口蹄疫病毒免疫原基因在番茄和拟南芥中的表达及转基因番茄动物免疫试验
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摘要
口蹄疫是当今世界上危害最严重的家畜传染病之一。疫苗接种是预防口蹄疫的主要措施。近年来,随着植物基因工程的发展,用转基因植物表达抗原相对于传统疫苗而言是一种更安全、更经济的表达系统。本研究将口蹄疫病毒免疫原基因整合进番茄基因组并得以表达,用转基因番茄叶片蛋白提取液经肌肉途径免疫豚鼠,根据豚鼠抗体水平的消长动态和免疫豚鼠抗强毒攻击的保护率进行植物疫苗免疫原性的研究,为FMD转基因疫苗的开发奠定基础。
此外,由于植物种子生产外源基因工程蛋白质的品质相当稳定,可作为饲料添加剂或口服疫苗,为优化表达体系,本研究构建了FMDV结构蛋白VP1基因的种子特异性表达载体。具体结果如下:
1)构建了O型FMDV China99株结构蛋白VP1基因的组成型植物双元表达载体pBin438/VP1及结构蛋白P1、非结构蛋白2A、3C以及部分2B基因的组成型植物双元表达载体pBin438/P12X3C。VP1基因及起始密码ATG、内质网引导多肽SEKDEL共660个核苷酸,编码220个氨基酸;P12X3C基因及起始密码ATG、内质网引导多肽SEKDEL共3018个核苷酸,编码1006个氨基酸,与亲本毒株相应核苷酸序列比较,同源性分别为100%和99.6%。
2)建立了番茄高频转化系统。高频转化系统以MS为基本培养基,附加ZT1.0mg/L和IAA0.1mg/L,选8～10日苗龄的子叶预培养2d,在活化的根癌农杆菌菌液里侵染3～5min,于暗处共培养2d后,直接在含卡那霉素再生培养基上筛选培养,形成抗性愈伤并分化出芽。转化率达18%。
3)转基因番茄PCR检测表明:FMDV免疫原基因VP1和P12X3C分别整合到番茄基因组中;RT-PCR结果证实目的基因在转基因番茄的转录水平表达;ELISA检测约40%的pBin438/VP1卡那抗性植株呈阳性,约25%的pBin438/P12X3C卡那抗性植株阳性:随机选取部分ELISA检测阳性植株进行Western blotting检测,重组蛋白能够与FMDV抗血清反应。表明FMDV免疫原基因VP1和P12X3C已分别转入番茄基因组并获得表达,且所表达的蛋白具有免疫反应性。
4)构建了FMDV结构蛋白VP1基因的种子特异性表达载体p7SBin438/VP1,浸花法转化拟南芥,分子检测表明,VP1基因已整合进拟南芥基因组并在荚中获得表达,为今后将FMDV免疫基因转入大豆等油料植物种子提供了实验依据。
5)转基因番茄叶片蛋白提取液免疫豚鼠,第一次免疫后10d ELISA检测不到FMD特异性抗体,第二次免疫后10d,豚鼠血清抗体水平显著提高并达到第一个高峰,第三次免疫后21d,抗体水平达到第二个高峰,其中pBin438/VP1转基因番茄第三次免疫豚鼠后21天血清效价最高可达1:64,攻毒后免疫豚鼠保护率达80%,pBin438/P12X3C转基因番茄第三次免疫后21天血清效价最高可达1:128,攻毒后免疫豚鼠保护率达100%。
Foot-and-mouth disease (FMD) is the etiological agent of an important disease of livestock. Vaccination is the major method to prevent FMD. But the conventional inactivated vaccines have many defects, such as the possibility of virus dissemination, and the virulent recovery of vaccine virus. Therefore, it is necessary to develop a safe, efficient and economic FMD vaccine.The use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as alternative methodology for the production of experimental vaccines. Antigens produced in transgenic plants are capable of invoking protective mucosal immune responses against important pathogens as it has already been demonstrated. To explore the feasibility of FMD edible vaccine, in this study, the immunogen gene of VP1 and P12X3C were transferred into the nuclear chromosomal DNA of the tomato plants respectively. In order to evaluate immune response of guinea pigs against FMDV, the extracts of transgenic leaves were injected into guinea pigs. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID_(50) FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants.Using plant seeds to produce target proteins could be used as food addition or oral vaccines. In this study, the structural protein VP1 gene was transformed into Arabidopsis thaliana and expression of VP1 in the genome of transgenic Arabidopsis thaliana was performed by molecular biology analysis. The main results presented in this thesis are as follows:1)The plant constitutive-expression vector pBin438/VP1 with VP1 and pBin438/P12X3C