猪传染性胃肠炎病毒纤突糖蛋白在转基因玉米中的表达

Expression of Spike Glycoprotein Gene of Porcine Transmissible Gastroenteritis Virus in Transgenic Maize

本研究拟构建携带猪传染性胃肠炎病毒纤突糖蛋白基因(TGEV-S)的转基因玉米植株并检测其表达情况。首先利用PCR方法自携带TGEV-S基因的重组质粒中扩增2.2kb的S基因片段,然后插入植物表达载体pBAC176中,该表达载体以编码除草剂草甘膦抗性的EPSP基因作为选择标记,目的基因以双增强子的CaMV35S(E35S)及其玉米Hsp70第一内含子为驱动。以授粉后10～12d约0.5～1mm大小的玉米幼胚为受体,用基因枪进行转化,经过愈伤组织诱导、草甘膦抗性筛选和分化再生培养,先后获得74株转化再生植株。利用PCR和Southern blot检测表明,T0代转基因苗有9株检测结果为阳性。T1代转基因玉米株系经PCR检测有14个转基因株系为阳性,初步确定了目的基因在这些转基因玉米中的稳定整合。进而通过间接ELISA分析初步确定目的基因在7个T1代转基因玉米株系获得了表达。研究结果为进一步研究表达TGEV-S转基因玉米的生物学活性奠定了基础。

The purpose of this study is constructing and measuring the expression of spike glycoprotein of porcine transmissible gastroenteritis virus in transgenic maize plant. To obtain transgenic maize expressing gene the spike glycoprotein of porcine transmissible gastroenteritis virus （TGEV-S）, the 2.2 kb TGEV-S gene fragment was successfully amplified from TGEV-S carrying recombinant plasmid by PCR and it was inserted into the expression vector pBAC176. The recombinant plasmid pBAC176 uses the glyphosate-resistant gene EPSP as the selective marker and E35S was used as the promoter for the target gene. The pBAC176 was transformed into immature embryos of maize by biolistic bombardment transformation. Following callus induction, glyphosate resistance selection and plant regeneration, 74 plants oftransgenic maize were obtained. TGEV-S gene was tested positive by PCR and Southern blot in 9 of the To generation transgenic plants. Further detection in T1 Transgenic maize showed that 14 transgenic lines were positive by PCR. The results showed preliminarily that the target gene displayed stable integration into the transgenic maize plants. The expression TGEV-S genes were detected in 7 lines of transgenic maize plants of T1 generation by indirect ELISA using the specific anti-TGEV-S antibody. The results from this study will provide basis for further studies on the immunogenicity of TGEV-S protein from transgenic maize, and its possibility as oral immunization.