H5N1禽流感病毒血凝素蛋白的表位预测及重组表达
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摘要
本实验室刘梅(2007)将禽流感病毒(AIV)血凝素HA1在苏云金芽胞杆菌表面展示,免疫雏鸡后产生针对AIV H5亚型的特异性抗体,但通过加大免疫剂量和增加免疫次数等措施,特异性抗体效价仅能升至3.8 log2,难以达到实际使用的免疫效果。其原因可能是表达量过少或者表面展示的抗原位点及构型未达到最佳水平。
本研究利用生物学软件对H5N1禽流感病毒的HA1蛋白表位进行预测,并通过常规克隆表达方法将不同构型的HA1表位展示在苏云金芽胞杆菌的S-层蛋白中,从而为研制热稳定高效的口服基因工程禽流感疫苗奠定基础。
1.H5N1禽流感病毒HA1蛋白表位的预测
应用SignalP3.0以及DNAStar等生物学软件对HA1蛋白进行二级结构预测,通过α-螺旋中心、β-折叠区、转角、无规则卷曲、柔韧性、亲水性、表面可能性和抗原性指数的综合分析,确定六个目标片段A、B、C、D、E和F。
2.H5N1禽流感病毒HA1蛋白表位的克隆
应用primer 5.0软件设计六对引物,以质粒pKG-HA1和pKG-HA1M为模板,进行常规PCR方法扩增,将获得的目标片段克隆到pGM-T载体,得到pGM-A、pGM-B、pGM-C、pGM-D、pGM-E和pGM-F的阳性克隆子。测定结果表明,PCR扩增产物序列与原序列相符,接头处的酶切位点也与设计的相符。
利用XbaⅠ+HincⅡ位点将扩增片段连接到质粒pBMB982-304中ctc基因相应的位点处,从而得到一系列含S-层融合蛋白基因的重组质粒pCTC-A、pCTC-B、pCTC-C、pCTC-D、pCTC-E和pCTC-F。利用EcoRⅠ位点将操纵元csaAB插入pCTC-A、pCTC-B和pCTC-E中,得到新的重组质粒pCSHA1A、pCSHA1B和pCSHA1E。
3.构建含有HA1表位基因的S-层融合蛋白的重组菌株
将重组质粒电脉冲转入苏云金芽胞杆菌BMB171中,分别得到BCA(含pCSHA1A)、BCB(含pCSHA1B)、BCE(含pCSHA1E)、BCCA(含pCTC-A和pMIL-CSA)、BCCB(含pCTC-B和pMIL-CSA)、BCCC(含pCTC-C和pMIL-CSA)、BCCD(含pCTC-D和pMIL-CSA)、BCCE(含pCTC-E和pMIL-CSA)和BCCF(含IoCTC-F和pMIL-CSA)。
4.检测外源蛋白在重组菌株细胞表面的表达情况
用血凝和血凝抑制试验检测重组菌株的蛋白表面展示情况。结果,部分重组菌株均表现出了预计的生物学活性,说明所构建的S-层融合蛋白基因在受体菌株中得到表达并展示到了细胞表面且具有活性。
S-layer protein CTC surface display system of Bacillus thuringiensis was used to display avian influenza virus hemagglutinin HA1 protein on the cell surface of Bacillus thuringiensis by Liu Mei(2007) in our laboratory,and the specific antibodies against AIV H5N1 subtype were producted after chickens were immunized with recombinant strains.But by increasing the immunization dose and frequency,the specific antibody titer was only up to 3.8log2,which was difficult to achieve the immune effect in practical application.The reason could be that the expression volume was too little or the configuration of the antigenic sites expressed did not meet the optimal levels.
In this study,epitopes of the HA1 protein of the H5N1 avian influenza virus were predicted by biology software.Then the epitopes of different configurations were cloned and expressed in the Bacillus thuringiensis S-layer protein.This study was the foundation for the development of the heat-stable and highly efficient genetic engineering vaccine about avian influenza.
1.The prediction of the HA1 protein epitopes of H5N1 avian influenza virus
The secondary structure of the HA1 protein of the H5N1 avian influenza virus was carried out by centralα-helix,β-folded area,corner,curl-free rules,flexibility, hydrophilicity,surface probability and antigenic index through the use of SignalP3.0 sever,DNAStar software,and so on.The six objective fragments called A,B,C,D,E and F were achieved after comprehensive analysis.
