【作者】 刘满杏；

【导师】 刘荣堂； 李霞；

【作者基本信息】 甘肃农业大学 ， 野生动植物保护与利用， 2010， 硕士

【摘要】 本实验利用胡萝卜作为生物反应器受体生产轮状病毒外壳蛋白VP6,VP7。通过PCR,RT-PCR,Western Blot,ELISA检测整合有轮状病毒外源基因的胡萝卜植株,即从基因水平到蛋白水平逐步检测。并将蛋白水平上表达有轮状病毒外壳基因的胡萝卜植株免疫小鼠,免疫方法采用注射免疫和灌胃免疫。1提取胡萝卜DNA进行PCR分析,表明VP6,VP7,VP7-U基因都已整合到胡萝卜植株基因组中。2转基因植株的RT-PCR分析显示部分植株进行了反转录。3对转录水平上有表达的转基因植株进行Western Blot和ELISA分析,结果表明转基因胡萝卜植株可以有效地表达VP7蛋白,并具有一定的免疫原性。4利用转VP7基因胡萝卜根部蛋白注射免疫小鼠,抗原免疫量为1μg左右,免疫周期为两周,免疫三次后采血并提取小鼠血清进行ELISA分析,结果表明注射阳性胡萝卜植株蛋白的小鼠体内IgG含量要高于注射阴性胡萝卜植株蛋白的小鼠及未免疫的小鼠。利用转VP7-U基因胡萝卜根部蛋白灌胃免疫小鼠,抗原量为20-30μg,免疫周期为一周,免疫四次后采血提取血清并进行ELISA检测,结果表明灌服VP7-U基因的胡萝卜植株蛋白的小鼠IgG含量要高于灌服阴性植株的小鼠或未灌服小鼠的IgG的含量。5同时将转VP7-U基因的植株抗原量与VP7基因植株抗原量进行比较,ELISA分析表明,转VP7-U基因的植株抗原量要高于VP7基因植株抗原量。更多还原

【Abstract】 Carrot was applied in the experiment as receptor of bioreactor to produce RV protein VP6, VP7. And transgenic carrots in different generations were screened with the methods of PCR,RT-PCR,Western Blot,ELISA. The transgenic carrots expressing RV protein were applied to immune mice.1 The PCR analysis showed that VP6,VP7,VP7-U gene had integrated to carrot genome.2 The result of RT-PCR demonstrated that only some transgenic carrots had reverse transcriptase procedure.3 Western Blot and ELISA showed that VP7 protein had been expressed effectively in transgenic carrots and the product had a certain immunogenicity.4 The root protein of transgenic carrot with VP7 gene was applied to immune mice through injection together with freund’s adjuvant and the dosage of antigen was about 1μg. The immunization period was two weeks and mice blood was got to extract serum after three times immunization. The ELISA analysis for serum showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non. The root protein of transgenic carrot with VP7-U gene was extracted to immune mice through filling stomach after analysises of PCR, RT-PCR,Western Blot,ELISA,and the antigen dosage was 20μg to 30μg. The immunization period was one week and mice blood was got to extract serum after four times immunization for ELISA analysis. The ELISA result showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non.5 Comparing with the carrots transformed VP7 gene, the antigen content in carrots transformed VP7-U gene was higher.更多还原