Expression And Enzyme Activity Analysis Of Esterase B1 With Binary Promoters Vectors

Pesticides as the primary support of prevention and control plant diseasesand elimination of pests has contribute to the increasing of agricultureproduction. But the following problems of the remain pollution has harmedseriously the environment and people's health. In order to solve it , muchresearch has been studied, this thesis is aimed at expressing the detoxificase(esterase B1)at high level .It will pave the way for utilization of esterase B1in reality. The choice of an expression system for the high-level production ofrecombinant proteins depends on many factors.Altogether two factors areclassified:Transcriptional regulation(promoter and transcriptional terminatorsare concluded)and Translational regulation(SD sequences and mRNAstability). This thesis is aimed at inreasing the expression of the detoxificase( esterase B1 ) by transcriptional regulation i.e. construction vectorscontaining two promoters.CaMV35s promoter was inserted in the vectorpRL439,so successfully constructing pRLdp which containing PpsbApromoter and CaMV35s promoter was obtained.Then the DNA fragment ofesterase B1 gene was cloned to MCS of plasmid vector pRLdp,The process was as follows :first,the plasmid pUC-B1 and pRLdp weredigested with restriction endonuclease EcoRI respectively.Then retrieved andpurified the 1.33kb fragment of pUC-B1 and 3.5kb fragment ofpRLdp through UNIQ-10 Column DNA gel Extraction Kit. After the purifiedexpression vector fragment was dephosphorylated , linking up the twofragments with T4 DNA ligase in vitro . After transferring the newrecombinant plasmid pRLdp-B1 into the host cell of E.coli DH5a, the positiveclones were screened on LB medium plate containing 100μg/mL Amp. And 3双启动子表达载体的构建及酯酶 B1 基因的表达then the recombinant plasmid was identified with restrictionendonuclease XbaI and BamHI. The engineering strain E.coli DH5a/pRLdp-B1 was aspepsis inoculatedin LB medium containing 50μg/mL Amp at 37℃,200rpm ,by enzyme activityanalysis of different inoculated mete,2% is best.In order to prove theexpression of esterase,2% inoculation was choosed, the engineering strainE.coli DH5a/pRLdp-B1 was inoculated at 37℃,200rpm for 15 hour,thencentrifuged,and the bacterial pellet with distilled liquor was sonicated timeafter time and centrifuged at 10000rpm for 15 minutes at 4℃.The solubleliquor was made to SDS-PAGE,it confirms that the objective protein wascorrectly expressed,and the amount of the expression was higher than thecontrol. The results of detoxifing experiment indicated that the esterase proteinexpressed by the engineering bacterial of E.coli DH5a/pRLdp-B1 exhibitshigh detoxificase activity in degrading the specific substrate such asa-naphthyl acetate(a-NA).Compare to the control,the muzzle velocity of newengineering bacterial is quicker and longer.