禽流感病毒M2e基因多拷贝串联体的原核表达及免疫原性研究
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摘要
高致病性禽流感(HPAI)是由H5和H7亚型禽流感病毒(AⅣ)引起的禽类急性高致病性传染病,被国际兽疫局(OIE)列为A类疾病,在我国被列为一类传染病。防控禽流感最有效的措施是免疫接种,但由于禽流感病毒容易变异,存在众多的血清亚型,并且各亚型之间没有交叉保护力,当前使用的疫苗必须在每年流行之前更新换代。因此,研制一种可以抵抗多种亚型禽流感病毒的“通用型”疫苗是当务之急。禽流感病毒基质蛋白2的膜外部分(M2e)在各个亚型之间高度保守,能够引起交叉保护,是研究“通用型”禽流感疫苗理想的靶抗原。
将人工合成M2e基因(H9N2)与质粒pET-32a-M2e-Fc(H5N1)中的M2e-Fc基因串联至psIMPLE载体中,并利用同尾酶将串联体进行多拷贝的重复,最终成功构建含1、2、3、4、5、6拷贝M2e串联体的克隆载体,并将多拷贝的串联体亚克隆至表达载体pET-32a中,经过酶切鉴定和测序,证实其插入的片断大小、读码框架正确,结果获得6个表达载体,分别命名pET-(H9M2e-H5M2e)_1-FcpET-(H9M2e-H5M2e)_2-Fc、pET-(H9M2e-H5M2e)_3-Fc、pET-(H9M2e-H5M2e)_4-Fc、pET-(H9M2e-H5M2e)_5-Fc、pET-(H9M2e-H5M2e)_6-Fc。将6种表达载体转化BL21感受态细胞,IPTG诱导表达,经SDS-PAGE分析,结果为6个重组质粒表达蛋白相对分子量分别约为40.9、47.0、53.1、59.3、65.3和71.4 kDa。将2、4和6拷贝基因重组质粒表达蛋白通过镍亲和层析,结果得到纯化的融合蛋白(H9M2e-H5M2e)_2-Fc、(H9M2e-H5M2e)_4-Fc、(H9M2e-H5M2e)_6-Fc,分别简写4M2e、8M2e和12M2e,经Western-blotting鉴定融合蛋白具有良好的抗原性,为研究重组蛋白的免疫原性奠定了基础。
利用纯化的3种重组蛋白4M2e、8M2e和12M2e分别与弗氏佐剂、白油佐剂和CpG佐剂进行乳化制备疫苗,取21日龄AⅣ阴性鸡分组进行动物免疫实验。其中三种蛋白分别与弗氏佐剂乳化分别为A、B和C组,蛋白12M2e与白油佐剂、CpG佐剂分别为D、E组,12M2e的全菌蛋白为F组,阴性组为G组,每组10只,免疫方式为肌肉注射,免疫剂量为50ug/只鸡,免疫两次,首免3周后加强免疫。首免后每周采血,用间接ELISA方法检测M2e特异性抗体滴度;分别在首免3周、加强免疫后2周与4周检测外周血淋巴细胞的CD4~+与CD8~+T细胞所占的比例;选取高滴度抗体试验组的血清进行了鸡胚中和试验、细胞病变试验和间接免疫荧光试验。结果显示,各实验组均产生了较高滴度的M2e特异性抗体,12M2e蛋白与弗式佐剂乳化疫苗组产生的抗体滴度最高,M2e特异性抗体在一定程度上能够中和H9N2亚型禽流感病毒感染鸡胚,并且这种抗体能够与表达在感染禽流感病毒的MDCK细胞表面的M2e发生反应,抑制MDCK细胞上病毒的释放。
Highly pathogenic avian influenza(HPAI) was a devastating disease of poultry leading to high mortality caused by some H5 and H7 subtypes influenza A viruses. HAPI was classified as one of the list A animal disease by OIE and China Ministry of Agriculture.Seasonal antigenic variation of the major influenza surface glycoproteins,hemagglutinin(HA) and neuraminidase(NA),pose a major obstacle to control the viral disease by vaccination,because immunization with currently available influenza vaccines induces antibodies only to the virus strains included in the vaccine.However,as the virus changes by mutations(drift) and gene re-assortment(shift),these antibodies loose step by step their efficacy to prevent the disease.Therefore,influenza infections can occur repeatedly throughout life and protection by vaccination requires annual administration with updated vaccines.We have developed a universal influenza A vaccine based on the extracellular domain of the third integral membrane protein,called M2e.M2-protein is scarcely present on virus particles,but occurs abundantly on virus infected cells.M2e is highly conserved in all influenza A virus strains.Therefore,the M2e based vaccine is expected to protect against any new epidemic or pandemic strain.
