禽流感病毒H5N1亚型基因工程疫苗的设计、表达制备及动物实验研究
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摘要
禽流感是一种禽类的烈性传染病，在世界范围内广泛流传，严重威胁着养禽业。尤其是高致病性禽流感的流行，病死率极高。禽流感的传染性极强，并可跨物种进行传播，近年发现，人类也是禽流感病毒宿主之一，自2003年底至2008年6月19日，亚、非、欧三大洲15个国家共报告了383例经实验室检查确诊的人H5N1禽流感病例，241例死亡。禽流感作为一种全新的病种，已成为人类健康和生命的严重威胁，而面对禽流感病毒的侵害，人类自身却毫无免疫防护。2004年初，世界卫生组织已就禽流感的流行向全世界提出警示，许多国家和地区更将禽流感列为头号监控对象。引起禽流感的病原体是禽流感病毒，该病毒具有非常高的变异性，理论上来讲，禽流感病毒的血清亚型可多达256个，并可与其他流感病毒进行重组，进而发生病毒变异和宿主的改变，因此，禽流感病毒复杂多变的基因组常常被许多学者看做是人类流感病毒的最大基因库。
由于禽流感病毒广泛存在于各种禽类和野生鸟类体内，因此禽流感的控制是一项长期艰巨的任务，疫苗的开发和使用是应对这项艰巨任务最实际、最有效和最经济的手段。理想的疫苗应该是在具备良好的安全性的前提下，不仅能够调动机体的体液免疫，产生病毒中和抗体，更能够充分调动机体的细胞免疫来充分发挥病毒清除机制。基于以上理念，本研究以亚洲地区主要流行的H5N1亚型禽流感病毒为研究对象，比对了2003年以来中国及周边国家，尤其是东南亚国家所发表的H5N1亚型禽流感病毒毒株的HA1和NP蛋白序列，筛选出了同源性较高的HA1和NP蛋白序列作为标准参考序列。通过计算机软件对禽流感病毒H5N1亚型的HA1蛋白进行了全面分析，优选了出了主要T细胞表位和B细胞表位，并对NP蛋白进行了全面分析，优选出了主要CTL表位。依据这些优选表位，我们设计了禽流感病毒H5N1亚型基因工程疫苗，该疫苗以含有主要保护性B细胞表位和覆盖多种DR类型的MHC II表位的H5N1病毒HA1蛋白做为主体骨架，并在该骨架C端引入了优选出的CTL细胞表位。在对该疫苗基因进行密码子和碱基组成优化后，采用人工合成的方法获得了编码该禽流感病毒H5N1亚型基因工程疫苗的基因，并将该基因克隆入大肠杆菌表达载体pRSET B中，在大肠杆菌中得到了良好表达，禽流感病毒H5N1亚型基因工程疫苗蛋白表达量占菌体总蛋白30%，表达形式为包涵体。
疫苗的使用是防治禽流感的最有效手段，同时也对疫苗生产过程中的产量和成本控制提出了很高的要求。本研究对禽流感病毒H5N1亚型基因工程疫苗工程菌的生长和表达的最理想pH值范围、碳源浓度、培养基配方以及诱导时长等条件进行了探讨并优化，建立了该工程菌的中试发酵工艺，并进行了高密度发酵培养，在保证禽流感病毒H5N1亚型基因工程疫苗蛋白30%表达量的前提下，使工程菌密度达到54OD600，禽流感病毒H5N1亚型基因工程疫苗理论产量约为3.4g/L。在此基础上，我们又进一步探索出了一条简洁高效的疫苗蛋白纯化和复性工艺，经过简单的包涵体洗涤和Ni+亲和柱层析即可使禽流感病毒H5N1亚型基因工程疫苗蛋白纯度达到95.5%，复性后疫苗蛋白原液浓度可达2.4mg/mL。
为评价该禽流感病毒H5N1亚型基因工程疫苗的免疫效力，本研究首先将该疫苗作为免疫原进行了小白鼠实验，验证了其在小白鼠体内的免疫应答。然后分别用高致病性禽流感病毒(HPAIV)A/Goose/GuangDong/1/96(H5N1)[Gs/GD/1／96(H5N1)毒株和低致病力禽流感病毒（LPAIV) A／Chicken／Huadong／87／20O6(Ck／HD／87／06（H9N2)对免疫后SPF鸡进行攻毒，确定该疫苗对两种不同禽流感病毒亚型的保护效果。两个靶动物试验均用低、中、高三个剂量组对SPF鸡进行免疫，并分别用两种毒株攻毒，其中H5N1亚型的保护试验通过临床反应症状、检测HI抗体水平、ELISA法检测抗体，以及建立RT-PCR方法检测攻毒后各组病毒载量，来说明禽流感H5N1亚型基因工程疫苗的免疫效力；H9N2亚型的保护试验通过检测免疫后HI抗体水平和攻毒后排毒情况来说明其免疫效力。结果表明，禽流感H5N1亚型基因工程疫苗能在小白鼠体内产生良好的免疫应答；不同剂量的免疫组对不同禽流感病毒亚型感染后SPF鸡均达到了不同程度的保护，根据两个病毒亚型试验的比较，说明该疫苗对禽流感病毒H5N1亚型的免疫效力略强于H9N2亚型。其中10μg/只剂量组免疫保护率达到了100%，因此可以说明，该禽流感H5N1亚型基因工程疫苗可以考虑作为防御禽流感疫病的疫苗。