2.Cloning of the HA1 protein epitopes of H5N1 avian influenza virus
Primer 5.0 software was used to design six pairs of primers,and pKG-HA1 and pKG-HA1M were used as templates of PCR in order to get the target fragments.Then, the target fragments were cloned into pGM-T vector to get the positive clones of pGM-A, pGM-B,pGM-C,pGM-D,pGM-E and pGM-F.The results showed that,the sequences of PCR products were in line with the original sequence,and the restriction sites were in line with the designed ones.
The target fragments were connected to the plasmid pBMB982-304 relatively by use of the restriction sites of XbaⅠand HincⅡ,which lead to the recombinant plasmids pCTC-A,pCTC-B,pCTC-C,pCTC-D,pCTC-E and pCTC-F.Using EcoRⅠsite,csaAB operon was inserted in pCTC-A,pCTC-B and pCTC-E to get new recombinant plasmids pCSHA1A,pCSHA1B and pCSHA1E.
3.Construction of recombinant strains which harboring target genes and S-layer fusion genes
Nine recombinant B.thuringiensis strains were constructed by transferring recombinant plasmids to receptor BMB171.The resulting strains included BCA (harboring pCSHA1A),BCB(harboring pCSHA1B),BCE(harboring pCSHA1E), BCCA(harboring pCTC-A and pMIL-CSA).BCCB(harboring pCTC-B and pMIL-CSA),BCCC(harboring pCTC-C and pMIL-CSA),BCCD(harboring pCTC-D and pMIL-CSA),BCCE(harboring pCTC-E and pMIL-CSA) and BCCF(harboring pCTC-F and pMIL-CSA).
4.Assaying the display of exogenous protein in recombinant strains
Hemagglutination assay showed that some recombinant proteins were displayed on the cell surface of respective recombinant strains.Hemagglutination inhibition assay showed recombinant proteins displayed on the cell surface of recombinant strains respectively were specific to standard positive serum of avian influenza virus H5N1.
本研究利用生物学软件对H5N1禽流感病毒的HA1蛋白表位进行预测,并通过常规克隆表达方法将不同构型的HA1表位展示在苏云金芽胞杆菌的S-层蛋白中,从而为研制热稳定高效的口服基因工程禽流感疫苗奠定基础。
1.H5N1禽流感病毒HA1蛋白表位的预测
应用SignalP3.0以及DNAStar等生物学软件对HA1蛋白进行二级结构预测,通过α-螺旋中心、β-折叠区、转角、无规则卷曲、柔韧性、亲水性、表面可能性和抗原性指数的综合分析,确定六个目标片段A、B、C、D、E和F。
2.H5N1禽流感病毒HA1蛋白表位的克隆
应用primer 5.0软件设计六对引物,以质粒pKG-HA1和pKG-HA1M为模板,进行常规PCR方法扩增,将获得的目标片段克隆到pGM-T载体,得到pGM-A、pGM-B、pGM-C、pGM-D、pGM-E和pGM-F的阳性克隆子。测定结果表明,PCR扩增产物序列与原序列相符,接头处的酶切位点也与设计的相符。
利用XbaⅠ+HincⅡ位点将扩增片段连接到质粒pBMB982-304中ctc基因相应的位点处,从而得到一系列含S-层融合蛋白基因的重组质粒pCTC-A、pCTC-B、pCTC-C、pCTC-D、pCTC-E和pCTC-F。利用EcoRⅠ位点将操纵元csaAB插入pCTC-A、pCTC-B和pCTC-E中,得到新的重组质粒pCSHA1A、pCSHA1B和pCSHA1E。
3.构建含有HA1表位基因的S-层融合蛋白的重组菌株
将重组质粒电脉冲转入苏云金芽胞杆菌BMB171中,分别得到BCA(含pCSHA1A)、BCB(含pCSHA1B)、BCE(含pCSHA1E)、BCCA(含pCTC-A和pMIL-CSA)、BCCB(含pCTC-B和pMIL-CSA)、BCCC(含pCTC-C和pMIL-CSA)、BCCD(含pCTC-D和pMIL-CSA)、BCCE(含pCTC-E和pMIL-CSA)和BCCF(含IoCTC-F和pMIL-CSA)。
4.检测外源蛋白在重组菌株细胞表面的表达情况
用血凝和血凝抑制试验检测重组菌株的蛋白表面展示情况。结果,部分重组菌株均表现出了预计的生物学活性,说明所构建的S-层融合蛋白基因在受体菌株中得到表达并展示到了细胞表面且具有活性。
S-layer protein CTC surface display system of Bacillus thuringiensis was used to display avian influenza virus hemagglutinin HA1 protein on the cell surface of Bacillus thuringiensis by Liu Mei(2007) in our laboratory,and the specific antibodies against AIV H5N1 subtype were producted after chickens were immunized with recombinant strains.But by increasing the immunization dose and frequency,the specific antibody titer was only up to 3.8log2,which was difficult to achieve the immune effect in practical application.The reason could be that the expression volume was too little or the configuration of the antigenic sites expressed did not meet the optimal levels.
