猪瘟病毒E2蛋白在大肠杆菌中的表达及免疫原性分析
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摘要
为制备具有免疫原性的猪瘟病毒E2蛋白,在大肠杆菌中表达E2蛋白,并对其进行纯化及免疫原性分析。将E2基因插入原核表达载体p ET-28a,构建重组质粒p ET-28a-E2,将其转化至大肠杆菌BL21(DE3)感受态细胞进行诱导表达,SDS-PAGE电泳和Western blot分析结果显示,成功表达了可溶性E2蛋白。对IPTG浓度及诱导温度和时间进行优化,结果显示,IPTG终浓度为0.1 mmol/L时,18℃条件下诱导表达16 h,E2蛋白的可溶性表达量最高。通过分子筛一步纯化E2蛋白,其终质量浓度为0.5 g/L。Western blot分析和间接ELISA结果显示,纯化后的E2蛋白在变性及非变性条件下都能够被猪瘟阳性血清特异性识别,表明纯化后的E2蛋白活性良好。将纯化后的E2蛋白添加弗氏佐剂乳化后免疫BALB/C小鼠,采集血清进行病毒中和试验,结果显示,免疫后56 d产生较高水平的中和抗体,表明E2蛋白免疫原性良好。综上,成功利用大肠杆菌BL21(DE3)菌株表达了可溶性E2蛋白,且纯化后的E2蛋白免疫原性良好。
The objective of this study was to obtain the immunogenic classical swine fever virus( CSFV) E2 protein using E. coli expression system,and then get the purified E2 protein,followed by estimation of its immunogenicity. The E2 gene was inserted into prokaryotic expression vector p ET-28 a to construct recombinant plasmid p ET-28 a-E2. The plasmid was then transformed into E. coli BL21( DE3) competent cells for expression. SDS-PAGE and Western blot results showed that E2 protein was successfully expressed in the cells and proved to be soluble. Through the optimization of IPTG concentration,inductiontemperature and time,it was found that the highest soluble expression of E2 protein was achieved at 18 ℃after induction with 0. 1 mmol/L IPTG for 16 hours. After one-step purification of size exclusion chromatography,the E2 protein was purified and the final concentration was 0. 5 g/L. Western blot and indirect ELISA results showed that the purified E2 protein with or without denaturation could be specifically recognized by the CSFV positive serum,indicating that the purified E2 protein was of good activity. The purified E2 protein was used to immunize BALB/C mice after emulsified with Freund 's adjuvant. Blood samples were collected at different time points for virus neutralization experiment. The results showed that the neutralizing antibody was detected after 56 days of immunization,indicating that the E2 protein had good immunogenicity. In conclusion,soluble envelope glycoprotein E2 could be expressed in E. coli BL21( DE3) competent cells,which represented good immunogenicity after purification.
The objective of this study was to obtain the immunogenic classical swine fever virus( CSFV) E2 protein using E. coli expression system,and then get the purified E2 protein,followed by estimation of its immunogenicity. The E2 gene was inserted into prokaryotic expression vector p ET-28 a to construct recombinant plasmid p ET-28 a-E2. The plasmid was then transformed into E. coli BL21( DE3) competent cells for expression. SDS-PAGE and Western blot results showed that E2 protein was successfully expressed in the cells and proved to be soluble. Through the optimization of IPTG concentration,inductiontemperature and time,it was found that the highest soluble expression of E2 protein was achieved at 18 ℃after induction with 0. 1 mmol/L IPTG for 16 hours. After one-step purification of size exclusion chromatography,the E2 protein was purified and the final concentration was 0. 5 g/L. Western blot and indirect ELISA results showed that the purified E2 protein with or without denaturation could be specifically recognized by the CSFV positive serum,indicating that the purified E2 protein was of good activity. The purified E2 protein was used to immunize BALB/C mice after emulsified with Freund 's adjuvant. Blood samples were collected at different time points for virus neutralization experiment. The results showed that the neutralizing antibody was detected after 56 days of immunization,indicating that the E2 protein had good immunogenicity. In conclusion,soluble envelope glycoprotein E2 could be expressed in E. coli BL21( DE3) competent cells,which represented good immunogenicity after purification.
引文
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[14]刘伯华.猪瘟病毒E2基因在大肠杆菌中的高效表达及其表达产物的制备[D].长春:中国人民解放军军需大学,2002.
[15]白羊,章永垒,邹有土,等.重组人βNGF在大肠杆菌中的可溶表达、纯化及活性鉴定[J].生物技术通报,2016,32(11):202-207.
[16]唐红慧.猪瘟兔化弱毒苗抗原量测定方法的探索及不同免疫程序的差异性比较研究[D].扬州:扬州大学,2010.
[17]Sorensen H P,Mortensen K K.Advanced genetic strategies for recombinant protein expression in Escherichia coli[J].Journal of Biotechnology,2005,115(2):113-128.
[18]杜立中,马霞,刘永录,等.黄芪多糖对猪瘟活疫苗免疫应答的佐剂作用[J].河南农业科学,2014,43(9):156-159.
[19]郑旭升.乳猪用猪瘟兔化苗免疫失败原因浅探[J].畜牧与兽医,1982(2):88.
[20]Yu X,Tu C,Li H,et al.DNA-mediated protection against classical swine fever virus[J].Vaccine,2001,19(11):1520-1525.
[21]Wang Z,Nie Y,Wang P,et al.Characterization of classical swine fever virus entry by using pseudotyped viruses:E1 and E2 are sufficient to mediate viral entry[J].Virology,2004,330(1):332-341.
[22]赵天生,张悦之,夏燕华.猪瘟病毒E(rns)蛋白RNase活性与免疫抑制功能的相关性研究[J].河南农业科学,2014,43(3):124-127,138.
