论文部分内容阅读
分别用两对引物PCR扩增BBTV cp、BBTV mp片段,混合各自PCR产物进行熔化退火可得到1/4两端含有Eco R玉和Bam H玉的PCR产物,分别克隆到酵母双杂交系统的p GBKT7诱饵载体中,并成功获得p GBKT7-cp和p GBKT7-mp。将重组质粒导入酵母Y2H菌株,转化成功后进行诱饵载体的自激活验证及毒性验证。结果表明,获得了正确的BBTV-cp和BBTV-mp基因片段,并成功克隆到p GBKT7诱饵载体中,同时在SD/-Trp营养缺陷平板上转化有诱饵载体的Y2H生长良好,说明诱饵载体的表达产物对酵母细胞无毒性,对报告基因也无自激活作用,最后自激活验证确定了p GBKT7-cp和p GBKT7-mp可用于该系统的互作蛋白筛选,为下一步利用酵母双杂交系统检测与CP、MP蛋白相互作用的寄主蛋白奠定基础。
PCR products of BBTV cp and BBTV mp were amplified by PCR using two pairs of primers respectively and the PCR products were mixed and annealed to obtain a PCR product containing Eco R and Bam H jade at both ends of the cDNA and cloned into the yeast two-hybrid system p GBKT7 decoy vector and pGBKT7-cp and pGBKT7-mp were successfully obtained. The recombinant plasmid was introduced into yeast Y2H strain, the transformation was successful bait vector self-activation verification and toxicity verification. The results showed that the correct BBTV-cp and BBTV-mp gene fragments were obtained and successfully cloned into the pGBKT7 bait vector while the Y2H transformed with the bait vector on the SD / -Trp auxotrophic plate grew well indicating that the bait vector The expression product was non-toxic to yeast cells and also had no self-activation effect on the reporter gene. Finally, pGBKT7-cp and pGBKT7-mp were identified as the interacting proteins of this system by self-activation verification. For the next step, yeast two-hybrid system Detection of CP, MP protein interaction of the host protein laid the foundation.