with multi-genes of FMDV were constructed respectively, multi-genes P12X3C encode for the FMDV O/China/99 genomic regions P1(1A,1B,1C,1D), 2A, 3C and a part of 2B. The sequence of VP1 contain 660nt including complete VP1, a start codon and microsomal retentional signal sequence SEKDEL. The construction of multi-genes P12X3C also contain 3 018nt including full length of P1, 2A, 3C and a part of 2B, a start codon and microsomal retentional signal sequence SEKDEL. The percent identity of VP1 and P12X3C to the same gene from GenBank of FMDV O/China/99 were 100% and 99.6% respectively.2)The optimal tomato transformation system that was mediated by Agrobacterium fumefaciens GV3101 was established. On the base of the efficient regeneration system, factors that affected transformation such as the Agrobacterium fumefaciens concentration, infiltration time and transform style were optimized. Our protocol of plant transformation system was as follow: from eight to ten days post-germination, cotyledon was infiltrated 3~5min and co-cultivated for 48h with an overnight grown culture of Agrobaterium Tumefaciens on medium of Murashige and Skoog (MS) adding 1.0mg/L ZT and 0.1mg/L IAA. Cotyledons post-culturation with Agrobacterium. Tumefaciens earring pBin438/VP1 and pBin438/P12X3C were directly placed onto the selective medium containing 50mg/L kanamycin and 500mg/L carbenicillin. After six weeks, kanamycin-resistant shoots were removed from calli, and transferred to the rooting medium. The frenquency of success transformations reached to 18%.
此外,由于植物种子生产外源基因工程蛋白质的品质相当稳定,可作为饲料添加剂或口服疫苗,为优化表达体系,本研究构建了FMDV结构蛋白VP1基因的种子特异性表达载体。具体结果如下:
1)构建了O型FMDV China99株结构蛋白VP1基因的组成型植物双元表达载体pBin438/VP1及结构蛋白P1、非结构蛋白2A、3C以及部分2B基因的组成型植物双元表达载体pBin438/P12X3C。VP1基因及起始密码ATG、内质网引导多肽SEKDEL共660个核苷酸,编码220个氨基酸;P12X3C基因及起始密码ATG、内质网引导多肽SEKDEL共3018个核苷酸,编码1006个氨基酸,与亲本毒株相应核苷酸序列比较,同源性分别为100%和99.6%。
2)建立了番茄高频转化系统。高频转化系统以MS为基本培养基,附加ZT1.0mg/L和IAA0.1mg/L,选8～10日苗龄的子叶预培养2d,在活化的根癌农杆菌菌液里侵染3～5min,于暗处共培养2d后,直接在含卡那霉素再生培养基上筛选培养,形成抗性愈伤并分化出芽。转化率达18%。
3)转基因番茄PCR检测表明:FMDV免疫原基因VP1和P12X3C分别整合到番茄基因组中;RT-PCR结果证实目的基因在转基因番茄的转录水平表达;ELISA检测约40%的pBin438/VP1卡那抗性植株呈阳性,约25%的pBin438/P12X3C卡那抗性植株阳性:随机选取部分ELISA检测阳性植株进行Western blotting检测,重组蛋白能够与FMDV抗血清反应。表明FMDV免疫原基因VP1和P12X3C已分别转入番茄基因组并获得表达,且所表达的蛋白具有免疫反应性。
4)构建了FMDV结构蛋白VP1基因的种子特异性表达载体p7SBin438/VP1,浸花法转化拟南芥,分子检测表明,VP1基因已整合进拟南芥基因组并在荚中获得表达,为今后将FMDV免疫基因转入大豆等油料植物种子提供了实验依据。
5)转基因番茄叶片蛋白提取液免疫豚鼠,第一次免疫后10d ELISA检测不到FMD特异性抗体,第二次免疫后10d,豚鼠血清抗体水平显著提高并达到第一个高峰,第三次免疫后21d,抗体水平达到第二个高峰,其中pBin438/VP1转基因番茄第三次免疫豚鼠后21天血清效价最高可达1:64,攻毒后免疫豚鼠保护率达80%,pBin438/P12X3C转基因番茄第三次免疫后21天血清效价最高可达1:128,攻毒后免疫豚鼠保护率达100%。
