of Dengue Jamacia virus .virology,1994,198—23 1
[11]. Rothman A L,Kurane I,Ennis F A,et a1.Multiple specificties in the murine CD4+ and CD8+ T-cell response to Dengue virus[J].J Virol,1996,70:6540-6546.
[12]. Bray M,Lai CJ.Dengue virus pre-membrane and membrane proteins elicit aprotective immune response[J].Virology,1991,185(1):505—508.
[13]. Bray M,Markoff B,Eckels KH,et a1.Mice immunized with recombinant vaccinia virus expressing Dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal Dengue virus encephalitis[J].J Virol,1989,63(6):2853—2856.
[14]. 汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000.29-34.
[15]. 司徒镇强,吴军正.细胞培养[M].世界图书出版社.1996,232
1.Castleberry JS,Mahon CR.Dengue fever in the Western Hemisphere.Clin Lab Sci,2003,16:34-38.
2. P. Philip Samuel ,B.K. Tyagi. Diagnostic methods for detection & isolation of Dengue viruses from vector mosquitoes. Indian J Med Res 123, May 2006, pp 615-628.
3.De Paula SO,Pires Neto PJ,Correa JA,et a1.The use of reverse transcription-polymerase chain reaction (RT-PCR) for the rapid detection and iDVtification of Dengue virus in an endemic region:a validation study.Trans R Soc Trop Med Hyg,2002,96:266-269.
4. Valéria CS Pinheiro, Wanderli P Tadei, Patrícia MSS Barros,Pedro FC Vasconcelos, Ana Cecília R Cruz. Detection of Dengue virus serotype 3 by reverse transcrip- tionpolymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas. Mem Inst Oswaldo Cruz, Rio de Janeiro,2005,Vol. 100(8): 833-839.
5.Li-Jung Chien, Tsai-Ling Liao, Pei-Yun Shu, Jyh-Hsiung Huang, Duane J. Gubler, and Gwong-Jen J. Chang. Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses. Journal Of Clinical Microbiology, Apr. 2006, p. 1295–1304.
6.de Oliveira Poersch C,Pavoni DP,Queiroz MH,et a1.Dengue virus infections:comparison of methods for diagnosing the acute disease.J Clin Virol,2005,32:272-277.
7. Mackay IM,ArDV KE,Nitsche A.Real-time PCR in virology.Nucleic Acids Res,2OO2,30(6):1292
8. Shu PY,Chang SF,Kuo YC,et a1.Development of group-and serotype- specific one-step SYBR Green I-based real-time reverse transcription-PCR assay for Dengue virus.J Clin Microbiol,2003,41(6):2408
9. Alessandra Cristina Gomes-Ruiz, Renata Teodoro Nascimento, Sérgio Oliveira de Paula, Benedito Ant?nio Lopes da Fonseca . SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of Dengue virus type 3 infections. Journal Of Medical Virology ,2006,Vol 78:760-763.
10.王云霞,黄庆,陈鸣,府伟灵.中华检验医学杂志,2006,4:379-380.
11.府伟灵,黄庆.基因芯片在临床疾病诊断中的应用.国外医学临床生物化学与检验学分册,2002,23:3-l1.
12.肖维威,马文丽,王洪敏,等.2型登革病毒检测基因芯片的研制.生命科学研究,2003,7:305-309.
13.AJ Baeumner.Biosensor for Dengue virus detection:sensitive,rapid.and serotype specific[J] .Anal Chem,2002,74(6):442-8.
14. Renard M,Betkadi L,Bedouelle H.Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors.J Mol Biol,2003,326:167-175.
[2]. Malavige GN,Fernando S,Fernando DJ.Dengue viral infections[J].Postgrad Med J,2004,80(948);588—601.
[3]. Gibbons RV,Vaughn DW.Dengue:an escalating problem[J].BMJ,2002,324:1563—1566.
[4]. 王竹晨,刘建中,李燕,等.人前脂肪细胞的原代培养[J].中山医科大学学报,2001,22(6):443.6.
[5]. World Health Organization.Dengue haemorrhagic fever:diagnosis,treatment,prevention and control[M],2nd ed.World Health Organization,Geneva,Switzerland.1997:205.
