hLMO3基因全长及截短序列原核表达载体的构建及蛋白诱导表达

Construction of prokaryotic plasmid of human LMO3 and deletions gene and identification of their recombinant proteins

目的构建hLMO3原核全长及5个截短表达载体并鉴定融合蛋白的表达。方法提取工具细胞HEK293的mRNA,反转录为cDNA,PCR扩增hLMO3全长及截短编码基因,亚克隆至GST融合表达载体pGEX-5X-2中。在E.coli BL21中诱导GST-hLMO3全长及截短融合蛋白表达,并经免疫印迹鉴定结果。结果 hLMO3全长及5个截短基因序列克隆到了原核表达载体pGEX-5X-2中,并经测序鉴定正确。Westernblot在E.coli Bl21中检测到了相应融合蛋白的表达。结论成功构建hLMO3全长及5个截短基因序列原核表达载体,在E.coli BL21中诱导表达出GST-LMO3全长及截短融合蛋白。

Objective To construct an prokaryotic expression vector of LMO3（LIM-domain only 3） full-length and deletion gene and identify their recombinant protein expressions induced by isopro-pyl-1-thio-b-Dgalactopyranoside（IPTG）.Methods Total RNA was extracted from HEK293,hLMO3 and deletion coding sequences were amplified by polymerase chain reaction（PCR）method and cloned into pGEX-5X-2.The expression of GST-hLMO3 fusion proteins was induced by IPTG and identified by Western blot.Results The coding sequence of hLMO3 full-length and deletion gene were cloned into the pGEX-5X-2 which was transformed into BL21 successfuly,and identified by double enzymes digestion and sequencing.The expression of GST-hLMO3 fusion proteins induced by IPTG was observed in Bl21.Conclusion The recombinant prokaryotic plasmids were constructed successfully into pGEX-5X-2 and the expression of GST-LMO3 full-length and deletion fusion proteins was identified.