马尔堡病毒糖蛋白RBD基因的原核表达、纯化及多克隆抗体的制备
|
摘要
原核表达并纯化马尔堡病毒(Marburg virus,MARV)糖蛋白(glycoprotein,GP)受体结合域(receptor binding domain,RBD)蛋白,并以此为抗原免疫家兔制备抗MARV-GP-RBD多克隆抗体。参照GenBank提供的MARV GP全基因序列,找到主要抗原表位区域,设计特异性引物,采用PCR方法扩增RBD基因,扩增产物经双酶切(EcoRⅠ/XhoⅠ)后定向克隆至原核表达载体pET-30a(+),构建重组表达质粒pET-30a(+)-GP-RBD,转化BL21(DE3)感受态表达宿主菌,在不同条件下(时间、IPTG浓度、温度)诱导表达目的蛋白,并用His-Band N+柱进行亲和层析纯化;以纯化的重组pET-30a(+)-GP-RBD蛋白免疫家兔,制备多克隆抗体。通过SDS-PAGE、Western blot和IFA鉴定重组蛋白的反应原性及免疫原性。结果显示:PCR扩增到长度为453bp的RBD基因片段;构建的重组质粒pET-30a(+)-GP-RBD经双酶切后得到与目的片段长度相同的特异性条带,测序结果显示没有突变;转化产物在培养7h、终浓度为0.4mmol/L IPTG和37℃条件下能够充分诱导目的蛋白表达,得到相对分子质量为25 000的重组蛋白,主要以包涵体形式存在,BCA试剂盒产量测定,每升诱导的重组菌可纯化约20mg纯度较高的目的蛋白;Western blot检测证实重组pET-30a(+)-GP-RBD蛋白能同时被抗His标签的单抗和兔源多抗识别并发生特异性反应,证明重组蛋白有良好的反应原性;IFA鉴定证实所制备的兔源多抗能够特异性识别表达MARV GP蛋白的重组杆状病毒rBacmid-GP-VP40,证明重组蛋白具有良好的免疫原性。结果表明:成功表达、纯化了MARV GP RBD蛋白,并完成了兔源多抗的制备,为MARV亚单位疫苗的制备和抗原、抗体检测方法的建立奠定基础。
Receptor binding domain(RBD)of GP protein of Marburg virus was expressed in prokaryotic cells and purified to produce the polyclonal antibody of rabbit against MARV-GP-RBD protein.According to the genes equences of MARV GP published in GenBank,apair of primers was designed for amplifying the RBD of MARV GP by PCR method.Double digested PCR products were cloned into the prokaryotic expression vector pET-30a(+)to generate the recombinant expression plasmid pET-30a(+)-GP-RBD,which was transformed into E.coli BL21(DE3)for expressing MARV-GP-RBD proteins under variously inducing conditions(time,IPTG concentration,temperature).Recombinant protein was purified using His-Band Ni+affinity chromatography and purified recombinant protein was used to immunize rabbit to produce polyclonal antibody.The immunogenicity and reactivity of the recombinant protein was identified by SDS-PAGE,Western blot and IFA.A fragment about 453 bp in length of RBD gene was successfully amplified,recombinant plasmid pET-30a(+)-GP-RBD was constructed,correctly inserted into BL21(DE3)after PCR restriction analysis and sequencing.SDS-PAGE indentified a 25 000 protein which was fully expressed as inclusion bodies with induction by 0.4mmol/L IPTGat 37℃for 7h.After purification by His-BandNi+,affinity chromatography,20 mg target protein was produced from 1Lof shake flask culture which determined by BCA protein assay kit.Western blot confirmed that the recombinant protein could be recognized by monoclonal antibody against His and polyclonal antibody from rabbit,showing agood immunoreactivity;IFA test showed that polyclonal antibodies could identify recombinant baculovirus rBacmid-GP-VP40 expressing MARV GP,proving agood immunogenicity.RBD protein of GP of Maburg virus was successfully expressed and purified.In addition,polyclonal antibody was prepared,which laied the foundation for the research on subunit vaccine and establishment of detection method of antigen and antibody for MARV.
