人层粘连蛋白α4链LG4-5组件的原核表达及多克隆抗体制备

Prokaryotic expression and polyclonal antiserum preparation of human laminin alpha4 LG4-5 module

目的利用基因工程技术克隆、表达人层粘连蛋白α4链LG4-5组件(hum an lam in in alpha4 LG4-5 modu le,hLNα4LG4-5)蛋白并制备其特异性多克隆抗体。方法采用RT-PCR方法从人胎盘组织扩增hLNα4LG4-5的cDNA片段,T/A克隆法将其插入pMD-18T载体进行测序。采用DNA重组技术构建原核表达载体pET-28 a-LG4-5,用BL21(DE3)/pET系统表达hLNα4LG4-5融合蛋白,以SDS-PAGE鉴定所表达的融合蛋白。用含融合蛋白的聚丙烯酰胺凝胶颗粒免疫家兔,制备抗hLNα4LG4-5多克隆抗体,W estern印迹法检测抗体的特异性。结果成功克隆了hLNα4LG4-5的cDNA片段,12%SDS-PAGE电泳检测显示,经IPTG诱导后BL21(DE3)/pET28 a-LG4-5总蛋白中出现一条分子质量为42×103的新蛋白带,制备了效价为1∶10 240的hLNα4LG4-5多克隆抗体,W estern印迹分析证实可检测到特异性目的蛋白带。结论克隆并原核表达了hLNα4LG4-5蛋白,制备了特异性抗hLNα4LG4-5的多克隆抗体,为进一步研究LG组件在疾病发生中所起的作用打下了基础。

Objective To clone and express human laminin alpha4 LG4-5 module（ hLNα4LG4-5 ） protein by gene engineering techniques, and to prepare specific polyclonal antibody against hLNα4LG4-5. Methods The cDNA encoding hLNα4LG4-5 was amplified by RT-PCR, then inserted into pMD-18T vector by T/A cloning and sequenced. Prokaryotic expression vector pET -28 a -LG4 -5 was constructed by recombinant DNA tech- nique. The hLNα4LG4-5 fusion protein was expressed in BL21 （DE3）/pET system and identified by SDSPAGE. Rabbits were immunized with the polyacrylamide gel particles containing the fusion protein for polyclonal antibody preparation and the sera were detected for antibody specificity by Western blotting. Results The cDNA fragment of hLNα4LG4-5 was cloned successfully. While BL21 （DE3）/pET28a-LG4-5 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 42 000 was shown on SDSPAGE profile. After rabbits were immunized, the high titer （ 1：10 240） polyclonal antiserum was isolated. The protein band of hLNα4LG3 -4 module expressed by BL21 （ DE3 ） was detected specifically by Western blotting in which the polyclonal antiserum of hLNα4LG4-5 was used as the first antibody. Conclusion The hLNα4LG4-5 protein was cloned and expressed successfully, its specific polyclonal antibody was prepared.