人视黄醇结合蛋白原核蛋白表达和兔多克隆抗体的制备

Prokaryotic expression of human retinol binding protein and preparation of rabbit polyclonal antibody

目的制备兔抗人视黄醇结合蛋白(RBP)多克隆抗体。方法逆转录PCR扩增人RBP基因,将扩增产物连接到原核表达载体pET-28a(+)上,构建重组质粒pET-28a(+)-RBP,转化至大肠杆菌中。用异丙基-β-D-硫代半乳糖苷(IPTG)诱导其表达,表达产物经SDS-PAGE分析鉴定出阳性克隆,扩大培养。用组蛋白标签(His-tag)纯化柱纯化重组蛋白。将纯化的重组蛋白皮下注射到新西兰白兔, 4次加强免疫得到抗血清。从抗血清中纯化出抗RBP多克隆抗体,并进行SDS-PAGE、 ELISA和Western blot法鉴定。结果成功构建了重组质粒pET-28a(+)-RBP,并获得高纯度的RBP重组蛋白, ELISA检测结果显示抗体效价达到1∶512 000。结论成功制备兔抗人RBP多克隆抗体。

Objective To produce rabbit polyclonal antibodies against human retinol-binding protein(RBP). MethodsRBP cDNA was amplified by reverse transcription polymerase chain reaction(RT-PCR) and then the amplified products were inserted into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-RBP. The established plasmid was then transformed into E. coli. Isopropylthio-β-D-thiogalactoside(IPTG) was used to induce the expression of recombinant protein His-RBP in E. coli. The expression products were identified by SDS-PAGE from different clones of E. coli to screen positive bacteria, followed by amplifying culture. His-RBP protein was purified from the expression products of positive clones. The purified recombinant His-RBP was used to immunize New Zealand white rabbits. Antisera were acquired after four times of booster immunization. The prepared purified polyclonal antibodies were identified by SDS-PAGE, ELISA and Western blotting. Results We successfully constructed the recombinant plasmid pET-28a(+)-RBP, and acquired recombinant protein His-RBP of high purity. ELISA showed that the antibody titer reached 1∶512 000. Conclusion The rabbit polyclonal antibodies against human RBP have been successfully prepared.