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目的 :制备转基因作物中选择标记基因潮霉素B磷酸转移酶 (hygromycinBphosphotransferase,hpt)基因表达产物的多克隆抗体 ,并探讨影响核酸免疫效果的因素及相应机制。方法 :以hpt的cDNA全长插入真核表达载体 pCDNA3中 ,并经限制性内切酶酶切及DNA测序鉴定。以纯化的重组质粒pCDNA3 HPT免疫BALB/c小鼠。用在E .coli中表达并纯化的 (His) 6 HPT进行ELISA ,检测免疫过程中小鼠血清抗体效价的增长状况 ,并用Westernblot检测抗血清的特异性。结果 :经 3次核酸免疫后 ,小鼠血清中未发现明显的抗HPT抗体。第 4次加强免疫时 ,将小鼠分为 3组 ,每组两只。第 1组改用去内毒素的质粒提取试剂盒提纯的重组质粒免疫 ,第2组用 (His) 6 HPT融合蛋白免疫 ,第 3组仍用原来提取的重组质粒免疫。结果发现 ,第 1组小鼠抗血清的效价有所上升 ,经ELISA检测达 1∶2 0 0 ;第 2组抗血清的滴度显著升高 ,达到 1∶2 0 0 0 ;而第 3组小鼠血清中仍未检测到明显的抗体。Westernblot证实 ,前两组抗血清均可与亲和层析纯化的GST HPT、(His) 6 HPT融合蛋白及其诱导表达的相应菌体总蛋白发生特异性结合。结论 :用DNA免疫法成功地制备了抗hpt基因表达产物的特异性抗体 ,但抗体效价不够理想。推测与hpt基因本身的性质及其在体内表达呈现的水?
OBJECTIVE: To prepare polyclonal antibodies against hygromycin Bphosphotransferase (hpt) gene in transgenic crops and to explore the factors that affect the effect of nucleic acid immunization and the related mechanisms. Methods: The full length cDNA of hpt was inserted into eukaryotic expression vector pCDNA3 and identified by restriction endonuclease digestion and DNA sequencing. BALB / c mice were immunized with the purified recombinant plasmid pCDNA3 HPT. ELISA was performed with (His) 6 HPT expressed and purified in E.coli to detect the growth of mouse serum antibody titer during the immunization, and the specificity of the antisera was detected by Western blot. Results: After three times of nucleic acid immunization, no significant anti-HPT antibodies were found in the serum of mice. At the 4th booster immunization, mice were divided into 3 groups of two. Group 1 was immunized with recombinant plasmids purified by endotoxin plasmid extraction kit. Group 2 was immunized with (His) 6 HPT fusion protein and group 3 was still immunized with the original recombinant plasmid. The results showed that the titers of antisera in group 1 increased slightly and reached 1: 200 by ELISA. The titers of antisera in group 2 increased significantly to 1: 200, while those in group 3 No significant antibody was detected in the serum of mice. Westernblot confirmed that both of the antiserums of the two groups could specifically bind to the purified total GST HPT, (His) 6 HPT fusion protein and the corresponding bacterial total proteins induced by affinity chromatography. Conclusion: The specific antibody against hpt gene expression product was successfully prepared by DNA immunization, but the antibody titer was not ideal. Speculate the nature of the hpt gene itself and its expression in the body of water?