Receptor binding domain(RBD)of GP protein of Marburg virus was expressed in prokaryotic cells and purified to produce the polyclonal antibody of rabbit against MARV-GP-RBD protein.According to the genes equences of MARV GP published in GenBank,apair of primers was designed for amplifying the RBD of MARV GP by PCR method.Double digested PCR products were cloned into the prokaryotic expression vector pET-30a(+)to generate the recombinant expression plasmid pET-30a(+)-GP-RBD,which was transformed into E.coli BL21(DE3)for expressing MARV-GP-RBD proteins under variously inducing conditions(time,IPTG concentration,temperature).Recombinant protein was purified using His-Band Ni+affinity chromatography and purified recombinant protein was used to immunize rabbit to produce polyclonal antibody.The immunogenicity and reactivity of the recombinant protein was identified by SDS-PAGE,Western blot and IFA.A fragment about 453 bp in length of RBD gene was successfully amplified,recombinant plasmid pET-30a(+)-GP-RBD was constructed,correctly inserted into BL21(DE3)after PCR restriction analysis and sequencing.SDS-PAGE indentified a 25 000 protein which was fully expressed as inclusion bodies with induction by 0.4mmol/L IPTGat 37℃for 7h.After purification by His-BandNi+,affinity chromatography,20 mg target protein was produced from 1Lof shake flask culture which determined by BCA protein assay kit.Western blot confirmed that the recombinant protein could be recognized by monoclonal antibody against His and polyclonal antibody from rabbit,showing agood immunoreactivity;IFA test showed that polyclonal antibodies could identify recombinant baculovirus rBacmid-GP-VP40 expressing MARV GP,proving agood immunogenicity.RBD protein of GP of Maburg virus was successfully expressed and purified.In addition,polyclonal antibody was prepared,which laied the foundation for the research on subunit vaccine and establishment of detection method of antigen and antibody for MARV.
引文
[1]SIEGERT R,SHU H,SLENCZKA H,et al.The aetiology of an unknown human infection transmitted by monkeys(preliminary communication)[J].German Med Monthly,1968,13(1):1-4.
[2]于双平,姜晓舜,王松俊,等.马尔堡出血热的研究进展[J].应用预防医学,2008,14(2):119-121.
[3]陆如山.马尔堡病毒病简介[J].医学情报工作,2005(3):38.
[4]SWANEPOEL R,SMIT S B,ROLLIN P E,et al.Studies of reservoir hosts for Marburg virus[J].Emerg Infect Dis,2007,13(2):1847-1851.
[5]MONATH T P.Ecology of Marburg and Ebola viruses:speculations and directions for future research[J].J Infect Dis,1999,179(1):127-138.
[6]LEFFEL E K,EED D S.Marburg and Ebola viruses as aerosol threats[J].Biosecur Bioterror,2004,2(3):186-191.
[7]FELDMANN H.Marburg hemorrhagic fever-the forgotten cousin strikes[J].N Engl J Med,2006,355(9):866-869.
[8]PAN Y,ZHANG W,CUI L,et al.Reston virus in domestic pigs in China[J].Arch Virol,2014,159(5):1129-1132.
[9]YUAN J,ZHANG Y,LI J,et al.Serological evidence of ebola virus infection in bats,China[J].Virol J,2012(9):236.
[10]HUANG Y,WEI H,WANG Y,et al.Rapid detection of filoviruses by real-time TaqMan polymerase chain reaction assays[J].Virol Sin,2012,27(5):273-277.
[11]NAKAYAMA E,YOKOYAMA A,MIYAMOTOH,et al.Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies[J].Clin Vaccine Immunol,2010,17(11):1723-1728.
[12]洪烨,郑夔,相大鹏,等.马尔堡病毒的实时荧光RT-PCR检测方法研究[J].中国国境卫生检疫杂志,2011,34(6):435-438.
[13]HUANG Y,ZHU Y J,YANG M S.Nucleoproteinbased indirect enzyme-linked immunosorbent assay(indirect ELISA)for detecting antibodies specific to Ebola virus and Marbug virus[J].Virol Sin,2014,29(6):372-380.
[14]钟璟皓,韩一芳,张晨,等.马尔堡病毒LAMP可视化快速检测方法的建立[J].中国病原生物学杂志,2016,11(4):320-324.