为评价该禽流感病毒H5N1亚型基因工程疫苗的抗原特异性和免疫效力，本研究分四个部分去验证。一是建立间接ELISA方法确定该疫苗抗原与禽流感H5（Re‐4株、Re‐5株）标准阳性血清、H9标准阳性血清、H7标准阳性血清的特异性反应，验证该疫苗的针对禽流感病毒型特异性；二是通过小白鼠实验，检测其抗体水平和细胞因子水平的变化，通过小白鼠实验模型，初步说明该疫苗抗原性；三是选择法国SEPPIC公司推荐的三种商业化佐剂，将该疫苗分别进行乳化，免疫SPF鸡，检测其抗体水平，根据不同时间内其抗体水平的比较，筛选最适合于本疫苗的禽类免疫的最佳佐剂MONTANIDETMISA50V2；四是用筛选的最佳佐剂乳化该疫苗抗原，免疫SPF鸡，通过检测其抗体水平的变化来验证该疫苗，得出最佳免疫剂量，并建立HI方法与ELISA方法在评价抗体水平方面的关联性，为攻毒实验提供前提条件。通过这四部分的研究，完成攻毒试验前的工作，确保后续的攻毒试验研究顺利进行。
Avian flu is a deadly infectious disease of poultry and serious threatto the poultry industry which spreads widely in the world. Especially theepidemic and high mortality causes important economic losses to countriesand people. A highly contagious Avian influenza virus (AIV) can spreadacross species. In recent years, the human also become one of the hostsof this virus. Since the end of2003to19June,2008, it has been reportedthat383cases in15countries of Asia, Africa and Europe were laboratoryconfirmed human H5N1avian influenza and241died. AIV is threatening tohuman health and life as a new bird flu disease, yet human beings haveno immune protection to it. In early2004, the World Health Organizationwarned the world of the AIV epidemic and many countries and regions putthe top priority on the control of avian influenza. Avian flu is causedby the pathogen bird flu virus which has a very high variability. AIVsubtypes can be up to256and can restructure with other influenza viruses,then place the virus mutation and change of the host, so many scholarstake the complex AIV genome as the greatest gene pool of human influenzavirus.
