猪传染性胃肠炎病毒纤突蛋白基因体外克隆表达及鉴定
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摘要
猪传染性胃肠炎(Porcine transmissible gastroenteritis,TGE),是由猪传染性胃肠炎病毒(TGEV)引起的一种急性、高度接触性肠道传染病。临床特征主要表现为呕吐、脱水和腹泻。不同品种和不同年龄的猪对该病均易感,2周龄以内的仔猪具有高度感染性,致死率可达到100%。目前该病广泛存在于世界各养猪国家,我国作为世界养猪大国也不断有该病发生和流行的报道,该病常常由于混合感染和细菌继发感染,难以确诊,缺乏有效疫苗,给养猪业带来严重危害。
本研究首先对长春市郊养猪户饲养猪群中一起疑似猪传染性胃肠炎病例进行分析,利用PK-15细胞从临床上表现为腹泻症状的病死仔猪肠系膜淋巴结材料中分离获得1株病毒,对该病毒进行病毒形态学、PCR检测与动物回归试验等系统鉴定后,证实该分离株为猪传染性胃肠炎病毒(TGEV),被命名为TGEV JL。对TGEV JL分离株的分离鉴定为进行针对猪传染性胃肠炎病毒的体外克隆表达及转植物疫苗研制提供基本条件。
已有报道,TGEV具有4种结构蛋白,分别为:纤突糖蛋白(S蛋白)、膜内在蛋白(M蛋白)、小膜蛋白(sM蛋白)和核衣壳蛋白(N蛋白)。而其中编码S蛋白的S基因全长约为4300bp,决定宿主的亲嗜性、血凝性和致病性,同时也是诱导机体产生中和抗体的最主要结构蛋白。本研究在获得TGEV JL株的基础上,应用RT-PCR技术扩增获得该病毒株的主要免疫原性基因TGEV S基因全序列,将其连接到pMD18-T载体,进行测序及序列分析,将该基因序列和推导的氨基酸序列与8个不同来源的TGEV毒株进行同源性比较分析,表明该分离株的TGEV S基因高度保守,可作为转植物疫苗研制的目的基因。然后将该基因插入植物表达载体pBI121的CaMV35S启动子下游,成功构建了高效植物表达载体pBI121-S。
玉米是我国主要的粮食及饲料作物之一。饲料消费是玉米最重要的消费渠道,约占消费总量的70%左右。玉米作为饲料消费在我国有两种情况。一是加工生产成配合饲料。二是传统的把玉米直接用于饲料的消费。本研究在获得pBI121-S的基础上,以玉米自交系“丹598”、“H99”和“丹988”的胚性愈伤组织为材料,通过愈伤组织对卡那霉素的敏感性试验,确定了卡那霉素40-56mg/L为愈伤组织适宜选择压。利用基因枪法将猪传染性胃肠炎病毒TGEV JL株S基因的植物表达载体pBI121-S导入玉米自交系中,并对基因枪法转化的条件进行了研究,对转化的愈伤组织进行分化诱导出苗后进行PCR和RT-PCR检测。PCR结果表明,外源目的基因已转入到玉米基因组中;通过RT-PCR试验证明,目的基因能在转基因玉米植株中获得转录。
同时将pBI121-S转入根癌农杆菌EHA101中,成功获得含有重组植物表达载体pBI121-S的农杆菌工程菌。利用农杆菌介导法将TGEV S基因导入玉米自交系中,并对农杆菌转化系统的条件进行了研究。结果表明,根癌农杆菌EHA101的菌液OD600nm值为0.5-0.6时,侵染20min为农杆菌转化的最适条件。对转化的愈伤组织进行分化诱导出苗后进行PCR、Southern blot和Northern杂交检测,证明外源目的基因S已转入到玉米基因组中。以上研究结果为进一步研究猪传染性胃肠炎病毒的转基因植物疫苗及其在兽医临床的应用提供了依据。
Porcine transmissible gastroenteritis(TGE)is an acute, highly contact intestinal infection,caused by porcine transmissible gastroenteritis virus (TGEV). Main clinical features are vomiting, diarrhea and dehydration. Different ages and different varieties pigs are susceptible with highly infectious, especiallly less than two week old piglets, and its mortality rate could reach 100%. Currently the disease spread in each country which raise pigs. China, being as the biggest pig breeding country, constantly had reports about happening and epidemic of the disease. Owing to the mixed infection and bacteria secondary infection, it is usually hard to diagnose,and lack of the effective vaccine .It brings a seriously harmful effect to the swine industry
The study firstly analyzed a suspected case of porcine transmissible gastroenteritis in changchun suburb. A TGE virus strain (TGEV JL) was successfully isolated from mesenteric lymphnodes of died piglets with clinical diarrhea symptom from a pig farm by passaging in PK-15 cells. The virus was identified as TGEV based on morphology, PCR results and animal regression test. The isolation of TGEV JL strain provide basic conditions for gene cloning of contagion enterogastrtisetc virus in vitro and plants vaccine development.
