重组人脑源性神经营养因子在大肠杆菌中的可溶性表达及纯化

Soluble Expression in E.coli and Purification of Recombinant Human Brain Derived Neurotrophic Factor

按照人脑源性神经营养因子(hBDNF)基因成熟肽编码序列设计合成引物,从人基因组DNA中扩增出360bp的片段,插入到改构载体pTIG-trx上,获得了pTIG-trx-BDNF原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为hBDNF基因成熟肽编码序列。将该重组质粒转化大肠杆菌BL21穴DE3雪,经IPTG诱导表达,在大肠杆菌表达系统中获得了高效可溶表达,并对表达产物进行了分离纯化,得到纯度大于83%的样品,Western杂交证实该蛋白具有hBDNF抗原活性。

The primers specific for the mature human brain derived neurotrophic factor(hBDNF) coding sequence was designed and synthesized. The hBDNF coding sequence was directly amplified from human genomic DNA by using PCR and inserted into vector pTIG-trx to obtain the recombinant expression plasmid pTIG-trx-BDNF. The restriction enzyme analysis and DNA sequence detect ion confirmed that the inserted fragment of clone pTIG-trx-BDNF is the mature BDNF coding sequence. The recombinant DNA was transformed into the host cells BL21(DE3). The E.coli BL21(DE3) transformed with pTIG-trx-BDNF was induced with IPTG. The fusion protein effective soluble expressed had been purified. The purity of the production reached 83%. The result of Western hybridization showed that this fusion protein reacted specifically to the antibodies to hBDNF.