草鱼出血病转基因植物疫苗表达载体的构建

Construction of Plant-Based Expression Vector of Transgenic Plant Vaccine Against the Grass Carp Hemorrhage

作者: 专业:水产养殖 导师:曾令兵 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Grass carp hemorrhage, Reovirus(GCRV), VP6 gene, E Coli, LTB gene, Plant-based expression vector, Construction, Ryegrass, Calli tissue culture

        草鱼(Ctenopharyngodon idellus)是我国重要的淡水养殖品种之一,在我国淡水渔业生产中占有举足轻重的地位。草鱼出血病是草鱼鱼种阶段最为严重的疾病,死亡率高达90%以上,造成重大的经济损失。目前对该病尚缺乏有效的防治方法。转基因植物疫苗是一种高新技术疫苗。利用现代分子克隆技术、植物基因工程技术与免疫学技术,构建外源基因植物表达载体并导入植物体内,表达外源基因编码的抗原蛋白,通过口服途径免疫,使人体或和动物获得特异性免疫能力。和其它传统疫苗相比,具有廉价、安全、有效等优点。目前,越来越多的人类与动物重大疾病病原的抗原蛋白在植物中得到了表达,有的已进入了动物和人体实验。本研究的主要研究任务是:构建表达草鱼出血病呼肠孤病毒VP6抗原蛋白的植物表达载体和培育植物胚性愈伤组织并进行分化,以期为草鱼出血病转基因植物疫苗研究奠定前期基础。本论文主要进行了以下几个方面的研究:1.根据草鱼呼肠孤病毒(GCRV)VP6抗原蛋白的全基因序列(GeneBankAF403394)设计特异性引物,采用RT-PCR技术扩增编码GCRV-VP6抗原蛋白的1.3Kbp读码框片段,将VP6基因编码片段与pCR2.1载体连接,经酶切、PCR扩增和序列测定确认正确后,再将其克隆到携带有绿色荧光蛋白(GFP)基因的植物表达载体pCAMBIA1302中。酶切、PCR扩增和序列测定显示,pCAMBIA1302-VP6含有1.3Kbp的草鱼呼肠孤病毒VP6基因片段,表明GCRV-VP6抗原基因已正确插入植物表达载体pCAMBIA1302中,成功构建了融合表达草鱼呼肠孤病毒VP6蛋白和绿色荧光蛋白的植物表达载体pCAMBIA1302-VP6。2.根据GeneBank中草鱼呼肠孤病毒VP6蛋白的全基因序列(AF403394)和大肠杆菌不耐热肠毒素B亚单位(LTB)的基因序列(M17874)设计并合成特异性引物,从感染草鱼呼肠孤病毒(GCRV)的草鱼肾细胞(CIK)中提取病毒核酸作为模板,进行RT-PCR扩增,得到约1.3Kbp的GCRV VP6基因读码框片段,同时提取大肠杆菌H44815全基因组作为模板,PCR扩增得到约370bp的LTB基因读码框片段。将获得的两目的片段分别克隆到pCR2.1载体上,经酶切、PCR扩增检测和序列测定确认正确后,将其克隆到携带绿色荧光蛋白标记的植物表达载体pCAMBIA1302上,成功构建了可将草鱼呼肠孤病毒VP6抗原蛋白、大肠杆菌不耐热肠毒素B亚单位、绿色荧光蛋白融合表达的植物载体pCAMBIA 1302-LTB-VP6。本研究为草鱼出血病可饲化转基因植物疫苗研制奠定了基础。3.以黑麦草(Lolium perenne)成熟胚为外植体材料,进行外植体的灭菌、愈伤组织的诱导、继代及植株再生。诱导出可以用于组织培养的黑麦草胚性愈伤组织。
    Grass carp(Ctenopharyngodon idellus), is a most popular commercial fish species in China. Grass Carp Hemorrhage is one of the most serious diseases of grass carp, causing more than 90% mortality and huge economic loss to the aquaculture industry. At present, no effective ways for prevention and treatment of the disease are available.Transgenic plant-derived vaccine is a kind of new vaccine that combines the technology of molecular cloning, plant gene engineering and immunology. It is prepared by constructing the pant-based expression vector and deliverring foreign genes into plants and then elicits the special immune response in the body of human or animal through oral approach. Compared with the traditional vaccines, transgenic plant-derived vaccine is cheaper, safer and more efficacious.Recently, more and more antigen proteins have been expressed in plants and tested in animals or human.In this study, plant-based expression vector containing the Grass carp reovirus VP6 antigen protein gene was constructed and the calli of ryegrass was induced. The present study has made a solid base for the innovation of edible transgenic plant vaccine against grass carp hemorrhage caused by reovirus through oral immunization. The study was carried out from the following aspects.1.The coding gene of grass carp reovirus (GCRV) VP6 antigen protein was amplified by RT-PCR from the viral RNA genome extracted in virus infected Ctenopharyngodon idellus Kidney (CIK) cells with primers based on the gene sequence in GeneBank (AF403394).The 1.3kb PCR product was ligated to pCR2.1 vector, and after identified with enzyme digestion, PCR detection and sequencing, the 1.3kb cording sequence of GCRV VP6 gene was cloned into the plant-based expression vector pCAMBIA1302, which had a green fluorescent protein (GFP) gene insert. Thus, a fusion-expression vector of grass carp reovirus VP6 gene and GFP gene was constructed.2.Specific primer pairs for amplifying the GCRV VP6 gene and the Escherichia Coli LTB gene were designed and synthesized based on the sequences of GCRV VP6 gene and Escherichia Coli LTB gene posted in GenBank. GCRV genome RNA was extracted from the virus infected CIK cells and the GCRV VP6 coding region was amplified by RT-PCR; The LTB coding region was amplified from Escherichia Coli H44815 genome.The PCR products were cloned into pCR2.1 vector respectively and then sequenced and subcloned into vector pCAMBIA 1302, which contained a green fluorescence protein gene marker. Fusion expression vector of LTB, grass carp reovirus VP6 gene and green fluorescence protein gene vector was successfully constructed.3.The calli induction, subculture, differentiation and plant regeneration of ryegrass were conducted in this study. The embryonic calli can be used to tissue culture.
        