As the avian influenza virus is in a wide variety of poultry and wildbird bodies, the bird flu control is a long-term and arduous task. Vaccinedevelopment and use is the most practical, effective and economicalmeasure to this challenge. The ideal vaccine would be the availabilityof good safety, and not only can stimulate the body's humoral immunityto produce antibodies, but also be able to fully help to regulate thecellular immunity to remove virus. Based on the above concept, we chose the H5N1subtype avian influenza virus which was mainly popular in Asiaas the research object, and selected a high homology HA1and NP proteinof H5N1subtype avian influenza virus strains which was used as standardreference sequence by comparing the protein sequences of Chinese AIV viruswith its neighboring countries. Through analysis of the HA1proteinsequence by computer software, we selected the major T cell epitopes andB cell epitopes. And then selected the main optimized CTL epitopes afterconducted a comprehensive analysis of the NP protein. According to thesepreferred epitopes, we designed the recombinant AIV H5N1subtype vaccine.This vaccine contains the major protective B cell epitopes and covers avariety of types of MHC II DR epitopes of H5N1virus HA1protein as themain skeleton. And at its C terminal we added the optimized CTL cellepitopes. After optimized the codon and base composition of the vaccinegenes, we obtained the genes coding the avian influenza virus subtype H5N1vaccine genes by using the synthetic method. We clone the vaccine genesinto the expression vector pRSET B in E. coli and got a good expressionthat the vaccine protein got to30%of total bacterial protein, and thetarget protein expressed in the form of inclusion bodies.
The use of vaccines is the most effective means for preventing theAIV disease. So the output and cost control in the vaccine production arehighly demanded. In this study, we explored and optimized the bestexpression of the growth and pH value range, carbon source concentration,culture medium, and induction conditions for the recombinant AIV subtypeH5N1vaccine. We established the high-density fermentation of theengineering strain and ensured that the engineering bacteria densityreached54OD600and the recombinant vaccine protein theoretical yieldachieved about3.4g/L on the premise of the vaccine protein expressionwas30%. On this basis, we further explored a simple and efficient vaccineprotein purification and recovery process. After a simple washing of inclusion bodies and Ni+affinity chromatography, the recombinant AIVsubtype H5N1vaccine could reach protein purity95.5%and refolded proteinvaccine stock solution concentration was up to2.4mg/mL.
To evaluate the avian influenza virus subtype specific and immuneeffects of the recombinant AIV H5N1subtype vaccine, this study dividedinto four parts. One is to establish an indirect ELISA method to determinethe specific reaction of the vaccine antigen and avian influenza H5(Re-4strain, Re-5strain) standard positive serum, H9standard positive serum,H7standard positive serum; Second, through the mouse experiments,tested the antibody levels and changes in cytokine levels.Through the miceexperimental model, we got the initial description of the vaccine antigen;Third, we selected the three recommended adjuvants of the French SEPPICcompany to emulsify with the vaccine and immuned SPF chickens. Bycomparing the antibody level, we chose the most suitable MONTANIDETMISA50V2for the best vaccine immune adjuvant; The last, we got the optimalimmunization dose by immuned SPF chickens. At the same time, we found theparallel relations between the ELISA method and the HI method. Throughthis four-part study, we completed the most primary work of the effectverifying test and ensured the smooth progress of the follow-up.