[23]田宏.猪水疱病和猪瘟基因工程亚单位疫苗的研究[D].杨凌:西北农林科技大学,2006.
[24]Wensvoort G.Topographical and funcitional mapping of epitopes on hog cholera virus with monoclonal antibodies[J].Journal of General Virology,1989,70(11):2865-2876.
[25]Dong X N,Chen Y H.Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2[J].Vaccine,2006,24(11):1906-1913.
[26]周景明,李鹏飞,张改平,等.猪瘟病毒E2蛋白在大肠杆菌中的表达及其可溶性分析[J].西北农业学报,2015,24(11):24-28.
[27]汪雪峰,董利阳,王钧,等.纳米材料与免疫系统的相互作用[J].江苏大学学报(医学版),2015,25(1):85-87,92.
[28]姚晨婕.纳米二氧化钛细胞毒性及免疫毒性机理研究[D].上海:上海大学,2016.
[2]许信刚,王丹,童德文.表达猪瘟病毒E2蛋白重组减毒鼠伤寒沙门氏菌活载体疫苗株的构建及动物免疫试验[J].中国兽医科学,2011,41(9):901-906.
[3]范学政,徐璐,赵启祖,等.猪瘟兔化弱毒E2基因重组杆状病毒的构建及抗体制备[J].中国兽药杂志,2015,49(1):6-9.
[4]Hammond J M,Mccoy R J,Jansen E S,et al.Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55(E2)gene protects pigs against classical swine fever[J].Vaccine,2000,18(11/12):1040-1050.
[5]Sun S Q,Yin S H,Guo H C,et al.Genetic typing of classical swine fever virus isolates from China[J].Transboundary and Emerging Diseases,2013,60(4):370-375.
[6]Sun Y,Liu D F,Wang Y F,et al.Generation and efficacy evaluation of a recombinant adenovirus expressing the E2protein of classical swine fever virus[J].Research in Veterinary Science,2010,88(1):77-82.
[7]Moormann R J,Van Gennip H G,Miedema G K,et al.Infectious RNA transcribed from an engineered full-length c DNA template of the genome of a pestivirus[J].Journal of Virology,1996,70(2):763-770.
[8]Lin M,Trottier E,Mallory M.Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection[J].Clinical and Diagnostic Laboratory Immunology,2005,12(7):877-881.
[9]范学政,王琴,陈振海,等.猪瘟兔化弱毒株E2基因的原核表达及间接ELISA的初步建立[J].中国病毒学,2005,20(3):253-256.
[10]李丽,陈弟诗,张毅等.猪瘟病毒及猪瘟疫苗的研究进展[J].当代畜牧,2014(24):48-49.
[11]Lee N H,Lee J A,Park S Y,et al.A review of vaccine development and research for industry animals in Korea[J].Clinical and Experimental Vaccine Research,2012,1(1):18-34.
[12]涂长春,王文成,余兴龙,等.猪瘟病毒DNA疫苗的田间免疫试验及环境生物安全评价[C]//中华预防医学会.2005全国第二届核酸疫苗研讨会论文集.北京:[出版者不详],2005.
[13]郭鹤.猪瘟病毒囊膜E2蛋白原核表达及初步应用研究[D].长春:吉林大学,2013.
[14]刘伯华.猪瘟病毒E2基因在大肠杆菌中的高效表达及其表达产物的制备[D].长春:中国人民解放军军需大学,2002.
[15]白羊,章永垒,邹有土,等.重组人βNGF在大肠杆菌中的可溶表达、纯化及活性鉴定[J].生物技术通报,2016,32(11):202-207.
[16]唐红慧.猪瘟兔化弱毒苗抗原量测定方法的探索及不同免疫程序的差异性比较研究[D].扬州:扬州大学,2010.
[17]Sorensen H P,Mortensen K K.Advanced genetic strategies for recombinant protein expression in Escherichia coli[J].Journal of Biotechnology,2005,115(2):113-128.
[18]杜立中,马霞,刘永录,等.黄芪多糖对猪瘟活疫苗免疫应答的佐剂作用[J].河南农业科学,2014,43(9):156-159.
[19]郑旭升.乳猪用猪瘟兔化苗免疫失败原因浅探[J].畜牧与兽医,1982(2):88.
[20]Yu X,Tu C,Li H,et al.DNA-mediated protection against classical swine fever virus[J].Vaccine,2001,19(11):1520-1525.
[21]Wang Z,Nie Y,Wang P,et al.Characterization of classical swine fever virus entry by using pseudotyped viruses:E1 and E2 are sufficient to mediate viral entry[J].Virology,2004,330(1):332-341.
[22]赵天生,张悦之,夏燕华.猪瘟病毒E(rns)蛋白RNase活性与免疫抑制功能的相关性研究[J].河南农业科学,2014,43(3):124-127,138.
[23]田宏.猪水疱病和猪瘟基因工程亚单位疫苗的研究[D].杨凌:西北农林科技大学,2006.
[24]Wensvoort G.Topographical and funcitional mapping of epitopes on hog cholera virus with monoclonal antibodies[J].Journal of General Virology,1989,70(11):2865-2876.
[25]Dong X N,Chen Y H.Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2[J].Vaccine,2006,24(11):1906-1913.
[26]周景明,李鹏飞,张改平,等.猪瘟病毒E2蛋白在大肠杆菌中的表达及其可溶性分析[J].西北农业学报,2015,24(11):24-28.
[27]汪雪峰,董利阳,王钧,等.纳米材料与免疫系统的相互作用[J].江苏大学学报(医学版),2015,25(1):85-87,92.
[28]姚晨婕.纳米二氧化钛细胞毒性及免疫毒性机理研究[D].上海:上海大学,2016.
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