草鱼出血病转基因植物疫苗表达载体的构建

摘要6-7
Abstract7
缩略语表8-9
第一章 文献综述9-19
    1 草鱼出血病9-11
        1.1 草鱼出血病的研究简史9-10
        1.2 草鱼出血病的症状及流行情况10
        1.3 草鱼呼肠孤病毒的生物学特性10-11
        1.4 草鱼出血病的防治方法11
    2 转基因植物可食性疫苗研究11-13
    3 渔用转基因植物疫苗研究13-14
    4 LTB基因及黑麦草遗传转化的研究14-17
        4.1 LTB基因研究14-15
        4.2 黑麦草遗传转化研究15-17
    五 植物表达载体PCAMBIA 130217-19
第二章 草鱼呼肠孤病毒VP6蛋白植物表达载体的构建19-35
    1 材料与方法20-28
        1.1 仪器与设备20-21
        1.2 实验材料与试剂21-23
        1.3 所用软件23
        1.4 实验方法23-28
    2 结果与分析28-33
        2.1 病毒滴度测定结果28-29
        2.2 聚丙烯酰胺凝胶电泳结果29-30
        2.3 GCRV VP6蛋白编码基因的克隆与鉴定30-32
        2.4 pCAMBIA 1302-VP6阳性重组质粒的鉴定32-33
    3 讨论33-35
第三章 草鱼呼肠孤病毒VP6蛋白与大肠杆菌LTB亚基融合植物表达载体的构建35-43
    1 材料与方法35-37
        1.1 实验材料与试剂35
        1.2 目的基因片段的PCR引物设计及扩增35-36
        1.3 重组质粒pCR 2.1-VP6和pCR 2.1-LTB构建36-37
        1.4 pCAMBIA 1302-LTB/VP6植物融合表达载体的构建37
    2 结果与分析37-41
        2.1 GCRV VP6蛋白与大肠杆菌LTB亚基编码基因的克隆及测序37-39
        2.2 pCAMBIA 1302-LTB/VP6阳性重组质粒的酶切和PCR鉴定39-40
        2.3 pCAMBIA 1302-LTB/VP6植物融合表达载体构建图40-41
    3 讨论41-43
第四章 黑麦草愈伤组织培养及遗传转化43-53
    1 材料与方法43-46
        1.1 仪器与设备43-44
        1.2 材料44-46
    2 方法46-48
        2.1 黑麦草愈伤组织培养46
        2.2 农杆菌工程菌种制备46-47
        2.3 农杆菌介导黑麦草的遗传转化47-48
    3 结果48-50
        3.1 黑麦草愈伤组织的诱导48-49
        3.2 黑麦草愈伤组织分化49
        3.3 阳性重组质粒转化农杆菌49-50
        3.4 农杆菌介导重组质粒转化黑麦草愈伤组织50
    4 讨论50-53
        4.1 转化受体的选择51
        4.2 菌液浓度51
        4.3 侵染方式与侵染时间51-52
        4.4 共培养时间52-53
结语53-54
参考文献54-64
致谢64
        


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