Construction Of Ribosome Display Human ScFv Library And Analysis Of Its Limitation On Selection Of ScFv Antibodies To Human LBP

With deep knowledge of gene structure and function of antibodies, researchers could produce antibodies with novel biological characteristics by gene engineering technology. And single chain variable fragments(scFv) are the most extensively studied antibodies because of their low molecular weight, high penetration of tissues and convenience in construction and expression. Phage display and ribosome display technology can be used for selection of high affinity scFv molecules, while phage display technology is the most widely used and mature system for panning of antibodies by gene engineering. Phage display technology is a high efficient, simple and quick method by which the phenotype and genotype of antibodies can be linked together by display of antibody proteins fused with N-terminal of coat protein gpⅢor gpⅧon the surface of phage particles, and after incubation of antibody library with solid-phase or liquid-phase immobilized antigen, specific clones can be enriched with several cycles of affinity binding-elution-amplification. Ribosome display is a relatively new in vitro technology platform for antibody isolation and evolution, which provide a linkage between the genotype and phenotype as mRNA-ribosome-antibody complexes utilizing in vitro expression system. Since ribosome display has the following advantages over phage display, it is becoming a potential technology platform for antibody research and production. Firstly, all the steps can be done in vitro; secondly, the antibody library is larger than phage display antibody library; thirdly, the procedures for construction of ribosome display antibody library and selection are easier and quicker than that of phage display and the affinity can be improved by introduction of mutations and evolution of antibody genes.Bacterial lipopolysaccharide(LPS)is the major component of endoxin, which is a significant factor for death in severe burn, trauma, and endotoxemia, sepsis, infectious shock and multiple organ dysfunction syndrome(MODS). Despite of progress of clinical therapy techniques in recent years,the death toll remains high due to the difficulties in treatment with complex pathophysiologic mechanisms. LBP(lipopolysaccharide binding protein) is a lipid transfer molecule catalyzing movement of LPS monomers from LPS aggregates to membrane CD14(mCD14),soluble CD14(sCD14) and phospholipids. It was reported that LBP can promote formation of LPS-LBP-CD14 complex and transduce the LPS signaling to activate monocyte/macrophage cells by mediating the interaction of LPS and CD14 on the surface of monocyte/macrophage cells, and finally promote development of inflammation. As LBP is the key molecular in activation of monocytes, utilization of specific, low toxicity, high affinity and stability antibodies to LBP by gene engineering aiming to block LBP/CD14-mediated binding of LPS to cells may be an effective strategy for the control of LPS-mediated pathophysiologic disorders.In this research, ribosome display and phage display technology were applied to select anti-LBP scFv antibodies, and a further modification work was planned to change the single chain Fv(scFv) into disulfied stabilized Fv(dsFv), which is more stable than scFv. The study on its antagonistic effect of LBP-LPS signaling induced augmentation of inflammation will provide useful information and enlightenment for endotoxin-related fundamental and application research.The main results and conclusions are as follows:1. White blood cells suspension of a healthy donor were collected and centrifuged to isolate mononuclear cells. And total RNA of mononuclear cells were isolated and reverse transcribed to construct scFv library by PCR and overlap extension PCR amplification. A second overlap extension PCR amplification was used to add T7 promoter, stem loop structure, ribosome binding sites to the 5- end and 6×His tag, spacer(geneⅢ), stem loop structure to the 3- end of scFv DNA sequence. In this way a large na?ve human scFv library containing >1013 members for ribosome display was successfully constructed, which provided a useful platform for selection of antibodies with high affinity and specificity.2.Microtiter plate was coated at 4℃overnight with recombinant LBP solution (10μg/ml in CBS,pH 9.0) for selection of anti-LBP scFv antibodies using ribosome display technology, but no positive bands were observed in the first round of RT-PCR to recover the specific scFv clones; in phage display experiments, 384 clones were picked after three cycles of biopanning, however,further identification by phage ELISA demonstrated non-specificity and weak binding to the plate coated with LBP. There were several possible reasons for the negative results of the bio-panning of ribosome display antibody.⑴The major possible reason was due to the inherent disadvantages of ribosome display technology. The insufficiency of transcription of scFv antibodies and the instability of antibody-ribosome-mRNA complex, the small amount of mRNA which was extremely prone to degradation by contaminating ribonucleases made it difficult for the recovery of the scFv mRNA, which may cause negative results, and no definite bands were observed in the agarose electrophoresis of RT-PCR products. Another possible reason may be the relative low affinity in the na?ve scFv antibody libraries which add to the difficulties for antibody isolation.⑵As to the phage display antibody technology, the XL1-Blue or TG1 need to be cultured and propagated to express the plasmid vector and help envelope of phage particles. And it was inevitable for the LPS to bind to the LBP which caused changes of epitope of LBP, so in phage ELISA the displayed scFv recognized LBP-LPS complex showed non-specificity and low affinity to LBP, which may bring about difficulties in selection for phage display antibody to epitope of LBP.⑶And the antigen concentration in biopanning for both ribosome display and phage display is lower than that of previous reports, which might add to the difficulties to successfully selection of needed antibody clones. Despite its preliminary character, the study can clearly indicate that both ribosome display and phage display antibody platform has limitations in selection for antibodies toward various antigens under different circumstances. The laboratory conditions and the researcher may also influence the results. Ribosome display and phage display technology are two efficient methods for antibody selection, but there exist some limitations or disadvantages and caution should be taken to consider the conditions of laboratory and antigen characteristics and avoid false positive or negative results in selecting antibodies with these technologies.3. Prokaryotic expression plasmids pET28a-NHLBP was constructed and transformed into E coli. BL21 cells and induced by IPTG for expression. The expressed product was analyzed by 12% SDS-PAGE and the antigenicity of the purified recombinant pET28a-NHLBP protein was identified by Westernblot test. It is feasible to produce antigen of N-terminal fragment of human LBP which could take place of purchased LBP protein in the beginning round of selection for anti-LBP scFv antibodies.