小反刍兽疫病毒N蛋白原核表达条件的优化及反应活性
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摘要
目的探讨小反刍兽疫病毒(Peste des Petits Ruminants Virus,PPRV)N蛋白原核表达、条件优化及反应活性。方法参照GenBank中PPRV (Nigeria/75/1)N基因序列,人工合成该基因,构建重组表达质粒pET-30a (+)-N,转化至BL21 (DE3)感受态细胞,IPTG诱导表达,进行SDS-PAGE电泳、Western-blot分析。在不同温度、时间、IPTG浓度条件下诱导表达N蛋白,确定最佳表达条件。结果 PPRV N蛋白(64.4kD)成功表达,能被羊PPRV免疫血清所识别。N蛋白主要以包涵体存在,其最佳诱导条件:诱导温度28℃、IPTG终浓度2.0mmol/L、诱导时间16h。结论原核表达的PPRV N蛋白具有良好的特异性和反应活性,为单克隆抗体的制备及快速诊断方法的建立提供了技术资料。
Objective To explore the prokaryotic expression of N protein of Peste des Petits Ruminants Virus,the optimal conditions and reactivity.Methods According to the sequence of PPRV(Nigeria/75/1)N gene published in GenBank,the gene was synthesized and cloned into the prokaryotic expression vector pET-30a(+).After the recombinant plasmid was obtained,it was transformed into BL21(DE3)competent cells.The expression of the recombinant protein was induced by IPTG,and identified by using SDS-PAGE electrophoresis and Western-blot.The expression of N protein was induced under different temperatures and IPTG concentrations and at different times to determine the optimal expression conditions.Results The PPRV N protein was successfully expressed.The fusion protein had a relative molecular mass of about64.4kD and could be identified by immunized sera from sheep PPRV.The induced expression of N protein was mainly in inclusion bodies.The optimal inducing conditions were a temperature at 18℃and a final concentration of IPTG at 2.0mmol/L with 16 hours of induction.Conclusion The prokaryotic expression of PPRV N protein has good specificity and reactivity,which lays a foundation for the preparation of monoclonal antibodies and the establishment of a rapid diagnostic method.
Objective To explore the prokaryotic expression of N protein of Peste des Petits Ruminants Virus,the optimal conditions and reactivity.Methods According to the sequence of PPRV(Nigeria/75/1)N gene published in GenBank,the gene was synthesized and cloned into the prokaryotic expression vector pET-30a(+).After the recombinant plasmid was obtained,it was transformed into BL21(DE3)competent cells.The expression of the recombinant protein was induced by IPTG,and identified by using SDS-PAGE electrophoresis and Western-blot.The expression of N protein was induced under different temperatures and IPTG concentrations and at different times to determine the optimal expression conditions.Results The PPRV N protein was successfully expressed.The fusion protein had a relative molecular mass of about64.4kD and could be identified by immunized sera from sheep PPRV.The induced expression of N protein was mainly in inclusion bodies.The optimal inducing conditions were a temperature at 18℃and a final concentration of IPTG at 2.0mmol/L with 16 hours of induction.Conclusion The prokaryotic expression of PPRV N protein has good specificity and reactivity,which lays a foundation for the preparation of monoclonal antibodies and the establishment of a rapid diagnostic method.
引文
[1]扈荣良,现代动物病毒学[M].北京:中国农业出版社,2014:1079.
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[11] PANG Wenjing,FU Mingzhe,ZHANG Qi,et al.Establishment and application of PPRV indirect ELISA detection method based on N protein[J].J Northwest A&F Univ(Nat Sci),2017,45(11):1-8.(in Chinese)庞文静,付明哲,张琪,等.基于N蛋白的PPRV间接ELISA检测方法的建立及应用[J].西北农林科技大学学报(自然科学版),2017,45(11):1-8.
[12] ZHANG Xue,JIA Yun,SUN Mingying,et al.Expression and bioactivity of nucleoprotein from Peste des Petits Ruminants Virus[J].Chin Agric Sci Bull,2016,32(23):27-31.(in Chinese)张雪,贾赟,孙铭英,等.小反刍兽疫病毒核蛋白的原核表达及免疫原性分析[J].中国农学通报,2016,32(23):27-31.
