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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。
OBJECTIVE: To clone mouse ficolin-A (ficolin-A) gene and construct it in eukaryotic and prokaryotic expression plasmids and to express and identify its protein in Escherichia coli and to prepare its antibody for further study on ficolin- A function. Methods: The cDNA of ficolin-A was amplified by RT-PCR from the liver of 7-day-old C57BL / 6 mice with GeneRacer kit and inserted into pVAX-1 eukaryotic expression vector and pGEX-KG prokaryotic expression vector In the vector, the fusion of the inserted gene is achieved and expressed in E. coli under the induction of IPTG. The expressed fusion protein was purified by GST-Sepharose 4B column, and the expressed product was identified by SDS-PAGE and Western blot. Polyclonal antibodies against ficolin-A were prepared and their potency determined. Results: The eukaryotic expression vector pVAX-1-ficolin-A and the prokaryotic expression vector pGEX-KG-ficolin-A were successfully constructed and expressed in Escherichia coli with high efficiency. The relative molecular mass (Mr) The values are consistent. Polyclonal antibodies were successfully prepared. CONCLUSION: The recombinant plasmids pVAX-1-ficolin-A and pGEX-KG-ficolin-A were successfully constructed and the ficolin-A protein was expressed in E.coli BL21, which provided the basis for further studies on the function of mouse ficolin-A The foundation.