Ficolin-A的克隆、蛋白表达和抗体的制备与鉴定

Molecular cloning,protein expression and preparation of polyclonal antibody of ficolin-A

目的:克隆小鼠ficolin-A(mouse ficolin-A)基因,构建在真核及原核表达载体,并在大肠杆菌中表达和鉴定其蛋白,并制备其抗体,以进一步用于研究小鼠ficolin-A的功能。方法:用RT-PCR的方法从新生7 d的C57BL/6小鼠肝脏中运用GeneRacer kit扩增ficolin-A cDNA片段,并将该片段分别插入pVAX-1真核表达载体及pGEX-KG原核表达载体中,实现插入基因的融合,在IPTG的诱导下在大肠杆菌中表达。用GST-Sepharose 4B的柱子对表达的融合蛋白进行纯化,用SDS-PAGE和Western blot对表达产物进行鉴定。制备ficolin-A的多克隆抗体并对其效价进行测定。结果:成功地构建了pVAX-1-ficolin-A真核表达载体及pGEX-KG-ficolin-A原核表达载体,并在大肠杆菌中获得高效的表达,表达产物的相对分子质量(Mr)同预期值相一致。并且成功制备了多克隆抗体。结论:成功地构建了重组表达载体pVAX-1-ficolin-A及pGEX-KG-ficolin-A,并在E.coliBL21中表达了ficolin-A蛋白,为下一步研究小鼠ficolin-A的功能奠定了基础。

AIM： To clone mouse ficolin-A cDNA, constutruct eukaryotic and prokaryotic expression vevtors and prepare polyclonal antibody of ficolin-A. METHODS： Full length of the ficolin-A cDNA fragment from the liver of newly born C57BL/6 mouse was amplified by RT-PCR by the use of GeneRacer kit. Then it was cloned into the eukaryotic vector pVAX-1 and prokaryotic vector pGEX-KG and expressed as a fusion protein in E. coli BL21 induced by IPTG. The expressed GST-ficolin-A fusion protein was purified via GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot. The antibody against ficolin-A was prepared and its titer was identified. RESULTS： The recombinant eukaryotic vector pVAX-1-ficolin-A and prokaryotic expression vector pGEX-KG-ficolin-A were successfully constructed. The recombinant GST-ficolin-A protein was overexpressed in E. coli. The relative molecular mass （Mt ） of the expressed product was identical with the predicted value. The antibody was prepared successfully. CONCLUSION： We have successfully got the constructions of the recombinant eukaryotic vector pVAX-1-ficolin-A, prokaryotic expression vector pGEX-KG-ficolin-A and the purified recombinant ficolin-A protein in E. coil BL.21. These will be helpful for the further investigation of the biological function of ficolin-A.