In this study,epitopes of the HA1 protein of the H5N1 avian influenza virus were predicted by biology software.Then the epitopes of different configurations were cloned and expressed in the Bacillus thuringiensis S-layer protein.This study was the foundation for the development of the heat-stable and highly efficient genetic engineering vaccine about avian influenza.
1.The prediction of the HA1 protein epitopes of H5N1 avian influenza virus
The secondary structure of the HA1 protein of the H5N1 avian influenza virus was carried out by centralα-helix,β-folded area,corner,curl-free rules,flexibility, hydrophilicity,surface probability and antigenic index through the use of SignalP3.0 sever,DNAStar software,and so on.The six objective fragments called A,B,C,D,E and F were achieved after comprehensive analysis.
2.Cloning of the HA1 protein epitopes of H5N1 avian influenza virus
Primer 5.0 software was used to design six pairs of primers,and pKG-HA1 and pKG-HA1M were used as templates of PCR in order to get the target fragments.Then, the target fragments were cloned into pGM-T vector to get the positive clones of pGM-A, pGM-B,pGM-C,pGM-D,pGM-E and pGM-F.The results showed that,the sequences of PCR products were in line with the original sequence,and the restriction sites were in line with the designed ones.
The target fragments were connected to the plasmid pBMB982-304 relatively by use of the restriction sites of XbaⅠand HincⅡ,which lead to the recombinant plasmids pCTC-A,pCTC-B,pCTC-C,pCTC-D,pCTC-E and pCTC-F.Using EcoRⅠsite,csaAB operon was inserted in pCTC-A,pCTC-B and pCTC-E to get new recombinant plasmids pCSHA1A,pCSHA1B and pCSHA1E.
3.Construction of recombinant strains which harboring target genes and S-layer fusion genes
Nine recombinant B.thuringiensis strains were constructed by transferring recombinant plasmids to receptor BMB171.The resulting strains included BCA (harboring pCSHA1A),BCB(harboring pCSHA1B),BCE(harboring pCSHA1E), BCCA(harboring pCTC-A and pMIL-CSA).BCCB(harboring pCTC-B and pMIL-CSA),BCCC(harboring pCTC-C and pMIL-CSA),BCCD(harboring pCTC-D and pMIL-CSA),BCCE(harboring pCTC-E and pMIL-CSA) and BCCF(harboring pCTC-F and pMIL-CSA).
4.Assaying the display of exogenous protein in recombinant strains
Hemagglutination assay showed that some recombinant proteins were displayed on the cell surface of respective recombinant strains.Hemagglutination inhibition assay showed recombinant proteins displayed on the cell surface of recombinant strains respectively were specific to standard positive serum of avian influenza virus H5N1.
引文
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56.Mesnage S,Fontaine T,Mignot T,Delepierre M,Mock M,Fouet A.Bacterial SLH domain proteins are non-covalently anchored to the surface via a conserved mechanism involving wall polysaccharide pyruvylation.EMBO J,2000,17:4473-4484
57.Mesnage S,Haustant M,Fouet A.A general strategy for identification of S-layer genes in the Bacillus cereus group;molecular characterization of such a gene in Bacillus thuringiensis subsp,galleriae NRRL 4045.Microbiology,2001,147:1343-1351
58.Mignot T,Denis B,Couture-Tosi E,Kolsto A-B,Mock M,Fouet A.Distribution of S-layers on the surface of Bacillus cereus strains;phylogenetic origin and ecological pressure.Environ Microbiol,2001,3:493-501
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