It’s reported that,TGEV had four types of structural proteins: the Spike glycoprotein (S), the inner membrane protein (M), small membrane protein (sM) and nucleocapsid protein (N). The length of S gene, which encoded S protein, was about 4300bp. It decided the host’s tropism, hemagglutination and pathogenicity, while it is the most important structure protein which induced the organism to produce the neutralizing antibody. On the basis of getting TGEV JL strain, the whole sequences of S gene (main immunogenicity gene) TGEV was obtained by RT-PCR amplification technology, which connected to the pMD18 - T vector.And sequenced and analyzed sequential in this research. Through compared the gene sequence and the amino acid sequence to seven different sources TGEV strains,and done some homology analysis, it showed that S gene of the isolate strain TGEV was highly conservative. It could be used as the purpose gene for the transfer of plant vaccines. Inserted this gene into plant expression vector pBI121, downstream of CaMV35S promoter , building high efficient plant expression vector pBI121-S.
Corn is the main food for human beings and animals. Feed consumption is the most important channel of corn consumption, accounts for about 70 percentages. Corn as feed consumption has two kinds of cases in China . One as compound feed and the other one is use to feed consumption. This research is in basis of acquired pBI121– S of Agrobacterium tumefaciens Corn and use the embryogenic calli of "Dan 598" and "H99" to make the experiment for kanamycin sensitive test. This study makes sure that 40-56mg/L is the best selection pressure. The pBI121-S ,TGEV S gene’s plant expression vector, was transferred into corn inbred line by using gene gun method. Conversion condition of the gene gun method had been studied. After being induced differentiation emergence, inversive callus was detected by PCR and RT-PCR.The result of PCR showed that exogenous gene had been transfered into the maize genome,while RT- PCR showed that the target gene could transcribe in the transgenic maize plants .
At the same time, through the means of mediated transferred the TGEVS gene of Agrobacterium tumefaciens into the maize inbred line, and studied its mediated condition. The research showed that when the value of the EHA101 Agrobacterium bacteria at 600nm OD was from 0.5 to 0.6 and 20min infection was the optimum condition for Agrobacterium tumefaciens transformation. The inversive callus, induced differentiation emergence, detected by PCR amplification, Sorthern and Nouthern blot.It turned out that the target gene had already been diverted to the maize genome.The research results provides a basis for further studying, not only the transgenic plant vaccine of transmissible gastroenteritis virus of swine,but also using it in veterinary clinic.
本研究首先对长春市郊养猪户饲养猪群中一起疑似猪传染性胃肠炎病例进行分析,利用PK-15细胞从临床上表现为腹泻症状的病死仔猪肠系膜淋巴结材料中分离获得1株病毒,对该病毒进行病毒形态学、PCR检测与动物回归试验等系统鉴定后,证实该分离株为猪传染性胃肠炎病毒(TGEV),被命名为TGEV JL。对TGEV JL分离株的分离鉴定为进行针对猪传染性胃肠炎病毒的体外克隆表达及转植物疫苗研制提供基本条件。
已有报道,TGEV具有4种结构蛋白,分别为:纤突糖蛋白(S蛋白)、膜内在蛋白(M蛋白)、小膜蛋白(sM蛋白)和核衣壳蛋白(N蛋白)。而其中编码S蛋白的S基因全长约为4300bp,决定宿主的亲嗜性、血凝性和致病性,同时也是诱导机体产生中和抗体的最主要结构蛋白。本研究在获得TGEV JL株的基础上,应用RT-PCR技术扩增获得该病毒株的主要免疫原性基因TGEV S基因全序列,将其连接到pMD18-T载体,进行测序及序列分析,将该基因序列和推导的氨基酸序列与8个不同来源的TGEV毒株进行同源性比较分析,表明该分离株的TGEV S基因高度保守,可作为转植物疫苗研制的目的基因。然后将该基因插入植物表达载体pBI121的CaMV35S启动子下游,成功构建了高效植物表达载体pBI121-S。
玉米是我国主要的粮食及饲料作物之一。饲料消费是玉米最重要的消费渠道,约占消费总量的70%左右。玉米作为饲料消费在我国有两种情况。一是加工生产成配合饲料。二是传统的把玉米直接用于饲料的消费。本研究在获得pBI121-S的基础上,以玉米自交系“丹598”、“H99”和“丹988”的胚性愈伤组织为材料,通过愈伤组织对卡那霉素的敏感性试验,确定了卡那霉素40-56mg/L为愈伤组织适宜选择压。利用基因枪法将猪传染性胃肠炎病毒TGEV JL株S基因的植物表达载体pBI121-S导入玉米自交系中,并对基因枪法转化的条件进行了研究,对转化的愈伤组织进行分化诱导出苗后进行PCR和RT-PCR检测。PCR结果表明,外源目的基因已转入到玉米基因组中;通过RT-PCR试验证明,目的基因能在转基因玉米植株中获得转录。
同时将pBI121-S转入根癌农杆菌EHA101中,成功获得含有重组植物表达载体pBI121-S的农杆菌工程菌。利用农杆菌介导法将TGEV S基因导入玉米自交系中,并对农杆菌转化系统的条件进行了研究。结果表明,根癌农杆菌EHA101的菌液OD600nm值为0.5-0.6时,侵染20min为农杆菌转化的最适条件。对转化的愈伤组织进行分化诱导出苗后进行PCR、Southern blot和Northern杂交检测,证明外源目的基因S已转入到玉米基因组中。以上研究结果为进一步研究猪传染性胃肠炎病毒的转基因植物疫苗及其在兽医临床的应用提供了依据。
