Construction Of Immune Antibody Phage Display Library And Generation Of Human TAT-Fab Antibody

Objective:Tetanus, which is caused by clostridium tetani infected the wound, is an acute infectious disease. It is primarily caused by exotoxin of clostridium tetani that combines with nerve tissue. At present, the unique effective therapy is to inject tetanus anti-toxin (TAT) or human tetanus immunoglobulin (TIG) into patient to neutralize the exotoxin. But the clinical application of TAT and TIG are limited because anaphylaxis is caused by TAT, blood resource is inadequate, and pathogen contamination is caused by TIG. In recent years, the development of phage antibody library technology makes it possible to prepare the human antibody. This kind of antibody can avoid the side effects of TAT or TIG. The objective of this thesis is to generate the human tetanus-anti-toxoin antibody using the immune antibody phage display library technology. At the present study, the genes of human Fab antibody were obtained from individual immunized with tetanus toxoid, and human Fab antibody phage display libary was constructed. The phages that displayed the human Fab antibody fragment to tetanus toxoid (the human TAT-Fab antibody) were selected by reaction specifically to tetanus toxoin. The soluble human TAT-Fab antibody was expressed in Escherichia coli. Furthermore, the animal experiment indicated that the soluble human TAT -Fab antibody could neutralize extoxoin Methods1. PBMC were isolated from individual immunized with tetanus toxoid. The total RNA was extracted from PBMC and transcribed reversely into cDNA. The genes of human Ig light chains (κ and λ chains) and heavy chains Fd (γ chains) were amplified by PCR.2. The vector pComb3 was extracted and purified from XL1-Blue containing phagemid pComb3.3. The purified pComb3 and the mixed genes of light chains were digested by XbaIand Sad, and then the digested products were ligated by T4 DNA ligase. The products of ligation were transfected into XL 1-Blue by electroporation. So the genes library of human Ig-light-chains (pComb3-L) was constructed. The genes library was identified using digestion of Xbal and Xhol.4. The purified pComb3-L and the mixed genes of heavy chains Fd were digested by Xhol and Spel, and then the digested products were ligated by T4 DNA ligase. The products of ligation were transfected into XL 1-Blue by electroporation. So the genes library of human Fab antibody (pComb3-HL) was constructed.5. The human Fab antibody phage display library was constructed, after the pComb3 containing genes library of human Fab antibody and help phage(VCSM13) were co-transfected into bacteria. The phages that displayed the human TAT-Fab antibody were selected by reaction specifically to tetanus toxoid, enriched by "adsorption-elution-enrichment", and identified.6. The recombinant phagemid containing the genes of the human TAT-Fab antibody was digested by Spel and Nhel to cut the phage GUI protein between heavy chain and light chain, and then the products of digestion were ligated by T4 DNA ligase again. The products of ligation were transfected into XL 1-Blue by electroporation. So the engineering bacteria expressing the soluble human TAT-Fab antibody were obtained7. The soluble human TAT-Fab antibody was expressed in XL 1-Blue and was demonstrated by SDS-PAGE and Western blot. The affinity of the soluble human TAT-Fab antibody to tetanus toxin was identified by ELISA.8. The soluble human TAT-Fab antibody was purified by protein affinity column which connected with the anti-human IgG Fab antibody. The biological activity of the human TAT-Fab antibody was identified by the animal experiment.Results1. Construction and identification of the genes library of human lg-tight-chainsPCR reactions were performed using 3 pairs of human Ig k chain primers and 3 pairs of human Ig X chain primers. The results showed that the specific bands(650bp) were obtained at different annealing temperature (54"C, 55°C, 56*C, 58"C) . The light chains genes (k and X chains) were subcloned into the vector pComb3, and the genes library of human Ig-light-chains CpComb3-L) was constructed. The size of the genes library was about 7.3 xlO5. The results by digestion indicated that this genes library contained human Ig-light-chains2. Construction and identification of the genes library of human Fab antibody The heavy chains Fd genes (700bp) were got by PCR reactions using 6 pairs of primer for human Ig y chain Fd at the annealing temperature of 58"C. The heavy chains Fd genes were subcloned into the vector pComb3-L, and the genes library of human antibody Fab was constructed. The size of the library was about 5*104. The results by restriction enzymes digestion indicated that this genes library contained human Ig heavy chains3. Selection and enrichment of the specific phages displayed the human TAT-Fab antibodyThe human Fab antibody phage display library was constructed after the recombinant pComb3 containing human Fab antibody and help phage vVCSM13) were co-transfected into bacteria. The tite of phage was 1.69x10" pfu/ml. After five times selection of "adsorption-elution-enrichment", the tite of the specific phages that displayed the human TAT-Fab antibody were 1.3xlO6 pfu/ml. The affinity of phages diaplaying human TAT-Fab antibody was detedcted by ELISA. One of 5 positive clones with highest expression, OD49o=1.68 was chosen to study further. The result from sequencing and comparation to the GenBank demonstrated that the genes encoding TAT-Fab antibody belong to k type light chain and they3 heavy chain.4. Expression and identification of the soluble human TAT-Fab antibody in XLl-BlueThe engineering bacteria containing the soluble human TAT-Fab antibody were constructed by above-mentioned clone. The soluble human TAT-Fab antibody was expressed in XLl-Blue and was detected by SDS-PAGE and Western blot. To enhance the yield of antibody expression, the cultural parametera of bacteriawere optimized. It demonstrated that highst expression of TAT-Fab was got after OD600=1.0 of the density of the engineering bacteria was induced by O.lmmolA of IPTG at 37"C for 14h. Under this condition the yield of antibody in bacterial soma was about 0.15IU/ml. and that in supernatant of bacteria cultures was O.lOIU/ml. The results from SDS-PAGE and western blot showed that the molecular weight of antibody was 50KD under non-reducing condition and 25KD under reducing condition5. Purification of the soluble human TAT-Fab antibody and identification of its biological activityThe soluble human TAT-Fab was purified by protein affinity column which connected with the anti-human IgG Fab antibody. It was showed that the purified human TAT-Fab antibody could neutralize partly exotoxin in the animal experiment. Conclusion and significance1. The immunonized human antibody phage display library to tetanus toxoid had been successfully set up2. The specific phages displaying human TAT-Fab antibody fragment were obtained by selection and enrichment3. A prokaryotic expression system expressing the soluble human TAT-Fab antibody had been established and the soluble human TAT-Fab antibody was generated in bacteria XL 1-Blue4. It had been demonstrated that soluble human TAT-Fab antibody purified by protein affinity column could neutralize partly exotoxin in the animal experiment.5. A new soluble human TAT-Fab expression system was established first time in China. This will lay solid foundation for production of soluble human TAT-Fab antibody by engineering method and application of it on treatment of tetanous disease in future.