论文部分内容阅读
采用基因重租技术,将野生型p16 cDNA通过中间载体pVL1392最后载入表达载体pGEX-5T,IPTG诱导重组质粒转化的大肠杆菌,蛋白质印迹证实42×10~3的融合蛋白GST-P16的表达。表达了GST-P16的重组菌经加热破菌后行SDS-PAGE,将GST-P16蛋白条带切下后经反复冻融于皮内多点注射免疫家兔。所收获的兔血清经蛋白质印迹法检测到抗P16抗体滴度为1:625。结果表明通过pGEX-5T融合蛋白表达系统能方便地提供制备抗P16抗血清的免疫原,该免疫原未经纯化并未影响抗P16抗血清的产生。
Recombinant plasmid pGEX-5T was transfected into wild type p16 cDNA using pVEX-5T. The expression of 42 × 10-3 fusion protein GST-P16 was confirmed by western blot. Recombinant bacteria expressing GST-P16 were broken by heating and SDS-PAGE. The GST-P16 protein band was excised and the rabbits were immunized by repeated freezing and thawing. The harvested rabbit serum was tested for anti-P16 antibody titer 1: 625 by Western blotting. The results show that it is convenient to provide immunogens for preparing anti-P16 antisera through the pGEX-5T fusion protein expression system without purification and without affecting the production of anti-P16 antisera.