骨形态发生蛋白诱导基因克隆和表达的实验研究

An experimental study on cloning and expression of bone morphogenetic protein-induced gene-3 kbs

目的:用基因工程技术在大肠杆菌中表达骨形态发生蛋白诱导基因(BMP-2-inducedgene3kbgene,BIG-3)所编码的蛋白。方法:依据Genbank中BIG-3的基因序列设计并合成引物,从新生小鼠颅骨组织中提取总RNA,通过反转录聚合酶链式反应(RT-PCR)得到BIG-3全长编码序列。将所得到的基因片段克隆至原核表达载体pGEX-4T-2的多克隆位点获得pGEX-4T-2-BIG-3重组表达载体,经测序证实后转化大肠杆菌BL21(DE3)菌株,挑选阳性克隆,经诱导表达后SDS-PAGE鉴定。结果:克隆得到BIG-3全长编码序列并在大肠杆菌中表达了GST-BIG-3融合蛋白,融合蛋白约占菌体总蛋白的45.3%。结论:通过RT-PCR从新生小鼠颅骨中克隆到BIG-3基因并在大肠杆菌中获得GST-BIG-3融合蛋白的高水平诱导表达。

AIM:To express the protein encoded by bone morphogenetic protein(BMP) 2 induced gene 3 kb(BIG 3) by using the gene engineering techniques. METHODS:Two primers were designed and synthesized according to the BIG 3 sequence reported by Genbank.The BIG 3 was cloned by reverse transcription polymerase chain reaction(RT PCR) from the total RNA which was extracted from calvarial tissue of new born mouse,and then cloned into the multiple cloning site of expression vector pGEX 4T 2 to obtain the carrier of recombinant expression,pGEX 4T 2 BIG 3.E.coli BL21(DE3) strain was transformed after confirmed by sequencing for selecting the positive cloning.The positive cloning was evaluated by SDS PAGE analysis after being induced and expressed. RESULTS:The full length sequences of BIG 3 were obtained by cloning and,GST BIG 3 fusion protein was expressed in E.coli BL21(DE3) successfully.The GST BIG 3 fusion protein accounted for 45.3%of the total bacterial proteins. CONCLUSION:By using the RT PCR,the BIG 3 gene was cloned from the alvarial tissue of new born mouse successfully and gained a high expression of the GST BIG 3 fusion protein in E.coli.