[13] Yadav V,Balamurugan V,Bhanuprakash V,et al.Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen[J].J Virolog Methods,2009,162(1-2):56-63.
[14] JIA Fengqin,LI Gang,LI Wei,et al.Expression and identification of Peste des petits ruminants virus N gene in Escherichia coli[J].J Agric Biotechnol,2009,17(2):195-200.(in Chinese)贾凤芹,李刚,李伟,等.小反刍兽疫病毒N基因在大肠杆菌中的表达及鉴定[J].农业生物技术学报,2009,17(2):195-200.
[15] RUAN Yang,QIN Junling,GAO Yuwei,et al.The prokaryotic expression of N gene from Peste des Petits Ruminants Virus[J],Prog Vet Med,2011,32(3):21-25.(in Chinese)阮洋,秦峻岭,高玉伟,等.小反刍兽疫病毒N基因的原核表达[J].动物医学进展,2011,32(3):21-25.
[16] LI Wentao,LI Zhen,DONG Shijuan,et al.Optimization of expression conditions of recombinant nucleocapsid protein of Peste des petits ruminant virus[J].Acta Agric Shanghai,2013,29(3):7-9.(in Chinese)李文涛,李震,董世娟,等.小反刍兽疫病毒核衣壳蛋白(N)重组表达条件的优化[J].上海农业学报,2013,29(3):7-9.
[17] SUN Yu,ZHAO Bailin,WANG Xiaoying,et al.Prokaryotic expression of N protein of peste des petits ruminants virus and development of indirect ELISA[J].Prog Vet Med,2017,38(1):6-10.(in Chinese)孙雨,赵柏林,王晓英,等.小反刍兽疫病毒N蛋白原核表达与间接ELISA检测方法的建立[J].动物医学进展,2017,38(1):6-10.
[18] HE Yapeng,BAO Changlei,ZHANG Qi,et al.Cloning,sequence analysis and prokaryotic expression of N and M gene of peste des petits ruminants virus vaccine strain[J].Acta Agric Boreali-Occidentalis Sinica,2017,26(2):179-184.(in Chinese)何亚鹏,鲍长磊,张琪,等.小反刍兽疫病毒疫苗株N和M基因的克隆、序列分析及原核表达[J].西北农业学报,2017,26(2):179-184.
[2] Kumar N,Maherchandani S,Kashyap SK,et al.Peste des petits ruminants virus infection of small ruminants:A comprehensive review[J].Viruses,2014,6(6):2287-2327.
[3] Al-Dubaib Ma.Peste des petitis ruminants morbillivirus infection in lambs and young goats at Qassim region,Saudi Arabia[J].Trop Anim Health Prod,2009,41(2):217-220.
[4] Hegde R,Gomes AR,Muniyellappah K,et al.A short note on peste des petits ruminants in Karnataka,India[J].Rev Sci Tech,2009,28(3):1031-1035.
[5] ZHAO Ya,MA Hailu,LI Yueyang,et al.Sequence analysis of N gene of peste des petits ruminants virus from Xinjiang province[J].Animal Husbandry&Vet Med,2016,48(2):17-23.(in Chinese)赵亚,马海璐,李岳洋,等.小反刍兽疫病毒新疆株的V基因序列分析[J].畜牧与兽医,2016,48(2):17-23.
[6] MIAO Shukui,REN Fang,WANG Ping,et al.Prokaryotic expression and bioinformatics analysis of N gene of Peste des Petits Ruminants Virus[J].Chin J Biolog,2017,30(8):806-810.(in Chinese)苗书魁,任方,汪萍,等.小反刍兽疫病毒N蛋白的原核表达及其生物信息学分析[J].中国生物制品学杂志,2017,30(8):806-810.
[7] Adombi CM,Lelenta M,Lanien CE,et al.Monkey CV1cell line expressing the sheep-goat SLAM protein:A highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens[J].J Virol Methods,2011,173(2):306-313.
[8] Balanurugan V,Sen A,Srrsvanan P,et al.One step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical sam ples[J].Vet Res Commun,2006,30(6):655-666.
