SPACR原核表达载体的构建和表达

Construction of prokaryotic expression vector and expression of SPACR

目的构建Sialoprotein associated with cones and rods(SPACR)原核表达载体并高效表达。方法根据基因文库中SPACR基因的cDNA序列设计引物,采用PCR方法扩增SPACR cDNA片段,并克隆进原核表达载体pGEX-6P-1中,转化于大肠杆菌BL21,测序鉴定后,用IPTG诱导表达出目的蛋白,经谷胱甘肽琼脂糖柱柱层析纯化,收获表达产物进行SDS-PAGE电泳分析和Western blot鉴定。结果 PCR后扩增得到的目的片段,经酶切鉴定及DNA序列测定,证实重组载体pGEX-6P-1-SPACR构建正确,表达蛋白分子量约为分别为21.2、22.6、36.2及20.8kDa。结论在大肠杆菌中获得了SPACR片段的高效表达,为研究其蛋白功能奠定了基础。

Objective To construct the prokaryotic expression vector of sialoprotein associated with cones and rods（SPACR） and induce its expression and purification.Methods Primers were designed by chicken SPACR cDNA in Genbank,cDNA segments were amplified by PCR,then ligated into pGEX6p-1 vector,and transformed into E.coli BL21 cells.After sequenced and identified,fusion proteins were induced,expressed and purified,the products were identified by SDS-PAGE and Western blot analysis.Results The objective fragments were obtained,the custom crafted expression vectors were confirmed by sequencing analysis that the sequence were coincided with the Genebank,fusion expressd proteins were about 21.2、22.6、36.2 and 20.8kDa in size.Conclusion The prokaryotic expression vectors were constructed successfully,and the SPACR protein fragments were obtained,which build a basis for research of SPACR biological function.