由于禽流感病毒广泛存在于各种禽类和野生鸟类体内，因此禽流感的控制是一项长期艰巨的任务，疫苗的开发和使用是应对这项艰巨任务最实际、最有效和最经济的手段。理想的疫苗应该是在具备良好的安全性的前提下，不仅能够调动机体的体液免疫，产生病毒中和抗体，更能够充分调动机体的细胞免疫来充分发挥病毒清除机制。基于以上理念，本研究以亚洲地区主要流行的H5N1亚型禽流感病毒为研究对象，比对了2003年以来中国及周边国家，尤其是东南亚国家所发表的H5N1亚型禽流感病毒毒株的HA1和NP蛋白序列，筛选出了同源性较高的HA1和NP蛋白序列作为标准参考序列。通过计算机软件对禽流感病毒H5N1亚型的HA1蛋白进行了全面分析，优选了出了主要T细胞表位和B细胞表位，并对NP蛋白进行了全面分析，优选出了主要CTL表位。依据这些优选表位，我们设计了禽流感病毒H5N1亚型基因工程疫苗，该疫苗以含有主要保护性B细胞表位和覆盖多种DR类型的MHC II表位的H5N1病毒HA1蛋白做为主体骨架，并在该骨架C端引入了优选出的CTL细胞表位。在对该疫苗基因进行密码子和碱基组成优化后，采用人工合成的方法获得了编码该禽流感病毒H5N1亚型基因工程疫苗的基因，并将该基因克隆入大肠杆菌表达载体pRSET B中，在大肠杆菌中得到了良好表达，禽流感病毒H5N1亚型基因工程疫苗蛋白表达量占菌体总蛋白30%，表达形式为包涵体。
疫苗的使用是防治禽流感的最有效手段，同时也对疫苗生产过程中的产量和成本控制提出了很高的要求。本研究对禽流感病毒H5N1亚型基因工程疫苗工程菌的生长和表达的最理想pH值范围、碳源浓度、培养基配方以及诱导时长等条件进行了探讨并优化，建立了该工程菌的中试发酵工艺，并进行了高密度发酵培养，在保证禽流感病毒H5N1亚型基因工程疫苗蛋白30%表达量的前提下，使工程菌密度达到54OD600，禽流感病毒H5N1亚型基因工程疫苗理论产量约为3.4g/L。在此基础上，我们又进一步探索出了一条简洁高效的疫苗蛋白纯化和复性工艺，经过简单的包涵体洗涤和Ni+亲和柱层析即可使禽流感病毒H5N1亚型基因工程疫苗蛋白纯度达到95.5%，复性后疫苗蛋白原液浓度可达2.4mg/mL。
为评价该禽流感病毒H5N1亚型基因工程疫苗的免疫效力，本研究首先将该疫苗作为免疫原进行了小白鼠实验，验证了其在小白鼠体内的免疫应答。然后分别用高致病性禽流感病毒(HPAIV)A/Goose/GuangDong/1/96(H5N1)[Gs/GD/1／96(H5N1)毒株和低致病力禽流感病毒（LPAIV) A／Chicken／Huadong／87／20O6(Ck／HD／87／06（H9N2)对免疫后SPF鸡进行攻毒，确定该疫苗对两种不同禽流感病毒亚型的保护效果。两个靶动物试验均用低、中、高三个剂量组对SPF鸡进行免疫，并分别用两种毒株攻毒，其中H5N1亚型的保护试验通过临床反应症状、检测HI抗体水平、ELISA法检测抗体，以及建立RT-PCR方法检测攻毒后各组病毒载量，来说明禽流感H5N1亚型基因工程疫苗的免疫效力；H9N2亚型的保护试验通过检测免疫后HI抗体水平和攻毒后排毒情况来说明其免疫效力。结果表明，禽流感H5N1亚型基因工程疫苗能在小白鼠体内产生良好的免疫应答；不同剂量的免疫组对不同禽流感病毒亚型感染后SPF鸡均达到了不同程度的保护，根据两个病毒亚型试验的比较，说明该疫苗对禽流感病毒H5N1亚型的免疫效力略强于H9N2亚型。其中10μg/只剂量组免疫保护率达到了100%，因此可以说明，该禽流感H5N1亚型基因工程疫苗可以考虑作为防御禽流感疫病的疫苗。
为评价该禽流感病毒H5N1亚型基因工程疫苗的抗原特异性和免疫效力，本研究分四个部分去验证。一是建立间接ELISA方法确定该疫苗抗原与禽流感H5（Re‐4株、Re‐5株）标准阳性血清、H9标准阳性血清、H7标准阳性血清的特异性反应，验证该疫苗的针对禽流感病毒型特异性；二是通过小白鼠实验，检测其抗体水平和细胞因子水平的变化，通过小白鼠实验模型，初步说明该疫苗抗原性；三是选择法国SEPPIC公司推荐的三种商业化佐剂，将该疫苗分别进行乳化，免疫SPF鸡，检测其抗体水平，根据不同时间内其抗体水平的比较，筛选最适合于本疫苗的禽类免疫的最佳佐剂MONTANIDETMISA50V2；四是用筛选的最佳佐剂乳化该疫苗抗原，免疫SPF鸡，通过检测其抗体水平的变化来验证该疫苗，得出最佳免疫剂量，并建立HI方法与ELISA方法在评价抗体水平方面的关联性，为攻毒实验提供前提条件。通过这四部分的研究，完成攻毒试验前的工作，确保后续的攻毒试验研究顺利进行。
Avian flu is a deadly infectious disease of poultry and serious threatto the poultry industry which spreads widely in the world. Especially theepidemic and high mortality causes important economic losses to countriesand people. A highly contagious Avian influenza virus (AIV) can spreadacross species. In recent years, the human also become one of the hostsof this virus. Since the end of2003to19June,2008, it has been reportedthat383cases in15countries of Asia, Africa and Europe were laboratoryconfirmed human H5N1avian influenza and241died. AIV is threatening tohuman health and life as a new bird flu disease, yet human beings haveno immune protection to it. In early2004, the World Health Organizationwarned the world of the AIV epidemic and many countries and regions putthe top priority on the control of avian influenza. Avian flu is causedby the pathogen bird flu virus which has a very high variability. AIVsubtypes can be up to256and can restructure with other influenza viruses,then place the virus mutation and change of the host, so many scholarstake the complex AIV genome as the greatest gene pool of human influenzavirus.