[15]BRAUBURGER K,HUME A J,HLBERGER E,et al.Forty-five years of Marburg virus research[J].Viruses,2012,4(10):1878-1927.
[16]BAUSCH D G,GEISBERT T W.Development of vaccines for Marburg hemorrhagic fever[J].Expert Rev Vaccines,2007,6(1):57-74.
[17]SANCHEZ A,GEISBERT T W,FELDMAN H,et al.Filoviridae:Marburg and Ebola viruses[M]//Knipe DM,Howley P M.Fields Virology,5th.Lippincott:Williams and Wilkins,2007.
[18]VOLCHKOV V E,VOLCHKOVA V A,STROHERU,et al.Proteolytic processing of Marburg virus glycoprotein[J].Virology,2000,268(1):1-6.
[19]GEISBERT T W,JAHRLING P B.Differentiation of filoviruses by electron microscopy[J].Virus Res,1999,39(2/3):129-150.
[20]MAR IANKOVA R F,GLUSHAKOVA S E,PYZHIK E V,et al.The penetration of the Marburg virus into eukaryotic cells[J].Vopr Virusol,1993,38(2):74-76.
[21]SCHOMBERG K,MATSUYAMA S,KABSCH K,et al.Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein[J].J Virol,2006,80(8):4174-4178.
[22]HEVEY M,NEGLEY D,GEISBERT J,et al.Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants[J].Virology,1997,239(1):206-216.
[23]FELDMANN H,VOLCHKOV V E,VOLCHKOVAV A,et al.Biosynthesis and role of filoviral glycoproteins[J].Gen Virol,2001,82(12):2839-2848.
[24]MITTLER E1,KOLESNIKOVA L,HERWIG A,et al.Assembly of the Marburg virus envelope[J].Cell Microbiol,2013,15(2):270-284.
[25]ECKERT D M,KIM P S.Mechanisms of viral membrane fusion and its inhibition[J].Ann Rev Biochem,2001,70:777-810.
[26]MEDINA M F,KOBINGER G P,UX J,et al.Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung[J].Mol Ther,2003,8(5):777-789.
[27]KUHN J H,RAOSHITZKY S R,GUTH A C,et al.Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavi-rus bind a common receptor[J].Biol Chem,2006,281(23):15951-15958.
[28]MARZI A,GRAMBERG T,SIMMONS G,et al.DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus[J].J Virol,2004,78(21):12090-12095.
[29]胡旭乐,温志远,宋坤,等.表达马尔堡病毒囊膜糖蛋白GP重组新城疫病毒的构建及免疫原性评估[J].中国预防兽医学报,2014,36(9):667-671.
[30]邵钰,王金良,刘任强.等.埃博拉和马尔堡病毒囊膜糖蛋白嵌合型重组水泡性口炎病毒的构建及鉴定[J].中国预防兽医学报,2016,38(6):438-442.
[31]唐以杰,陈清池.利用大肠杆菌获取重组蛋白的研究进展[J].广东教育学院学报,2001,21(2):60-63.
[2]于双平,姜晓舜,王松俊,等.马尔堡出血热的研究进展[J].应用预防医学,2008,14(2):119-121.
[3]陆如山.马尔堡病毒病简介[J].医学情报工作,2005(3):38.
[4]SWANEPOEL R,SMIT S B,ROLLIN P E,et al.Studies of reservoir hosts for Marburg virus[J].Emerg Infect Dis,2007,13(2):1847-1851.
[5]MONATH T P.Ecology of Marburg and Ebola viruses:speculations and directions for future research[J].J Infect Dis,1999,179(1):127-138.
[6]LEFFEL E K,EED D S.Marburg and Ebola viruses as aerosol threats[J].Biosecur Bioterror,2004,2(3):186-191.
[7]FELDMANN H.Marburg hemorrhagic fever-the forgotten cousin strikes[J].N Engl J Med,2006,355(9):866-869.
[8]PAN Y,ZHANG W,CUI L,et al.Reston virus in domestic pigs in China[J].Arch Virol,2014,159(5):1129-1132.
[9]YUAN J,ZHANG Y,LI J,et al.Serological evidence of ebola virus infection in bats,China[J].Virol J,2012(9):236.
