| Glutathione peroxidase(GPX) is an important member of antioxidative enzyme systems in vivo, and is a kind of protein containing selenium. GPX can protect organisms from the damage coming from active oxygen species through scavenging hydrogen peroxide and other hydroperoxidase. GPX has strong antioxidant ability and it is important for treating and preventing Keshan disease, angiocardiopathy, inflammation and cancer. GPX deficiency is related to many kinds of diseases. Due to the limitations associated with native GPX, such as instability, limited availability, big molecular weight and immunogenicity, many scientists have made a great deal of efforts to study the GPX mimics.The key of GPX mimics is the GSH specific binding site, it is necessary to obtain the high activity mimics.Due to the lack of the GSH binding site, the early GPX mimics have generally rather low activities. The development of abzyme preparation technology made a new way for the mimics of enzmes. Abzyme, also is called catalytic antibody, is a kind of immunoglobulin with catalytic activity. And it unifies the high selectivity and amazing diversity as antibodies and highly catalytic activities as enzmes. It can bind any molecule because of its diversity as antibodies. Due to the characteristic of antibodies and antigens'binding, we can obtain the specific mimics binding with the target antigen.The research of the antibody has experienced three phases: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies.With the developing of molecular biology, antibody technology has a succession from cell engineered antibody to genetically engineered antibody. But due to the big molecular weight, the whole antibody is not suitable for pharmaceutical development. It has some drawbacks for use as drugs, such as the big molecular size led to the difficulty for transport in organism and penetration into cells. In recent years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-lives in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte.Phage antibody library technology includes, amplify the full set variable region gene of antibody by PCR, connect the DNA fragments of antibody molecule with phage coat protein pâ…¢or pâ…§gene, and then let the fusion protein be expressed on phage surface. This technology connects genotype with phenotype in one single virus particle and unifies the ability which can identify antigen and phage's amplification. Phage display technology has already become one of the key techniques of antibody engineering. To obtain specific antibody against any antigens without cell fusion and immunity by screening from phage display antibody library of human .These antibodies can be used for human directly, because of no immunogenicity and anaphylaxis.At the present time, there are many expression system of antibody. For example: bacterium, yeast, cell of insect, cell of plant and mammalian cell.Every expression system has many virtues and drawbacks. Due to no use to decorate expression protein of scFv, we can select the expression syetem of Ecoli which has many virtues, such as : the simpleness of gene operating, the low cost, the speedy breed.Escherichia coli is the most widely used host for high-level production of single-chain Fv fragment. There are two expression forms of the scFv expression in E.coli. The scFvs are expressed in cytoplasm and periplasm. However, in the cytoplasm proteins are expressed in biologically inactive and aggregated forms or inclusion bodies. Although refolding procedures exist for obtaining proteins in soluble form, these are lengthy processes, often inefficient and not generalizable. We performed soluble and functional expression of the scfvs in E.coli.We have carried out these works:1.The single chain fragments of antibodies (scFvs) against GSH were screened from phage display antibody library using GSH-S-DNP as target antigen by phage display technique. Specific antibody is enriched and screened by three times' combining-eluting-amplifying. These scFvs are expressed on phage surface. The positive antibodies were identified by ELISA.2.The phagemid of positive clone was extracted .We have applied PCR method to amplify the target fragments using the common primers of the antibody library from the phagemid , which contain the gene of anti-GSH single chain antibody, screened from the phage display antibody library of human. The products of PCR were cleaved with the two enzymes of NcoI and NotI to acquire the target gene fragments, then the target gene fragments were linked to the plasmid vector pPelB cleaved with the same two enzymes. The recombinant plasmids were transformed to E.coli.Rosetta, and further induced soluble expression by IPTG under proper conditions. Harvested bacterial bodies to perform SDS-PAGE and Western blot.Conclusion1. After three times' combining-eluting-amplifying, the phage antibodies were screened and enriched. The phage antibodies identified by ELISA. Fourty randomly selected clones were tested for the presence of anti-GSH scFvs,7 clones showed positive.2. The fragment (about 750bp) of target gene was released from the products of PCR by the two enzymes digestion. The recombinant plasmids were transformed to E.coli, sequenced the plasmids of the positive bacterium, the result indicated that the coding sequence of VL,VH,linker and leader were all correct. The target gene fragment of about 750bp was released by the two enzymes digestion, which could suggest that there was no mutation in the processes of the construction of vector and the transformation of E.coli by recombinant plasmids. SDS-PAGE and Western blot were performed on the positive bacterial bodies induced to express, the result showed that the positive bacterium expressed the anti-GSH scFv, whose molecular weight is about 31kD. We successfully expressed the anti-GSH scFv in E.coli, and laid a foundation for the following studies .
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