【摘要】 目的:克隆小鼠TIM-3基因,构建原核表达载体,制备相应的多克隆抗体并初步鉴定。方法:以小鼠脾细胞总RNA为模板,用RT-PCR方法,扩增得到TIM-3基因编码区,构建pRSET-B-TIM-3原核表达载体;经IPTG诱导表达并纯化重组蛋白;然后常规免疫家兔,制备多克隆抗体;用ELISA方法检测抗体的效价,用Western blot、免疫组化、流式细胞术检测抗体的特异性。结果:成功构建的原核表达载体pRSET-B-TIM-3在大肠杆菌中诱导后可以高效表达TIM-3蛋白;免疫获得的多克隆抗体经过ELISA检测,抗体效价为1∶320 000,经Western blot、免疫组化和流式细胞术等鉴定,抗体的特异性较好。结论:成功克隆出小鼠的TIM-3基因,构建了原核表达载体,制备的兔抗小鼠TIM-3多克隆抗体具有较高的效价和良好的特异性。更多还原

【Abstract】 Objective:To construct a prokaryotic expression vector of mouse Tim-3 molecule and prepare the polyclonal antibody against mouse TIM-3.Methods:The extracellular TIM-3 gene was cloned from the spleen cells of BALB/c mice by RT-PCR and inserted into the vector pRSET-B in order to construct prokaryotic expression vector pRSET-B-TIM-3.Mouse TIM-3 protein was expressed by IPTG induction and purified by Ni-NTA column.Polyclonal antibody against TIM-3 protein was generated by immunizing rabbit with the routine method.The specificity and sensitivity of this antibody was confirmed by ELISA,Western blot,immunohistochemistry assay and FACS.Results:The fragment of 510 bp extracellular DNA sequence was obtained from the spleen cells of BALB/c mice by RT-PCR,and the prokaryotic expression vector pRSET-B-TIM-3 was constructed successfully.Induced by IPTG,the recombinant TIM-3 protein could be expressed effectively.After immunization of rabbit with the recombinant TIM-3 protein,the polyclonal antibody against mouse TIM-3 protein with a high affinity and specificity was obtained.Conclusion:The TIM-3 gene is cloned and the prokaryotic expression vector pRSET-BTIM-3 is constructed successfully.The polyclonal antibody with high affinity and specificity is generated successfully.It can be used to study the mouse TIM-3 effectively.更多还原