[6]. De Paula SO,Pires Neto Pd,Correa JA,et a1.The use of reverse Transcription-polymerase chain reaction (RT—PCR) for the rapid detection and iDVtification of Dengue virus in an endemic region;avalidation study.Trans R Soc Trop Med Hyg,2002,96:266-269.
[7]. 李志军,王树声。登革病毒快速检测鉴定技术研究进展。广西预防医学,l997,3:40_43.
[8]. Rey F A,Heinz F X,Mandel C,et a1.The envelope glycoprotein from tick-brone encephalitis at 2A resolution [J]. Nature,1995,375:291—298.
[9]. 闻玉梅,现代医学微生物学[M].上海:上海医科大学出版社.1999.1ll7.1142
[10]. Rochrig J T, Risi P A, Mathews J H .T-Helper cell epitopes on the E-glycoprotein of Dengue Jamacia virus .virology,1994,198—23 1
[11]. Rothman A L,Kurane I,Ennis F A,et a1.Multiple specificties in the murine CD4+ and CD8+ T-cell response to Dengue virus[J].J Virol,1996,70:6540-6546.
[12]. Bray M,Lai CJ.Dengue virus pre-membrane and membrane proteins elicit aprotective immune response[J].Virology,1991,185(1):505—508.
[13]. Bray M,Markoff B,Eckels KH,et a1.Mice immunized with recombinant vaccinia virus expressing Dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal Dengue virus encephalitis[J].J Virol,1989,63(6):2853—2856.
[14]. 汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000.29-34.
[15]. 司徒镇强,吴军正.细胞培养[M].世界图书出版社.1996,232
1.Castleberry JS,Mahon CR.Dengue fever in the Western Hemisphere.Clin Lab Sci,2003,16:34-38.
2. P. Philip Samuel ,B.K. Tyagi. Diagnostic methods for detection & isolation of Dengue viruses from vector mosquitoes. Indian J Med Res 123, May 2006, pp 615-628.
3.De Paula SO,Pires Neto PJ,Correa JA,et a1.The use of reverse transcription-polymerase chain reaction (RT-PCR) for the rapid detection and iDVtification of Dengue virus in an endemic region:a validation study.Trans R Soc Trop Med Hyg,2002,96:266-269.
4. Valéria CS Pinheiro, Wanderli P Tadei, Patrícia MSS Barros,Pedro FC Vasconcelos, Ana Cecília R Cruz. Detection of Dengue virus serotype 3 by reverse transcrip- tionpolymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas. Mem Inst Oswaldo Cruz, Rio de Janeiro,2005,Vol. 100(8): 833-839.
5.Li-Jung Chien, Tsai-Ling Liao, Pei-Yun Shu, Jyh-Hsiung Huang, Duane J. Gubler, and Gwong-Jen J. Chang. Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses. Journal Of Clinical Microbiology, Apr. 2006, p. 1295–1304.
6.de Oliveira Poersch C,Pavoni DP,Queiroz MH,et a1.Dengue virus infections:comparison of methods for diagnosing the acute disease.J Clin Virol,2005,32:272-277.
7. Mackay IM,ArDV KE,Nitsche A.Real-time PCR in virology.Nucleic Acids Res,2OO2,30(6):1292
8. Shu PY,Chang SF,Kuo YC,et a1.Development of group-and serotype- specific one-step SYBR Green I-based real-time reverse transcription-PCR assay for Dengue virus.J Clin Microbiol,2003,41(6):2408
9. Alessandra Cristina Gomes-Ruiz, Renata Teodoro Nascimento, Sérgio Oliveira de Paula, Benedito Ant?nio Lopes da Fonseca . SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of Dengue virus type 3 infections. Journal Of Medical Virology ,2006,Vol 78:760-763.
10.王云霞,黄庆,陈鸣,府伟灵.中华检验医学杂志,2006,4:379-380.
11.府伟灵,黄庆.基因芯片在临床疾病诊断中的应用.国外医学临床生物化学与检验学分册,2002,23:3-l1.
12.肖维威,马文丽,王洪敏,等.2型登革病毒检测基因芯片的研制.生命科学研究,2003,7:305-309.
13.AJ Baeumner.Biosensor for Dengue virus detection:sensitive,rapid.and serotype specific[J] .Anal Chem,2002,74(6):442-8.
14. Renard M,Betkadi L,Bedouelle H.Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors.J Mol Biol,2003,326:167-175.