一种乙肝表面抗原结合蛋白（SBP）在Pichia Pastoris中的表达，纯化工艺及功能研究
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摘要
乙型肝炎(hepatitis B,HB)是世界范围内流行的一种严重危害人类健康的疾病,由乙型肝炎病毒(HBV hepatitis B virus)引起.我国是乙肝高流行区,大约有1.12亿人慢性感染HBV,占全世界感染者的30%以上.乙肝疫苗是预防感染乙型肝炎和降低乙肝病毒携带率的最有效措施.但是,5-10%的个体在按标准程序接种乙肝疫苗后不能产生足够的抗体以抵抗乙肝病毒的感染.
HBsAg-BP(Hepatitis B surface antigen binding protein,SBP)是本室以血清来源的乙肝表面抗原(HBsAg)为探针,通过印迹免疫技术从人肝cDNA噬菌体表达库中筛选获得,其特征是可以与乙肝表面抗原HBsAg特异性的相互作用.在本室前期的研究工作中,我们构建了原核表达载体pBV-SBP,用Ecoli大量表达重组SBP,并证实其具有增强乙肝疫苗免疫效果的作用.
本文将SBP表达框克隆到在毕赤酵母表达载体pPICZαA中,通过电击法转化毕赤酵母菌株GS115,获得能够分泌表达SBP的GS115转化菌株;对培养工艺进行了优化,发现SBP表达菌株在pH为6.0,诱导剂甲醇浓度为1.0-1.5%的BMMY中诱导96小时,达到最佳表达水平,可以加入终浓度为50μg/ml的Amp和15μg/ml的Kan以防止染菌及2mM的EDTA以抑制降解且不影响蛋白表达;在最优条件下诱导表达SBP并收集培养液,通过超滤和亲和层析的方法纯化获得了一定量的重组SBP蛋白,ELISA法证实重组SBP具有与HBsAg特异性结合的能力,并用多种方法计算了二者间亲和常数,在小鼠免疫实验中证实重组SBP具有乙肝疫苗免疫增效作用.
The current HBV vaccine performed certain protective role in preventing the infection of HBV. However, still about 5-10% people are under no protection because no response is detected after inoculation.
In our previous study, we have found a protein named HBsAg Binding Protein (SBP), which can specifically interact with HBsAg. Several experiments have proved its ability on enhancing the immuno-response of HBV vaccine using E.coli expressed SBP.
In the present study, SBP gene was cloned into the eukaryotic expression vector pPICZαA and transformed into Pichia pastoris strain GS115. The protein gene was placed downstream of the methanol inducible AOX1 promoter and theα-mating Factor signal sequence so the protein was expressed into the culture media. We studied on several factors which play important parts on SBP expression level, and found that when SBP expression was induced 72 hours under MeOH concentration of 1.0-1.5%, high expression level can be acquired. The using of 50μg/ml Amp and 15μg/ml Kan to prevent contamination and 2mM EDTA to prevent degradation had no effect on expression level. Subsequently we purified SBP through ultra-filtration and affinity chromatography.
After identification through Western Blot and mass spectrometry analyses, we demonstrated that SBP has the ability to bind specifically with HBsAg and calculated the affinity constant value using two different methods. The function of SBP in enhancing HBV vaccine performance was also testified in vivo.
HBsAg-BP(Hepatitis B surface antigen binding protein,SBP)是本室以血清来源的乙肝表面抗原(HBsAg)为探针,通过印迹免疫技术从人肝cDNA噬菌体表达库中筛选获得,其特征是可以与乙肝表面抗原HBsAg特异性的相互作用.在本室前期的研究工作中,我们构建了原核表达载体pBV-SBP,用Ecoli大量表达重组SBP,并证实其具有增强乙肝疫苗免疫效果的作用.