[10]HUANG Y,WEI H,WANG Y,et al.Rapid detection of filoviruses by real-time TaqMan polymerase chain reaction assays[J].Virol Sin,2012,27(5):273-277.
[11]NAKAYAMA E,YOKOYAMA A,MIYAMOTOH,et al.Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies[J].Clin Vaccine Immunol,2010,17(11):1723-1728.
[12]洪烨,郑夔,相大鹏,等.马尔堡病毒的实时荧光RT-PCR检测方法研究[J].中国国境卫生检疫杂志,2011,34(6):435-438.
[13]HUANG Y,ZHU Y J,YANG M S.Nucleoproteinbased indirect enzyme-linked immunosorbent assay(indirect ELISA)for detecting antibodies specific to Ebola virus and Marbug virus[J].Virol Sin,2014,29(6):372-380.
[14]钟璟皓,韩一芳,张晨,等.马尔堡病毒LAMP可视化快速检测方法的建立[J].中国病原生物学杂志,2016,11(4):320-324.
[15]BRAUBURGER K,HUME A J,HLBERGER E,et al.Forty-five years of Marburg virus research[J].Viruses,2012,4(10):1878-1927.
[16]BAUSCH D G,GEISBERT T W.Development of vaccines for Marburg hemorrhagic fever[J].Expert Rev Vaccines,2007,6(1):57-74.
[17]SANCHEZ A,GEISBERT T W,FELDMAN H,et al.Filoviridae:Marburg and Ebola viruses[M]//Knipe DM,Howley P M.Fields Virology,5th.Lippincott:Williams and Wilkins,2007.
[18]VOLCHKOV V E,VOLCHKOVA V A,STROHERU,et al.Proteolytic processing of Marburg virus glycoprotein[J].Virology,2000,268(1):1-6.
[19]GEISBERT T W,JAHRLING P B.Differentiation of filoviruses by electron microscopy[J].Virus Res,1999,39(2/3):129-150.
[20]MAR IANKOVA R F,GLUSHAKOVA S E,PYZHIK E V,et al.The penetration of the Marburg virus into eukaryotic cells[J].Vopr Virusol,1993,38(2):74-76.
[21]SCHOMBERG K,MATSUYAMA S,KABSCH K,et al.Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein[J].J Virol,2006,80(8):4174-4178.
[22]HEVEY M,NEGLEY D,GEISBERT J,et al.Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants[J].Virology,1997,239(1):206-216.
[23]FELDMANN H,VOLCHKOV V E,VOLCHKOVAV A,et al.Biosynthesis and role of filoviral glycoproteins[J].Gen Virol,2001,82(12):2839-2848.
[24]MITTLER E1,KOLESNIKOVA L,HERWIG A,et al.Assembly of the Marburg virus envelope[J].Cell Microbiol,2013,15(2):270-284.
[25]ECKERT D M,KIM P S.Mechanisms of viral membrane fusion and its inhibition[J].Ann Rev Biochem,2001,70:777-810.
[26]MEDINA M F,KOBINGER G P,UX J,et al.Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung[J].Mol Ther,2003,8(5):777-789.
[27]KUHN J H,RAOSHITZKY S R,GUTH A C,et al.Conserved receptor-binding domains of Lake Victoria marburgvirus and Zaire ebolavi-rus bind a common receptor[J].Biol Chem,2006,281(23):15951-15958.
[28]MARZI A,GRAMBERG T,SIMMONS G,et al.DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus[J].J Virol,2004,78(21):12090-12095.
[29]胡旭乐,温志远,宋坤,等.表达马尔堡病毒囊膜糖蛋白GP重组新城疫病毒的构建及免疫原性评估[J].中国预防兽医学报,2014,36(9):667-671.
[30]邵钰,王金良,刘任强.等.埃博拉和马尔堡病毒囊膜糖蛋白嵌合型重组水泡性口炎病毒的构建及鉴定[J].中国预防兽医学报,2016,38(6):438-442.
[31]唐以杰,陈清池.利用大肠杆菌获取重组蛋白的研究进展[J].广东教育学院学报,2001,21(2):60-63.