L-ficolin突变体原核表达载体的构建及表达产物的鉴定

Construction of Prokaryotic Expression Vector L-ficolin Mutant and Identification of Expressed Protein

目的构建L-ficolin突变体的原核表达载体，为研究L-ficolin的糖结合位点奠定基础。方法设计L-ficolin突变体基因上、下游引物，分别引入EcoRⅠ和XhoⅠ酶切位点。以插入L—ficolin M2（Val144Phe）和M6（Glu 307Asp）点突变的完整编码区基因的质粒pCDNA3-L-ficolin为模板，通过PCR扩增获得突变体的基因片段，将其克隆入原核表达载体pGEX—KG．然后通过酶切和序列测定对其进行鉴定；将此表达载体转化入表达菌BL21（DE3）pLvSs诱导表达。并采用SDS—PAGE对表达产物进行鉴定。结果经酶切及序列分析表明．成功地构建了重组原核表达载体pGEX-KG—L—ficolin—M2、pGEX-KG-L-ficolin—M6，SDS—PAGE显示目的蛋白大量表达。结论L—ficolin突变体融合蛋白可以在大肠杆菌中大量表达。为研究L-ficolin的糖结合位点打下基础。

Object To construct a recombinant prokaryotic plasmid of L-ficolin mutant that can express L-ficolin mutant GST-protein. Method The L-ficolin mutant gene derived from the plasmid pCDNA3-L-ficolin by polymerase chain reaction （PCR） was cloned into the prokaryotic expression vector pGEX-KG. The recombinant expression plasmids were transformed into BL21 （DE3） pLySs and expressed. The expression products were analyzed by SDS-PAGE. Result L-ficolin mutant gene was rightly inserted into the vector by restruction enzyme analysis and sequence analysis. The proteins were expressed at high level respectively according to the SDSPAGE results. Conclusinon： The prokaryotic expression vector with L-ficolin mutant gene was efficiently expressed in BL21 （DE3） pLySs bacteria. sites. The result would contribute to further studies of L-ficolin Sugar binding sites.