本文将SBP表达框克隆到在毕赤酵母表达载体pPICZαA中,通过电击法转化毕赤酵母菌株GS115,获得能够分泌表达SBP的GS115转化菌株;对培养工艺进行了优化,发现SBP表达菌株在pH为6.0,诱导剂甲醇浓度为1.0-1.5%的BMMY中诱导96小时,达到最佳表达水平,可以加入终浓度为50μg/ml的Amp和15μg/ml的Kan以防止染菌及2mM的EDTA以抑制降解且不影响蛋白表达;在最优条件下诱导表达SBP并收集培养液,通过超滤和亲和层析的方法纯化获得了一定量的重组SBP蛋白,ELISA法证实重组SBP具有与HBsAg特异性结合的能力,并用多种方法计算了二者间亲和常数,在小鼠免疫实验中证实重组SBP具有乙肝疫苗免疫增效作用.
The current HBV vaccine performed certain protective role in preventing the infection of HBV. However, still about 5-10% people are under no protection because no response is detected after inoculation.
In our previous study, we have found a protein named HBsAg Binding Protein (SBP), which can specifically interact with HBsAg. Several experiments have proved its ability on enhancing the immuno-response of HBV vaccine using E.coli expressed SBP.
In the present study, SBP gene was cloned into the eukaryotic expression vector pPICZαA and transformed into Pichia pastoris strain GS115. The protein gene was placed downstream of the methanol inducible AOX1 promoter and theα-mating Factor signal sequence so the protein was expressed into the culture media. We studied on several factors which play important parts on SBP expression level, and found that when SBP expression was induced 72 hours under MeOH concentration of 1.0-1.5%, high expression level can be acquired. The using of 50μg/ml Amp and 15μg/ml Kan to prevent contamination and 2mM EDTA to prevent degradation had no effect on expression level. Subsequently we purified SBP through ultra-filtration and affinity chromatography.
After identification through Western Blot and mass spectrometry analyses, we demonstrated that SBP has the ability to bind specifically with HBsAg and calculated the affinity constant value using two different methods. The function of SBP in enhancing HBV vaccine performance was also testified in vivo.
引文
[1]Guy B.The perfect mix:recent progress in adjuvant research[J].Nature Rev.Microbiol.,2007,5(7):505-517.
[2]Aguilar J.C.,Rodriguez E.G.Vaccine adjuvants revisited[J].Vaccine,2007,25(19):3752-3762.
[3]Gaston J.S.Heat shock proteins and innate immunity[J].Clin.Exp.Immunol.,2002,127(1):72-77.
[4]傅安静,岳华,刘树玲等.免疫佐剂的研究进展[J].西南民族大学学报自然科学版,2005,73-77.
[5]Mata-Haro V.,Cekic C.,Martin M.,et al.The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4[J].Science,2007,316(5831):1628-1632.
[6]章岩,边传周,郑纯宁.新型免疫佐剂研究进展[J].河南农业科学,2005,7,103-104.
[7]Suzue K.,Zhou X.,Eisen H.N.,et al.Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class Ⅰ presentation pathway[J].Proc.Natl.Acad.Sci.U.S.A.,1997,94(24):13146-13151.
[8]Wu Y.,Wan T.,Zhou X.,et al.Hsp72-1ike protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8+ CTL response by Dendritic cell vaccine[J].Cancer Res.,2005,65(11):4947-4954.
[9]Li D.,Wang H.,Li L.,et al.Gene fusion of molecular adjuvant C3d to hCGb enhances the anti-hCGb antibody response in DNA immunization[J].J.Reprod.Immunol.,2003,60(2):129-141.
[10]Dempsey P.W.,Allison M.E.,Akkaraju S.,et al.C3d of complement as a molecular adjuvant:bridging innate and acquired immunity[J].Science,1996,271(5247):348-350.
[11]Green T.D.,Newton B.R.,Rota P.A.,et al.C3d enhancement of neutralizing antibodies to measles hemagglutinin[J].Vaccine,2002,20(1-2):242-248.
[12]Ross T.M.,Xu Y.,Bright R.A.,et al.C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge[J].Nat.Immunol.,2000,1(2):127-131.