The H9M2e and H5M2e were synthesized based on avain influenza m2e gene and E coli condon bias.The two gene was inserted into plasmid pSIMPLE and the multidermer copy M2e clone plasmid was constructed based on the fact isocaudamer enzyme existed.After that,the target gene was subcloned into an expression vector pET-32a(+).Then the recombinant expression plasmid was was digested by BglⅡ/XhoⅠand sequenced.The results showed that 6 expression plasmid included 1、2、3、4、5 and 6 copies of M2e tandem of avain influenza respectively were successfully constructed.The positive recombinant plasmid was named as pET-(H9M2e-H5M2e)_1-Fc、pET-(H9M2e-H5M2e)_2-Fc、pET-(H9M2e-H5M2e)_3-Fc、pET-(H9M2e-H5M2e)_4-Fc、pET-(H9M2e-H5M2e)_5-Fc、pET-(H9M2e-H5M2e)_6-Fc. Then the positive recombination plasmid was transformed into E.coli BL21(DE3), and induced with IPTG..The expression for the fusion proteins which named as 2M2e、4M2e、6M2e、8M2e、10M2e、12M2e were analyzed by SDS-PAGE.Results indicated that the relative molecular mass of the fusion proteins were measured to be 40.9、47.0、53.1、59.3、65.3 and 71.4 kDa.Three fusion proteins 4M2e、8M2e 12M2e were purified by Ni-sepharose affinity chromatography,which could be applied to learn its immunogenicity,a specific antigenicity of the three purified protein were demonstrated by Western blotting.
21-days-old unimmunized chicken were divided into 7 groups.The three fusion proteins 4M2e、8M2e and 12M2e with Freund' adjuvant were divied as group A、B、C,the protein 12M2e with vash oil and CpG adjuvant were divied as group D and E, the protein 12M2e unpurified with vash oil was devided as group F,and the negative group was devied as group G.The protein was injected intramuscularly at the dose of 150ug. Animals were boostered three weeks post the primary immunization.Blood samples were Collected every weeks after first immunization and M2e specific antibodies in the serum were detected with indirect ELISA established.The expression of CD3, CD4 and CD8 on the periphera blood lymphocytes surface was evaluated by monitoring fluorimetric changes of the corresponding FITC/PE conjugated monoclonal antibodyby flow cytometry on 3 weeks,5 weeks and 7 weeks after the fist immunization.Results showed that the fusion protein 12M2e with Freund's adjuvant could induce the highest level of antibodies.This serum was used in Viral neutralization test accomplished on SPF egg embryos and Madin-Darby canine kidney(MDCK) cell,at the same time,indirected fluorescence assay(IFA) was tested on MDCK infected by H9N2 viruses.M2 protein-specific antibodies were able to inhibit virus adsorption and penetration of virions,and were able to inhibit the replication of influenza virus.Fluorescence microscopy showed that the anti-M2e sera could bind the M2 expressed on the surface of the infected MDCK cells.
将人工合成M2e基因(H9N2)与质粒pET-32a-M2e-Fc(H5N1)中的M2e-Fc基因串联至psIMPLE载体中,并利用同尾酶将串联体进行多拷贝的重复,最终成功构建含1、2、3、4、5、6拷贝M2e串联体的克隆载体,并将多拷贝的串联体亚克隆至表达载体pET-32a中,经过酶切鉴定和测序,证实其插入的片断大小、读码框架正确,结果获得6个表达载体,分别命名pET-(H9M2e-H5M2e)_1-FcpET-(H9M2e-H5M2e)_2-Fc、pET-(H9M2e-H5M2e)_3-Fc、pET-(H9M2e-H5M2e)_4-Fc、pET-(H9M2e-H5M2e)_5-Fc、pET-(H9M2e-H5M2e)_6-Fc。将6种表达载体转化BL21感受态细胞,IPTG诱导表达,经SDS-PAGE分析,结果为6个重组质粒表达蛋白相对分子量分别约为40.9、47.0、53.1、59.3、65.3和71.4 kDa。将2、4和6拷贝基因重组质粒表达蛋白通过镍亲和层析,结果得到纯化的融合蛋白(H9M2e-H5M2e)_2-Fc、(H9M2e-H5M2e)_4-Fc、(H9M2e-H5M2e)_6-Fc,分别简写4M2e、8M2e和12M2e,经Western-blotting鉴定融合蛋白具有良好的抗原性,为研究重组蛋白的免疫原性奠定了基础。
利用纯化的3种重组蛋白4M2e、8M2e和12M2e分别与弗氏佐剂、白油佐剂和CpG佐剂进行乳化制备疫苗,取21日龄AⅣ阴性鸡分组进行动物免疫实验。其中三种蛋白分别与弗氏佐剂乳化分别为A、B和C组,蛋白12M2e与白油佐剂、CpG佐剂分别为D、E组,12M2e的全菌蛋白为F组,阴性组为G组,每组10只,免疫方式为肌肉注射,免疫剂量为50ug/只鸡,免疫两次,首免3周后加强免疫。首免后每周采血,用间接ELISA方法检测M2e特异性抗体滴度;分别在首免3周、加强免疫后2周与4周检测外周血淋巴细胞的CD4~+与CD8~+T细胞所占的比例;选取高滴度抗体试验组的血清进行了鸡胚中和试验、细胞病变试验和间接免疫荧光试验。结果显示,各实验组均产生了较高滴度的M2e特异性抗体,12M2e蛋白与弗式佐剂乳化疫苗组产生的抗体滴度最高,M2e特异性抗体在一定程度上能够中和H9N2亚型禽流感病毒感染鸡胚,并且这种抗体能够与表达在感染禽流感病毒的MDCK细胞表面的M2e发生反应,抑制MDCK细胞上病毒的释放。