Foot-and-mouth disease (FMD) is the etiological agent of an important disease of livestock. Vaccination is the major method to prevent FMD. But the conventional inactivated vaccines have many defects, such as the possibility of virus dissemination, and the virulent recovery of vaccine virus. Therefore, it is necessary to develop a safe, efficient and economic FMD vaccine.The use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as alternative methodology for the production of experimental vaccines. Antigens produced in transgenic plants are capable of invoking protective mucosal immune responses against important pathogens as it has already been demonstrated. To explore the feasibility of FMD edible vaccine, in this study, the immunogen gene of VP1 and P12X3C were transferred into the nuclear chromosomal DNA of the tomato plants respectively. In order to evaluate immune response of guinea pigs against FMDV, the extracts of transgenic leaves were injected into guinea pigs. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID_(50) FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants.Using plant seeds to produce target proteins could be used as food addition or oral vaccines. In this study, the structural protein VP1 gene was transformed into Arabidopsis thaliana and expression of VP1 in the genome of transgenic Arabidopsis thaliana was performed by molecular biology analysis. The main results presented in this thesis are as follows:1)The plant constitutive-expression vector pBin438/VP1 with VP1 and pBin438/P12X3C with multi-genes of FMDV were constructed respectively, multi-genes P12X3C encode for the FMDV O/China/99 genomic regions P1(1A,1B,1C,1D), 2A, 3C and a part of 2B. The sequence of VP1 contain 660nt including complete VP1, a start codon and microsomal retentional signal sequence SEKDEL. The construction of multi-genes P12X3C also contain 3 018nt including full length of P1, 2A, 3C and a part of 2B, a start codon and microsomal retentional signal sequence SEKDEL. The percent identity of VP1 and P12X3C to the same gene from GenBank of FMDV O/China/99 were 100% and 99.6% respectively.2)The optimal tomato transformation system that was mediated by Agrobacterium fumefaciens GV3101 was established. On the base of the efficient regeneration system, factors that affected transformation such as the Agrobacterium fumefaciens concentration, infiltration time and transform style were optimized. Our protocol of plant transformation system was as follow: from eight to ten days post-germination, cotyledon was infiltrated 3~5min and co-cultivated for 48h with an overnight grown culture of Agrobaterium Tumefaciens on medium of Murashige and Skoog (MS) adding 1.0mg/L ZT and 0.1mg/L IAA. Cotyledons post-culturation with Agrobacterium. Tumefaciens earring pBin438/VP1 and pBin438/P12X3C were directly placed onto the selective medium containing 50mg/L kanamycin and 500mg/L carbenicillin. After six weeks, kanamycin-resistant shoots were removed from calli, and transferred to the rooting medium. The frenquency of success transformations reached to 18%.
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