[13]Haas K.M.,Toapanta F.R.,Oliver J.A.,et al.Cutting edge:C3d functions as a molecular adjuvant in the absence of CD21/35 expression[J].J.Immunol.,2004,172(10):5833-5837.
[14]Valenzuela P.,Medina A.,Rutter W.J.,et al.Synthesis and assembly of hepatitis B virus surface antigen particles in yeast[J].Nature,1982,298(5872):347-350.
[15]胡兴斌,尹文,徐志凯.增强乙型肝炎疫苗免疫效果的研究进展[J].生命科学研究,2004,8(4Suppl.):127-130.
[16]Townsend K.,Saillberg M.,O' Del J.,et al.Characterization of CD8 cytotoxic T-lymphocyte responses after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B virus core and e antigens[J].J.Virol.,1997,71(5):3365-3374.
[17]Shi T.D.,Wu Y.Z.,Jia Z.C.,et al.Therapeutic polypeptides based on HBcAg 18-27 CTL epitope can induce antigen-specific CD8+ CTL-mediated cytotoxicity in HLA-A2 transgenic mice[J].World J.Gastroenterol.,2004,10(8):1222-1226.
[18]何萍,吕凤林,任建敏等.铝佐剂的机制有其纳米化前景[J].世界化人消化杂志,2003,11(11):1764-1768.
[19]王四清,田淑芳,许洪林等.CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响[J1.病毒学报,2002,18(2):108-112.
[20]刘建勋,石浩,杨建国等.应用白介素-2诱导乙肝疫苗免疫无应答者特异性免疫反应的研究[J].河南预防医学杂志,1999,10(3):138-139,142.
[21]Chow Y.H.,Huang W.L.,Chi W.K.,et al.Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2[J].J.Virol.,1997,71(1):169-178.
[22]Arrington J.,Braun R.P.,Dong L.C.,et al.Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin form Escherichia coli are strong adjuvants for DNA vaccines[J].J.Virol.,2002,76(5):4536-4546.
[23]冯立,胡显文,陈惠鹏.治疗性乙型肝炎疫苗研究进展[J].生物技术通讯,2004,15(3):289-291.
[24]刘爱顺,徐道振,张剑平等.治疗性乙型肝炎疫苗的免疫效应[J].中华肝脏病杂志,2006,14(2):89-92.
[25]秦刚,施光峰.治疗性乙型肝炎疫苗研究进展[J].中国抗感染化疗杂志,2005,5(2):124-127.
[26]陈媛媛,朱乃硕.一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究[J].中国生物化学与分子生物学报,2005,21(1):53-60.
[27]Cereghino J.,Cregg J.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Microbiol.Rev.,2000,24:45-66.
[28]Cregg J.,Vedvick T.,Raschke W.Recent advances in the expression of foreign genes in pichia pastoris.Biotechnica,1993,11:905-910.
[29]李晶,赵晓祥,沙长青等.甲醇酵母基因表达系统的研究进展[J].生物工程进展,1999,19(2):17-20.
[30]欧阳立明,张惠展,张嗣同.巴斯德毕赤酵母的基因表达系统研究进展.生物化学与生物物理进展,2000,27(2):151-154
[31]Macauley-Patrick S.,Fazenda M.L.,McNeil B.,et al.Hetertologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-270.
[32]陈媛媛,朱乃硕.一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究[J].中国生物化学与分子生物学报,2005,21(1):53-60.
[33]Beatty J.D.,Baetty B.G.,Vlahos W.G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].J Immunol.Methods,1987,100(122):173
[34]陈莹,袁汉英,李海等.毕赤酵母YPS1基因的失活对减少重组人血清白蛋白降解的作用[J].复旦学报(自然科学版),2008,47(3):311-317.
[35]Macauley-Patrick S.,Fazenda M.L.,McNeil B.,et al.Heterologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-270.