Highly pathogenic avian influenza(HPAI) was a devastating disease of poultry leading to high mortality caused by some H5 and H7 subtypes influenza A viruses. HAPI was classified as one of the list A animal disease by OIE and China Ministry of Agriculture.Seasonal antigenic variation of the major influenza surface glycoproteins,hemagglutinin(HA) and neuraminidase(NA),pose a major obstacle to control the viral disease by vaccination,because immunization with currently available influenza vaccines induces antibodies only to the virus strains included in the vaccine.However,as the virus changes by mutations(drift) and gene re-assortment(shift),these antibodies loose step by step their efficacy to prevent the disease.Therefore,influenza infections can occur repeatedly throughout life and protection by vaccination requires annual administration with updated vaccines.We have developed a universal influenza A vaccine based on the extracellular domain of the third integral membrane protein,called M2e.M2-protein is scarcely present on virus particles,but occurs abundantly on virus infected cells.M2e is highly conserved in all influenza A virus strains.Therefore,the M2e based vaccine is expected to protect against any new epidemic or pandemic strain.
The H9M2e and H5M2e were synthesized based on avain influenza m2e gene and E coli condon bias.The two gene was inserted into plasmid pSIMPLE and the multidermer copy M2e clone plasmid was constructed based on the fact isocaudamer enzyme existed.After that,the target gene was subcloned into an expression vector pET-32a(+).Then the recombinant expression plasmid was was digested by BglⅡ/XhoⅠand sequenced.The results showed that 6 expression plasmid included 1、2、3、4、5 and 6 copies of M2e tandem of avain influenza respectively were successfully constructed.The positive recombinant plasmid was named as pET-(H9M2e-H5M2e)_1-Fc、pET-(H9M2e-H5M2e)_2-Fc、pET-(H9M2e-H5M2e)_3-Fc、pET-(H9M2e-H5M2e)_4-Fc、pET-(H9M2e-H5M2e)_5-Fc、pET-(H9M2e-H5M2e)_6-Fc. Then the positive recombination plasmid was transformed into E.coli BL21(DE3), and induced with IPTG..The expression for the fusion proteins which named as 2M2e、4M2e、6M2e、8M2e、10M2e、12M2e were analyzed by SDS-PAGE.Results indicated that the relative molecular mass of the fusion proteins were measured to be 40.9、47.0、53.1、59.3、65.3 and 71.4 kDa.Three fusion proteins 4M2e、8M2e 12M2e were purified by Ni-sepharose affinity chromatography,which could be applied to learn its immunogenicity,a specific antigenicity of the three purified protein were demonstrated by Western blotting.
21-days-old unimmunized chicken were divided into 7 groups.The three fusion proteins 4M2e、8M2e and 12M2e with Freund' adjuvant were divied as group A、B、C,the protein 12M2e with vash oil and CpG adjuvant were divied as group D and E, the protein 12M2e unpurified with vash oil was devided as group F,and the negative group was devied as group G.The protein was injected intramuscularly at the dose of 150ug. Animals were boostered three weeks post the primary immunization.Blood samples were Collected every weeks after first immunization and M2e specific antibodies in the serum were detected with indirect ELISA established.The expression of CD3, CD4 and CD8 on the periphera blood lymphocytes surface was evaluated by monitoring fluorimetric changes of the corresponding FITC/PE conjugated monoclonal antibodyby flow cytometry on 3 weeks,5 weeks and 7 weeks after the fist immunization.Results showed that the fusion protein 12M2e with Freund's adjuvant could induce the highest level of antibodies.This serum was used in Viral neutralization test accomplished on SPF egg embryos and Madin-Darby canine kidney(MDCK) cell,at the same time,indirected fluorescence assay(IFA) was tested on MDCK infected by H9N2 viruses.M2 protein-specific antibodies were able to inhibit virus adsorption and penetration of virions,and were able to inhibit the replication of influenza virus.Fluorescence microscopy showed that the anti-M2e sera could bind the M2 expressed on the surface of the infected MDCK cells.
引文
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