As the avian influenza virus is in a wide variety of poultry and wildbird bodies, the bird flu control is a long-term and arduous task. Vaccinedevelopment and use is the most practical, effective and economicalmeasure to this challenge. The ideal vaccine would be the availabilityof good safety, and not only can stimulate the body's humoral immunityto produce antibodies, but also be able to fully help to regulate thecellular immunity to remove virus. Based on the above concept, we chose the H5N1subtype avian influenza virus which was mainly popular in Asiaas the research object, and selected a high homology HA1and NP proteinof H5N1subtype avian influenza virus strains which was used as standardreference sequence by comparing the protein sequences of Chinese AIV viruswith its neighboring countries. Through analysis of the HA1proteinsequence by computer software, we selected the major T cell epitopes andB cell epitopes. And then selected the main optimized CTL epitopes afterconducted a comprehensive analysis of the NP protein. According to thesepreferred epitopes, we designed the recombinant AIV H5N1subtype vaccine.This vaccine contains the major protective B cell epitopes and covers avariety of types of MHC II DR epitopes of H5N1virus HA1protein as themain skeleton. And at its C terminal we added the optimized CTL cellepitopes. After optimized the codon and base composition of the vaccinegenes, we obtained the genes coding the avian influenza virus subtype H5N1vaccine genes by using the synthetic method. We clone the vaccine genesinto the expression vector pRSET B in E. coli and got a good expressionthat the vaccine protein got to30%of total bacterial protein, and thetarget protein expressed in the form of inclusion bodies.
The use of vaccines is the most effective means for preventing theAIV disease. So the output and cost control in the vaccine production arehighly demanded. In this study, we explored and optimized the bestexpression of the growth and pH value range, carbon source concentration,culture medium, and induction conditions for the recombinant AIV subtypeH5N1vaccine. We established the high-density fermentation of theengineering strain and ensured that the engineering bacteria densityreached54OD600and the recombinant vaccine protein theoretical yieldachieved about3.4g/L on the premise of the vaccine protein expressionwas30%. On this basis, we further explored a simple and efficient vaccineprotein purification and recovery process. After a simple washing of inclusion bodies and Ni+affinity chromatography, the recombinant AIVsubtype H5N1vaccine could reach protein purity95.5%and refolded proteinvaccine stock solution concentration was up to2.4mg/mL.
To evaluate the avian influenza virus subtype specific and immuneeffects of the recombinant AIV H5N1subtype vaccine, this study dividedinto four parts. One is to establish an indirect ELISA method to determinethe specific reaction of the vaccine antigen and avian influenza H5(Re-4strain, Re-5strain) standard positive serum, H9standard positive serum,H7standard positive serum; Second, through the mouse experiments,tested the antibody levels and changes in cytokine levels.Through the miceexperimental model, we got the initial description of the vaccine antigen;Third, we selected the three recommended adjuvants of the French SEPPICcompany to emulsify with the vaccine and immuned SPF chickens. Bycomparing the antibody level, we chose the most suitable MONTANIDETMISA50V2for the best vaccine immune adjuvant; The last, we got the optimalimmunization dose by immuned SPF chickens. At the same time, we found theparallel relations between the ELISA method and the HI method. Throughthis four-part study, we completed the most primary work of the effectverifying test and ensured the smooth progress of the follow-up.
引文
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