[9] ZENG Shaoling,RUAN Zhouxi,TANG Jinming,et al.Prediction of B cell epitopes,synthesis and development of indirect ELISA for N protein of peste des petits ruminants virus[J].Prog Vet Med,2015,36(8):40-44.(in Chinese)曾少灵,阮周曦,唐金明,等.小反刍兽疫病毒N蛋白B细胞表位预测、合成及间接ELISA方法的建立[J].动物医学进展,2015,36(8):40-44.
[10] WANG Dongfang,LIU Ye,MI Lijuan,et al.Prokaryotic expression and purification of Peste des Petits Ruminants Virus N protein and development of indirect ELISA method for its antibody[J].Chin J Biolog,2017,30(8):838-842.(in Chinese)王东方,刘晔,米立娟,等.小反刍兽疫病毒N蛋白的原核表达、纯化及其抗体间接ELISA检测方法的建立[J].中国生物制品学杂志,2017,30(8):838-842.
[11] PANG Wenjing,FU Mingzhe,ZHANG Qi,et al.Establishment and application of PPRV indirect ELISA detection method based on N protein[J].J Northwest A&F Univ(Nat Sci),2017,45(11):1-8.(in Chinese)庞文静,付明哲,张琪,等.基于N蛋白的PPRV间接ELISA检测方法的建立及应用[J].西北农林科技大学学报(自然科学版),2017,45(11):1-8.
[12] ZHANG Xue,JIA Yun,SUN Mingying,et al.Expression and bioactivity of nucleoprotein from Peste des Petits Ruminants Virus[J].Chin Agric Sci Bull,2016,32(23):27-31.(in Chinese)张雪,贾赟,孙铭英,等.小反刍兽疫病毒核蛋白的原核表达及免疫原性分析[J].中国农学通报,2016,32(23):27-31.
[13] Yadav V,Balamurugan V,Bhanuprakash V,et al.Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen[J].J Virolog Methods,2009,162(1-2):56-63.
[14] JIA Fengqin,LI Gang,LI Wei,et al.Expression and identification of Peste des petits ruminants virus N gene in Escherichia coli[J].J Agric Biotechnol,2009,17(2):195-200.(in Chinese)贾凤芹,李刚,李伟,等.小反刍兽疫病毒N基因在大肠杆菌中的表达及鉴定[J].农业生物技术学报,2009,17(2):195-200.
[15] RUAN Yang,QIN Junling,GAO Yuwei,et al.The prokaryotic expression of N gene from Peste des Petits Ruminants Virus[J],Prog Vet Med,2011,32(3):21-25.(in Chinese)阮洋,秦峻岭,高玉伟,等.小反刍兽疫病毒N基因的原核表达[J].动物医学进展,2011,32(3):21-25.
[16] LI Wentao,LI Zhen,DONG Shijuan,et al.Optimization of expression conditions of recombinant nucleocapsid protein of Peste des petits ruminant virus[J].Acta Agric Shanghai,2013,29(3):7-9.(in Chinese)李文涛,李震,董世娟,等.小反刍兽疫病毒核衣壳蛋白(N)重组表达条件的优化[J].上海农业学报,2013,29(3):7-9.
[17] SUN Yu,ZHAO Bailin,WANG Xiaoying,et al.Prokaryotic expression of N protein of peste des petits ruminants virus and development of indirect ELISA[J].Prog Vet Med,2017,38(1):6-10.(in Chinese)孙雨,赵柏林,王晓英,等.小反刍兽疫病毒N蛋白原核表达与间接ELISA检测方法的建立[J].动物医学进展,2017,38(1):6-10.
[18] HE Yapeng,BAO Changlei,ZHANG Qi,et al.Cloning,sequence analysis and prokaryotic expression of N and M gene of peste des petits ruminants virus vaccine strain[J].Acta Agric Boreali-Occidentalis Sinica,2017,26(2):179-184.(in Chinese)何亚鹏,鲍长磊,张琪,等.小反刍兽疫病毒疫苗株N和M基因的克隆、序列分析及原核表达[J].西北农业学报,2017,26(2):179-184.