H5N1亚型禽流感病毒HA1基因在大肠杆菌中的表达与纯化
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摘要
禽流感是目前影响世界养禽业的最严重的疫病。血凝素蛋白(HA)是禽流感病毒最重要的结构蛋白，占囊膜蛋白的90％，是诱导体液免疫的主要靶抗原，并且还能诱导细胞毒性T细胞作用，另外HA各部分的抗原性和免疫原性也有差别，研究表明，HA1的免疫原性以及抗原性强于HA2。
本研究利用RT-PCR技术扩增和克隆了A／Chicken／Guangdong／1／00(H5N1)亚型禽流感病毒的HA1基因，对HA1基因核苷酸序列和编码的氨基酸序列与基因库中已发表的序列A／Goose／Guangdong／1／96(H5N1)及A／Hongkang／156／97(H5N1)进行比较分析。结果表明同源性分别达到98％和97％，并且在HA1切割位点有多个碱性氨基酸的插入序列，证明其为强毒株。
将HA1基因克隆至原核表达载体pGEX-4T-2，经酶切鉴定及PCR鉴定，证明成功构建了融合表达载体pGEX-HA1。将构建好的融合表达载体，在1mmol／L的IPTG诱导下，在大肠杆菌BL21中得到大量表达，经过4h表达量既达到高峰。融合蛋白GST-HA1的分子量为62KD，以包涵体形式存在。Western-Blot免疫印记分析，融合蛋白与H5亚型AIV阳性血清发生特异性反应，表明该重组蛋白具有良好的抗原性。
用含有2mol／L和4mol／L尿素的包涵体洗涤液洗涤包涵体，在37℃条件下，洗去了大部分菌体蛋白及其它核酸物质。用8mol／L尿素作为变性剂溶解包涵体，包涵体在8mol／L尿素中的溶解性非常好。用梯度透析法对变性的融合蛋白GST-HA1进行复性，在低蛋白浓度的条件下，获得了很好的复性结果，复性前蛋白浓度为1.02mg／ml，复性后蛋白浓度为0.875mg／ml。分别用亲和层析以及电洗脱两种方法对GST-HA1融合蛋白进行了纯化，都获得了理想的纯化结果。
采用ELISA方法检测纯化后的融合蛋白的抗原性。结果表明，该融合蛋白能同H5亚型阳性血清发生特异性结合，并且同H7、H9亚型AIV阳性血清没有交叉反应。说明该蛋白具有很好的抗原性和特异性。
Avian influenza (AI) is a serious disease of poultry throughout the world. Hemagglutinin(HA) is the major surface glycoprotein of AIV, representing 90% of the envelope protein. It is the most important antigen in developing immunity against the virus. In addition, antigenicity and immunogenicity may very depend on the region of HA expressed, and HA1 is the most suitable expression region.
In this study, The HA1 gene of A/Chicken/Guangdong/2/97(H5Nl) AIV were cloned by reverse transcription polymerase chain reaction (RT-PCR) using primers designed and the sequences and amino acids were analyzed with molecular softpakage. The homogeneity of the HA1 of A/chicken/guangdong/2/97(H5N 1 )gene with the published gene of A/Goose/Guangdong/1/9 6(H5N1) and A/Hongkang/156/97(H5Nl) is 98% and 97%, and they all had an insert of basic amino acids at the cleavage site , meaning the H5N1 isolate virus is a highly pathogenic virus.
The HA 1 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing HA 1 gene were identified by restriction enzyme analysis and PCR mathod.
The recombinant fusion protein was highly expressed in E.coli BL21 induced by Immol/L IPTG in the form of inclusion bodies. The molecular weights of GST-H A1 is 62KD. Western-blot analysis with AIV antibodies against H5 subtype showed the recombinant protein shared good antigencity and specificity.
The inclusion bodies of recombinant protein were purified with washing buffer consisting of various urea (2mol/L and 4mol/L) for several times, and then dissolve the fusion protein in the denature buffer using 8mol/L urea as denaturant. After that a good renature result was obteined using gradient with lower protein concentration.
The purity of the recombinant protein by affinity-purification of Glutathione Sepharose 4B and electrodialysis method was also attained perfect result.
The antigenicity of the GST-HA1 protein were identified using ELISA mathod. The recombinant protein exhibited specific binding to antiviral antibodies of H5 subtype and has no cross-reaction with H7 and H9 subtype. The result show that the purified protein has excellent antigenicity and specificity.