[36]Dou W.F.,Lei J.Y.,Zhang L.F.,et al.Expression,purification,and characterization of recombinant human serum albumin fusion protein with two human glucagons-like peptide-1 mutants in Pichia pastoris[J].Protein Expr.Purif.,2008,61(1):45-49.
[37]Anup K.U.,Da le E.E.Characterization of detergent purified recombinant rat liver monoarnine oxidase B expressed in Pichia pastoris[J].Protein Expr.Purif.,2008,59(2):349-356
[38]Cereghino J.L.,Cregg J.M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Microbiol.Rev.,2000,24(1):45-66.
[39]Vervecken W.,Kaigorodov V.,Callewaert N.,et al.In vivo synthesis of mammalian-like,hybrid-type N-glycans in Pichia pastoris[J].Appl.Environ.Microbiol.,2004,70(5):2639-2646.
[40]Chiba Y.,Suzuki M.,Yoshida S.,et al.Production of human compatible high mannose-type sugar chains in Saccharomyces cerevisiae[J].J.Biol.Chem.,1998,273(41):26298-26304.
[2]Aguilar J.C.,Rodriguez E.G.Vaccine adjuvants revisited[J].Vaccine,2007,25(19):3752-3762.
[3]Gaston J.S.Heat shock proteins and innate immunity[J].Clin.Exp.Immunol.,2002,127(1):72-77.
[4]傅安静,岳华,刘树玲等.免疫佐剂的研究进展[J].西南民族大学学报自然科学版,2005,73-77.
[5]Mata-Haro V.,Cekic C.,Martin M.,et al.The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4[J].Science,2007,316(5831):1628-1632.
[6]章岩,边传周,郑纯宁.新型免疫佐剂研究进展[J].河南农业科学,2005,7,103-104.
[7]Suzue K.,Zhou X.,Eisen H.N.,et al.Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class Ⅰ presentation pathway[J].Proc.Natl.Acad.Sci.U.S.A.,1997,94(24):13146-13151.
[8]Wu Y.,Wan T.,Zhou X.,et al.Hsp72-1ike protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8+ CTL response by Dendritic cell vaccine[J].Cancer Res.,2005,65(11):4947-4954.
[9]Li D.,Wang H.,Li L.,et al.Gene fusion of molecular adjuvant C3d to hCGb enhances the anti-hCGb antibody response in DNA immunization[J].J.Reprod.Immunol.,2003,60(2):129-141.
[10]Dempsey P.W.,Allison M.E.,Akkaraju S.,et al.C3d of complement as a molecular adjuvant:bridging innate and acquired immunity[J].Science,1996,271(5247):348-350.
[11]Green T.D.,Newton B.R.,Rota P.A.,et al.C3d enhancement of neutralizing antibodies to measles hemagglutinin[J].Vaccine,2002,20(1-2):242-248.
[12]Ross T.M.,Xu Y.,Bright R.A.,et al.C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge[J].Nat.Immunol.,2000,1(2):127-131.
[13]Haas K.M.,Toapanta F.R.,Oliver J.A.,et al.Cutting edge:C3d functions as a molecular adjuvant in the absence of CD21/35 expression[J].J.Immunol.,2004,172(10):5833-5837.
[14]Valenzuela P.,Medina A.,Rutter W.J.,et al.Synthesis and assembly of hepatitis B virus surface antigen particles in yeast[J].Nature,1982,298(5872):347-350.
[15]胡兴斌,尹文,徐志凯.增强乙型肝炎疫苗免疫效果的研究进展[J].生命科学研究,2004,8(4Suppl.):127-130.
[16]Townsend K.,Saillberg M.,O' Del J.,et al.Characterization of CD8 cytotoxic T-lymphocyte responses after genetic immunization with retrovirus vectors expressing different forms of the hepatitis B virus core and e antigens[J].J.Virol.,1997,71(5):3365-3374.