Porcine transmissible gastroenteritis(TGE)is an acute, highly contact intestinal infection,caused by porcine transmissible gastroenteritis virus (TGEV). Main clinical features are vomiting, diarrhea and dehydration. Different ages and different varieties pigs are susceptible with highly infectious, especiallly less than two week old piglets, and its mortality rate could reach 100%. Currently the disease spread in each country which raise pigs. China, being as the biggest pig breeding country, constantly had reports about happening and epidemic of the disease. Owing to the mixed infection and bacteria secondary infection, it is usually hard to diagnose,and lack of the effective vaccine .It brings a seriously harmful effect to the swine industry
The study firstly analyzed a suspected case of porcine transmissible gastroenteritis in changchun suburb. A TGE virus strain (TGEV JL) was successfully isolated from mesenteric lymphnodes of died piglets with clinical diarrhea symptom from a pig farm by passaging in PK-15 cells. The virus was identified as TGEV based on morphology, PCR results and animal regression test. The isolation of TGEV JL strain provide basic conditions for gene cloning of contagion enterogastrtisetc virus in vitro and plants vaccine development.
It’s reported that,TGEV had four types of structural proteins: the Spike glycoprotein (S), the inner membrane protein (M), small membrane protein (sM) and nucleocapsid protein (N). The length of S gene, which encoded S protein, was about 4300bp. It decided the host’s tropism, hemagglutination and pathogenicity, while it is the most important structure protein which induced the organism to produce the neutralizing antibody. On the basis of getting TGEV JL strain, the whole sequences of S gene (main immunogenicity gene) TGEV was obtained by RT-PCR amplification technology, which connected to the pMD18 - T vector.And sequenced and analyzed sequential in this research. Through compared the gene sequence and the amino acid sequence to seven different sources TGEV strains,and done some homology analysis, it showed that S gene of the isolate strain TGEV was highly conservative. It could be used as the purpose gene for the transfer of plant vaccines. Inserted this gene into plant expression vector pBI121, downstream of CaMV35S promoter , building high efficient plant expression vector pBI121-S.
Corn is the main food for human beings and animals. Feed consumption is the most important channel of corn consumption, accounts for about 70 percentages. Corn as feed consumption has two kinds of cases in China . One as compound feed and the other one is use to feed consumption. This research is in basis of acquired pBI121– S of Agrobacterium tumefaciens Corn and use the embryogenic calli of "Dan 598" and "H99" to make the experiment for kanamycin sensitive test. This study makes sure that 40-56mg/L is the best selection pressure. The pBI121-S ,TGEV S gene’s plant expression vector, was transferred into corn inbred line by using gene gun method. Conversion condition of the gene gun method had been studied. After being induced differentiation emergence, inversive callus was detected by PCR and RT-PCR.The result of PCR showed that exogenous gene had been transfered into the maize genome,while RT- PCR showed that the target gene could transcribe in the transgenic maize plants .
At the same time, through the means of mediated transferred the TGEVS gene of Agrobacterium tumefaciens into the maize inbred line, and studied its mediated condition. The research showed that when the value of the EHA101 Agrobacterium bacteria at 600nm OD was from 0.5 to 0.6 and 20min infection was the optimum condition for Agrobacterium tumefaciens transformation. The inversive callus, induced differentiation emergence, detected by PCR amplification, Sorthern and Nouthern blot.It turned out that the target gene had already been diverted to the maize genome.The research results provides a basis for further studying, not only the transgenic plant vaccine of transmissible gastroenteritis virus of swine,but also using it in veterinary clinic.
引文
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