The studies provided fundamental data and materials not only for the development of monoclonal antibody (MAb) , but also for the preparation and development of special diagnosis kits of H5 AIV subtypes.
本研究利用RT-PCR技术扩增和克隆了A／Chicken／Guangdong／1／00(H5N1)亚型禽流感病毒的HA1基因，对HA1基因核苷酸序列和编码的氨基酸序列与基因库中已发表的序列A／Goose／Guangdong／1／96(H5N1)及A／Hongkang／156／97(H5N1)进行比较分析。结果表明同源性分别达到98％和97％，并且在HA1切割位点有多个碱性氨基酸的插入序列，证明其为强毒株。
将HA1基因克隆至原核表达载体pGEX-4T-2，经酶切鉴定及PCR鉴定，证明成功构建了融合表达载体pGEX-HA1。将构建好的融合表达载体，在1mmol／L的IPTG诱导下，在大肠杆菌BL21中得到大量表达，经过4h表达量既达到高峰。融合蛋白GST-HA1的分子量为62KD，以包涵体形式存在。Western-Blot免疫印记分析，融合蛋白与H5亚型AIV阳性血清发生特异性反应，表明该重组蛋白具有良好的抗原性。
用含有2mol／L和4mol／L尿素的包涵体洗涤液洗涤包涵体，在37℃条件下，洗去了大部分菌体蛋白及其它核酸物质。用8mol／L尿素作为变性剂溶解包涵体，包涵体在8mol／L尿素中的溶解性非常好。用梯度透析法对变性的融合蛋白GST-HA1进行复性，在低蛋白浓度的条件下，获得了很好的复性结果，复性前蛋白浓度为1.02mg／ml，复性后蛋白浓度为0.875mg／ml。分别用亲和层析以及电洗脱两种方法对GST-HA1融合蛋白进行了纯化，都获得了理想的纯化结果。
采用ELISA方法检测纯化后的融合蛋白的抗原性。结果表明，该融合蛋白能同H5亚型阳性血清发生特异性结合，并且同H7、H9亚型AIV阳性血清没有交叉反应。说明该蛋白具有很好的抗原性和特异性。
Avian influenza (AI) is a serious disease of poultry throughout the world. Hemagglutinin(HA) is the major surface glycoprotein of AIV, representing 90% of the envelope protein. It is the most important antigen in developing immunity against the virus. In addition, antigenicity and immunogenicity may very depend on the region of HA expressed, and HA1 is the most suitable expression region.
In this study, The HA1 gene of A/Chicken/Guangdong/2/97(H5Nl) AIV were cloned by reverse transcription polymerase chain reaction (RT-PCR) using primers designed and the sequences and amino acids were analyzed with molecular softpakage. The homogeneity of the HA1 of A/chicken/guangdong/2/97(H5N 1 )gene with the published gene of A/Goose/Guangdong/1/9 6(H5N1) and A/Hongkang/156/97(H5Nl) is 98% and 97%, and they all had an insert of basic amino acids at the cleavage site , meaning the H5N1 isolate virus is a highly pathogenic virus.
The HA 1 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing HA 1 gene were identified by restriction enzyme analysis and PCR mathod.
The recombinant fusion protein was highly expressed in E.coli BL21 induced by Immol/L IPTG in the form of inclusion bodies. The molecular weights of GST-H A1 is 62KD. Western-blot analysis with AIV antibodies against H5 subtype showed the recombinant protein shared good antigencity and specificity.
The inclusion bodies of recombinant protein were purified with washing buffer consisting of various urea (2mol/L and 4mol/L) for several times, and then dissolve the fusion protein in the denature buffer using 8mol/L urea as denaturant. After that a good renature result was obteined using gradient with lower protein concentration.
The purity of the recombinant protein by affinity-purification of Glutathione Sepharose 4B and electrodialysis method was also attained perfect result.
The antigenicity of the GST-HA1 protein were identified using ELISA mathod. The recombinant protein exhibited specific binding to antiviral antibodies of H5 subtype and has no cross-reaction with H7 and H9 subtype. The result show that the purified protein has excellent antigenicity and specificity.
The studies provided fundamental data and materials not only for the development of monoclonal antibody (MAb) , but also for the preparation and development of special diagnosis kits of H5 AIV subtypes.
引文
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