[17]Shi T.D.,Wu Y.Z.,Jia Z.C.,et al.Therapeutic polypeptides based on HBcAg 18-27 CTL epitope can induce antigen-specific CD8+ CTL-mediated cytotoxicity in HLA-A2 transgenic mice[J].World J.Gastroenterol.,2004,10(8):1222-1226.
[18]何萍,吕凤林,任建敏等.铝佐剂的机制有其纳米化前景[J].世界化人消化杂志,2003,11(11):1764-1768.
[19]王四清,田淑芳,许洪林等.CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响[J1.病毒学报,2002,18(2):108-112.
[20]刘建勋,石浩,杨建国等.应用白介素-2诱导乙肝疫苗免疫无应答者特异性免疫反应的研究[J].河南预防医学杂志,1999,10(3):138-139,142.
[21]Chow Y.H.,Huang W.L.,Chi W.K.,et al.Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2[J].J.Virol.,1997,71(1):169-178.
[22]Arrington J.,Braun R.P.,Dong L.C.,et al.Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin form Escherichia coli are strong adjuvants for DNA vaccines[J].J.Virol.,2002,76(5):4536-4546.
[23]冯立,胡显文,陈惠鹏.治疗性乙型肝炎疫苗研究进展[J].生物技术通讯,2004,15(3):289-291.
[24]刘爱顺,徐道振,张剑平等.治疗性乙型肝炎疫苗的免疫效应[J].中华肝脏病杂志,2006,14(2):89-92.
[25]秦刚,施光峰.治疗性乙型肝炎疫苗研究进展[J].中国抗感染化疗杂志,2005,5(2):124-127.
[26]陈媛媛,朱乃硕.一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究[J].中国生物化学与分子生物学报,2005,21(1):53-60.
[27]Cereghino J.,Cregg J.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Microbiol.Rev.,2000,24:45-66.
[28]Cregg J.,Vedvick T.,Raschke W.Recent advances in the expression of foreign genes in pichia pastoris.Biotechnica,1993,11:905-910.
[29]李晶,赵晓祥,沙长青等.甲醇酵母基因表达系统的研究进展[J].生物工程进展,1999,19(2):17-20.
[30]欧阳立明,张惠展,张嗣同.巴斯德毕赤酵母的基因表达系统研究进展.生物化学与生物物理进展,2000,27(2):151-154
[31]Macauley-Patrick S.,Fazenda M.L.,McNeil B.,et al.Hetertologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-270.
[32]陈媛媛,朱乃硕.一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究[J].中国生物化学与分子生物学报,2005,21(1):53-60.
[33]Beatty J.D.,Baetty B.G.,Vlahos W.G.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].J Immunol.Methods,1987,100(122):173
[34]陈莹,袁汉英,李海等.毕赤酵母YPS1基因的失活对减少重组人血清白蛋白降解的作用[J].复旦学报(自然科学版),2008,47(3):311-317.
[35]Macauley-Patrick S.,Fazenda M.L.,McNeil B.,et al.Heterologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-270.
[36]Dou W.F.,Lei J.Y.,Zhang L.F.,et al.Expression,purification,and characterization of recombinant human serum albumin fusion protein with two human glucagons-like peptide-1 mutants in Pichia pastoris[J].Protein Expr.Purif.,2008,61(1):45-49.
[37]Anup K.U.,Da le E.E.Characterization of detergent purified recombinant rat liver monoarnine oxidase B expressed in Pichia pastoris[J].Protein Expr.Purif.,2008,59(2):349-356
[38]Cereghino J.L.,Cregg J.M.Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J].FEMS Microbiol.Rev.,2000,24(1):45-66.
[39]Vervecken W.,Kaigorodov V.,Callewaert N.,et al.In vivo synthesis of mammalian-like,hybrid-type N-glycans in Pichia pastoris[J].Appl.Environ.Microbiol.,2004,70(5):2639-2646.
[40]Chiba Y.,Suzuki M.,Yoshida S.,et al.Production of human compatible high mannose-type sugar chains in Saccharomyces cerevisiae[J].J.Biol.Chem.,1998,273(41):26298-26304.