A组轮状病毒G1型中国株VP7基因转化番茄的研究
摘要
轮状病毒(Rotaviruses，RV)是世界范围内引起婴幼儿病毒性腹泻的主要病原体。迄今，对轮状病毒感染最有力的预防和控制措施，仍是发展有效的疫苗。VP7是轮状病毒的主要外壳蛋白和中和抗原，是发展基因工程疫苗的首选。研究表明，将细菌性和病毒性病原体抗原的编码基因导入植物细胞，能够表达出较好保留天然免疫原性的抗原，这为利用转基因植物生产疫苗提供了良好的基础。因此，将VP7基因导入番茄，获得具有免疫原性的轮状病毒植物口服疫苗，将为防治轮状病毒引起的小儿腹泻提供新型疫苗，具有潜在的社会和经济意义。
本研究分别以组成型启动子CaMV35S和番茄果实特异表达启动子TFP构建了VP7基因的两种植物表达载体pBIVP7和pEVP7。利用农杆菌介导法，转化番茄叶盘，经卡那霉素(Kan)多重筛选，共获得了18株抗性植株。PCR分析结果为阳性的植株有17株(转pBIVP7抗性植株10株，转pEVP7抗性植株7株)。随机选取生长良好的9株PCR检测阳性植株(5株转pBIVP7，4株转pEVP7)进行PCR-Southern blot和Southern blot分析，结果表明：有8株(5株转pBIVP7，3株转pEVP7)为转基因植株。RT-PCR分析证明，在组成型启动子CaMV35S控制下的5株转基因植株的叶片和果实中，有4株VP7基因在转录水平上表达；而在果实特异表达启动子TFP控制下的3株转基因植株，VP7基因仅在果实中获得转录水平上表达。此外，从两种转基因植株中分别选取一自交株进行T_1代遗传分析，其Kan抗性基因分离比皆为3:1，T_1代抗性番茄植株的PCR结果也呈阳性。
本研究为研究和开发新型轮状病毒口服疫苗提供一条新的途径和方法，具有一定的理论意义和应用前景。
Rotaviruses (RV) are the most common cause of severe diarrhea in children all over the world,VP7 is major outer capsid protein and is a primary candidate for inclusion in a subunit or recombinant. Advances in genetic engineering in the past decade have accelerated the expression, in plant, of foreign proteins with pharmaceutical value. Antigens from infectious bacterial or viral diseases have been introduced into plants. So introducing VP7 gene into tomato to develop edible plant vaccine of rotaviruses would change the traditional means of production of vaccines and the cost of vaccine production would be reduced greatly. It is provided with commercial meaning and applicative prospect.
In this study, two plant expression vectors pBIVP7 and pEVP7 were constructed. In pBIVPT, it is under control of CaMV35S promoter. In pEVP7, it is under control of tomato-fruit-specific TFP promoter. After co-cultivation with Agrobacterium Tumefaciens EHA105(containing pBIVP7 or pEVP7) and several times kanamycin-resistant selection, eighteen regenerated tomato plantlets were obtained . One plantlet was ruled out by PCR , then 9 of 17 plantlets selected randomly were analyzed further by PCR-Southern blot and Southern blot, eight plantlets were confirmed that the VP7 gene had been integrated into plant genome by Southern hybridization. The result of RT-PCR of transgenic plants showed: in transcriptional level, VP7 gene was expressed in leaves and fruits of 4 transgenic tomato plants under control of CaMV35S promoter; VP7 gene was expressed in the tomato fruits not in the leaves of 3 transgenic tomato plants under control of fruit-specific TFP promoter.
One TO offspring (T1) was selected for further genetic analysis from two types of self-pollinated transgenic plants and screened for the presence of the recombinant gene by the PCR respectively. The result suggested that the resistance to kanamycin can be a stable heredity by genetic analysis, separation ratio of T1 was 3:1 and T1 kanamycin-resistant tomato plants can carry VP7 gene.
本研究分别以组成型启动子CaMV35S和番茄果实特异表达启动子TFP构建了VP7基因的两种植物表达载体pBIVP7和pEVP7。利用农杆菌介导法，转化番茄叶盘，经卡那霉素(Kan)多重筛选，共获得了18株抗性植株。PCR分析结果为阳性的植株有17株(转pBIVP7抗性植株10株，转pEVP7抗性植株7株)。随机选取生长良好的9株PCR检测阳性植株(5株转pBIVP7，4株转pEVP7)进行PCR-Southern blot和Southern blot分析，结果表明：有8株(5株转pBIVP7，3株转pEVP7)为转基因植株。RT-PCR分析证明，在组成型启动子CaMV35S控制下的5株转基因植株的叶片和果实中，有4株VP7基因在转录水平上表达；而在果实特异表达启动子TFP控制下的3株转基因植株，VP7基因仅在果实中获得转录水平上表达。此外，从两种转基因植株中分别选取一自交株进行T_1代遗传分析，其Kan抗性基因分离比皆为3:1，T_1代抗性番茄植株的PCR结果也呈阳性。
本研究为研究和开发新型轮状病毒口服疫苗提供一条新的途径和方法，具有一定的理论意义和应用前景。
Rotaviruses (RV) are the most common cause of severe diarrhea in children all over the world,VP7 is major outer capsid protein and is a primary candidate for inclusion in a subunit or recombinant. Advances in genetic engineering in the past decade have accelerated the expression, in plant, of foreign proteins with pharmaceutical value. Antigens from infectious bacterial or viral diseases have been introduced into plants. So introducing VP7 gene into tomato to develop edible plant vaccine of rotaviruses would change the traditional means of production of vaccines and the cost of vaccine production would be reduced greatly. It is provided with commercial meaning and applicative prospect.
In this study, two plant expression vectors pBIVP7 and pEVP7 were constructed. In pBIVPT, it is under control of CaMV35S promoter. In pEVP7, it is under control of tomato-fruit-specific TFP promoter. After co-cultivation with Agrobacterium Tumefaciens EHA105(containing pBIVP7 or pEVP7) and several times kanamycin-resistant selection, eighteen regenerated tomato plantlets were obtained . One plantlet was ruled out by PCR , then 9 of 17 plantlets selected randomly were analyzed further by PCR-Southern blot and Southern blot, eight plantlets were confirmed that the VP7 gene had been integrated into plant genome by Southern hybridization. The result of RT-PCR of transgenic plants showed: in transcriptional level, VP7 gene was expressed in leaves and fruits of 4 transgenic tomato plants under control of CaMV35S promoter; VP7 gene was expressed in the tomato fruits not in the leaves of 3 transgenic tomato plants under control of fruit-specific TFP promoter.
One TO offspring (T1) was selected for further genetic analysis from two types of self-pollinated transgenic plants and screened for the presence of the recombinant gene by the PCR respectively. The result suggested that the resistance to kanamycin can be a stable heredity by genetic analysis, separation ratio of T1 was 3:1 and T1 kanamycin-resistant tomato plants can carry VP7 gene.
引文
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