Establishment Of The BSH Gen From Lactobacillus Plantarum In Prokaryotic And P.pastoris Expression System

Bile salt hydrolase is one kind of endocellular enzyme which is codogenic with the BSH gen.The BSH enzyme is the metabolic product generated during the growth and reproduction of microorganism.This enzyme is mainly generated from Bacterium which can switch hydrolysis conjunctive aminoethylsulfonic acid bile salt and aminoacetic acid,to amino acids and Lib cholalic acid. Absorption efficiency of De-conjuncted bile salt is lower than conjuncted bile salt,to result in copious De-conjuncted bile salt is discarded as dejecture.It needs more cholesterol to to make up the loss,so to lower the serum cholesterol.The prokaryotic and P.pastoris expression vector were constructed and expressed in E.coli and P.pastoris in order to research the expression of the BSH gens and prokaryotic expression system.The primers were designed according to the sequences of BSH gene in the GenBank and the multiple clone site of the sequence of pET-30a(+)vector.Extract the genome DNA of Lactobacillus plantarum.Amplify BSH gene from Lactobacillus by PCR and clone into expression vector pMD18-T,transform the constructed recombinant plasmid pMD18-BSH to E.coli DH5α′,Through PCR,double digesting,T4-ligase Iigating,blue-whites Potsereening,the fragment of BSH gen was subcloned inio the pET30a(+),expression vector so called pET30a(+)-BSH.The ideniified pET30a(+)-BSH was transfeeted into E.coli BL21(DE3) under induction of IPTG.Purify the expressed product by electroeluting and identify by SDS-PAGE and Western bloting;The BSH gene was amplified with the template of pMD18-BSH by PCR.The Primers were designed with the multiple clone site in the expression vector pGAPZαA and the BSH gene,The exactly ideniifled pMD18-BSH was digested with EcoRⅠ,XhoⅠ,then ligated with the digested pGAPZαA.The BSH fragment was subeloned into the pGAPZαA,expression vector so called pGAPZαA-BSH.Then pGAPZαA-BSH was transfected inio E.coli DH5α′.The results of PCR and enzyme digestion of plasmid proved that recombination vector was obtained so called pGAPZαA-BSH- SMD1168.After ligation of the plasmids with AvrⅡ,integrated into the genome of host yeast P.pastoris SMD1168 by electroporation,sereening the converter by ZeocinTM, The results of PCR of plasmid proved that recombination vector was obtained (named pGAPZαA-BSH-SMD1168),detected the Immunologic competence with SDS-PAGE and Western bloting.In this research,the BSH gen from Lactobacillus plantarum was cloned.There construeted Plasmid pET-30a-BSH was construeted suceessfully,SDS-PAGE analyzing indicated a molecular weight of 41kDa(with 6 his tag) protein was obtained at inducing 3 hours under induction of 1.0mmoL/L IPTG.Prokaryotic express system have been aequired,the expressed protein reached a purity of more than 92% after initial purification.The fused Protein was ideniified by westem blot with antigenicity.In addition,pGAPZαA–BSH was construeted as well and the BSH gene was successfully expressed in P.pastoris SMD1168.SDS-PAGE and Western bloting analysis showed that a high-level expression of 35kDa of architectural gene of BSH with immunologic competence,higher purity of the purified protein can be used for follow-up test.