Study On Transformation And Application Of Human Anti-HBs Fab Expression System

Chimeric antibodies and humanized antibodies produced by recombinant genetic engineering techniques have also become available for use in preclinical and clinical studies. Human antibodies are the necessary style in clinical application in the future, eliminated murine components, which refrain from eliciting HAMA or other complications when used in human patients. Fully human antibodies are rapidly becoming the norm, and which are low immunogenicity and retain their specificity for antigen and biologic properties is important to allow application. A number of technologies exist which enable the development of 100% human antibodies.Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al[1]. Generally,utilized various phage display vectors and gene fusion techniques, along with PCR amplification of gene for Ab subunits, allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunization.The bottleneck of preparation of human antibody is that obtained effective expression and production with higher activity. As a continuation of our earlier study, pComb3 expression vector have a higher amount the background expression of Fab genes that interfered with cell growth could not be completely prevented, and Fab expression from PBAD can be more tightly repressed than that of Plac.[2]We describe a new strategy for preparation anti-HBs Fab based on changing expression vector to subclone anti-HBs Fab genes with pBAD/gIII vector. The most important characteristics of this system are that it can be used to (i)achieve very low levels of uninduced expression,(ii).obtain moderately high levels of expression in the presence of inducer, (iii).modulate expression over a wide range of inducer concentrations, and(iv).expression from this system was tightly regulated[3].On the other hand, culture condition and the induction of expression have profound effects on the way the recombinant protein purification. The optimal salt and/or imidazole concentrations for the washes will vary slightly for various proteins and must be determined.The research work comprise following two parts:Part 1.Construction of prokaryotic vector expressing the anti-HBs Fab in periplasmic spaceUsing pComb3/anti-HBsFab[4] plasmid as template, the Fd amd Lc DNAs were amplified in the presence of the corresponding 3'-specific primers of Fd and Lc genes by polymerase chain reaction(PCR) techniques[5]. Fd PCR products were combined and digested with Xho I and Bgl II ,and Lc PCR were products combined digested with pst I and Xba I to yield two populations of digested antibody DNAs for gel purification. These fragments were cloned into the pBAD/gIII vector separately. Following bacterial transformation, the two single chain combinatorial libraries were identified with digestion. Then the DNAs that includes Lc DNA, araBAD promoter and leader peptide segment were amplified by PCR. After purification, performing digestion with EcoR I and Xba I and cloning them into Fd-pBAD vector, constructed a double promoter transcribed directional secretion of Fd, Lc peptides to periplasmic space of Top 10 E.coli.Analysis of restriction enzyme digestion of recombinant double cloned vector confirmed the correct Fab gene clone. Single clones were picked from the resulting transformation, grown in 100ml of LB broth with ampicillin (100μg/L) to an OD600 of 1.0, and induced with 0.02% arabinose for 6hrs at 37 ℃.Human HBs Fab fragments from arabinose-induced 5ml culture supernatants were concentrated and separated in a 12% SDS-PAGE. The proteins were transferred to nitrocellulose, block, incubated with HRP-conjugated goat anti-human Fab. The molecular weight of the purified protein was about 50Kd and the protein showed well bioactivity with western blot.The new constructed anti-HBs Fab expression system show better and economic Fab productio...