人类CD252基因片段在大肠杆菌中的表达、纯化及免疫原性分析

Expression and Purification of Human CD252 Gene and Analysis of Immunogenicity of Recombinant Protein

目的优化表达含人类CD252胞外段的融合蛋白,纯化目的蛋白,为单克隆抗体的制备打下良好的基础。方法将测序正确的pET32a(+)-CD252胞外段重组质粒转入表达菌株BL21感受态细菌中,用SDS-PAGE电泳鉴定重组菌的诱导表达情况,通过改变pET32a(+)-CD252阳性菌株的诱导时间、温度,以优化出最佳表达条件。采用包涵体纯化法纯化目的蛋白。用纯化的重组蛋白免疫BalB/C小鼠,采用ELISA法测定血清中抗体效价。结果成功获得表达pET32a(+)-CD252的阳性菌株。转化有pET32a(+)-CD252胞外段重组质粒的表达菌经IPTG诱导后,在蛋白分子量标准的31.0～42.7kD之间出现了新的蛋白条带,新的蛋白主要存在于包涵体中。优化表达条件分析表明,在诱导4h、温度37 ℃、IPTG浓度为1.0mmol/L时,融合蛋白的表达量最高。包涵体洗涤纯化后,经SDS-PAGE电泳证明可得到较纯的目的蛋白。免疫小鼠后测得的平均抗体效价为1:3200。结论pET32a(+)-CD252胞外段原核表达载体能够表达CD252蛋白,并获取了目的蛋白的优化表达条件,表达蛋白具有良好的免疫原性。

Objective To optimize the expression of the fusion protein with the human CD252 extracellular segment,purify the interest protein and prepare the monoclonal antibody of human CD252. Construct a solid basis for further studying. Methods The pET32a-CD252 recombinant plasmid sequenced correctly was transformed into a kind of strain which express BL21 （ DE3 ）. The recombinant protein was induced by IPTG and identified by SDS-PAGE. In order to offer better parameters,the positive clone＇s expression condition was optimized by adjusting the time and temperature of the induction. Purifying the interest protein using inclusion body depuration and SDS-PAGE. The BAlB/c mouse was immuued by the purified recombination protein. The titer of polyclonal antibody was identified by ELISA. Results The positive stain that expressed the pET32a-CD252 was obtained successfully. After induced by IPTG,SDS-PAGE showed that the product of interest protein and the new protein existed in inclusion body of bacterial. SDS-PAGE showed that fusion protein expressed highly when induced with 1.0mmol/L IPTG for 4 hours at the temperature of 37℃. After purifying the interest protein with inclusion body,the purified interest protein was obtained. The titer of polyclonal antibody was 1 ： 3200 by indirect ELISA. Conclusion The CD252 protein can be expressed by prokaryotic expression vector of human CD252 extracellular gene,and the good condition of fusion protein was obtained. Furthermore,the expression protein posses excellent immunogenicity.