Construction, Expression And Characterization Of Recombinant Escherichia Coli Heat-labile Enterotoxin, It's Mutants And It's B Subunit

Heat-labile enterotoixin(LT), a hexmeric protein produced by EnterotoxigenicEshchirichia(ETEC), is a causative reagent of diarrhea in humans and farm animals.Besides potent toxicity, LT possesses strong immunogenicity and can assist otherantigens delivered through mucosal pathway to produce specific antibodies, thus, LT,especially the non-toxic or low toxic mutant, is a good mucosal immunization adjuvantand has significant medical and economic values in mucosal immunization research anddevelopment of vaccine for mucosal immunization. Composed of an enzymaticallyactive A subunit and five identical B subunits that constitute the binding portion, LT is acomplicate oligomer and mutation of amino acid residue in different sites may diverselyaffect the three dimensional structure and function. Thus, by researching the mutants ofLT, not only candidate mucosal immunization adjuvants could be screened out but alsoour knowledge about structure and function relationship of protein could be broadened. Objective: To obtain efficiently expression systems for recombinant LT and LTBproducing on pilot scale. To find optimal method for purifying LT. To find optimalmethod for storing LT. Preliminarily screen out LT mutant(s) with lower toxicity andhigher mucosal immunization adjuvanticity. Theoretically explore the effect of mutationof amino acid(s) on the structure and function relationship of LT. Methods: Modified genome extraction method was used to obtain gene encodingheat-labile enterotoxin from wild type ETEC. Techniques of genetic engineering wereemployed to recombine gene encoding LT into expression vectors and then transformrecombinant plasmids into the host bacteria. Method that comparing the production rateof recombinant LT or LTB expressed in different expression systems grown in shakingflask was used to screen out the optimal expression systems. Methods that changing thepH and component of equilibration buffer and elution buffer for immobilizedD(+)-galactose affinity chromatography column were applied to optimize thepurification strategy. The optimal storage method of LT was determined by comparingthe stabilities of LTs stored under different conditions and the stabilities were denotedby A280nm recorded during LTs elution in chromatography column in HPLC. Thesite-directed mutagenesis was used to construct LT mutants. LT and LT mutants wereapplied to chromatography column in HPLC and eluted by buffer with 1%SDS. A280nmrecorded during elution were used to judge the differences of stabilities among LT and III重庆大学博士学位论文LT mutants. The amounts of LTA1 fragments from LT and LT mutants nicked by trypsinwere used to judge the trypsin sensitivity. The toxicities were estimated by the dosagesand degrees causing cell distortion from CHO toxicity assay and by the amounts of fluidaccumulation in intestine of mice from patent-mouse in vivo toxicity assay. Theadjuvanticities of LT mutants were appraised as follows: BALB/c mice were orallyimmunized by B subunit of Helicobacter pylori(Hp) urease(UREB) with LT mutants asadjuvants. ELISA was used to assay UREB specific antibodies of sera and mucosal;ELISPOT was used to observe the antibody secreting cell in Pyer's patch; RT-PCR wasused to estimate the expression of cytokines. Compare the adjuvanticities of LT mutantswith other Hp vaccine. Results: 1, Plasmid carrying lt gene was obtained from wild type ETEC by using modified genome extraction method. 2, Recombinant LT was efficiently expressed in E.coli BL21(DE3) carrying recombinant plasmid pET11c-LT grown in modified M9-CAA culture medium. The production rate is about 46mg/L culture medium. 3, Purification immediately followed by freeze-drying, LT powder stored in 4℃ may maintain the intact structure of hexamer for long term storage. 4, LTK63(S->K), LTR72(A->R) and LTKR(S63K and A72R) were construc...