Cloning And Procaryotic Expression Of Gene Of Retinol-Binding Protein

Retinol-binding protein (RBP) is a carrier transporting vitamin A . It plays an important role in the distribution of vitamin A . To satisfy the need for cell differentiation ,vision maintenance , reproduction and individual maturity , vitamin A is transported to target cell in a highly specific and regulated manner by combination with RBP and transthyretin. The fluctuation of RBP in blood circulation or urine could be observed due to some diseases. So RBP is regarded as sensitive biomarker for the clinical diagnose of these diseases . it can also reveal the early change of nutrition state of protein.Concentration of RBP in body is low . Its half-life peried is much shorter . And it easily degraded . So it's difficult to isolate and purify RBP from human and animal plasma and urine of kidney patients. In order to study the structure-function of RBP, we produced RBP from Escherichia coli by constructing engineering strain which can express RBP . On the bases of designing a primer pair, we obtained the coding domain sequence of RBP by PCR from cDNA library .Then the gene was cloned into pGEM-T vector, The DNA sequencing showed that the cloned gene was in agreement with the reported sequence .Then the tageted gene of RBP was further subcloned into a procaryotic expression vector pBV220 and constructed recombinant plasmids was named pBV220-RBP. In order to expresse RBP in procaryotic cell efficiently, the recombinant plasmids was introduced into E.coliDHS a straints. Expression of RBP was induced by temperature induction . Production of RBP was identified by SDS -PAGE . Through these experiments we realized cloning of the gene of RBP and its expression in procaryotic cell. That have established the foundations for the later study of RBP.