E.coli expression system non-glycosylated scFv have certain advantages, but the expression of the characteristics of the production depends on the scFv. Have been reported in E. coli expression system to express several different types of scFv, including monovalent and multivalent scFv, scDbs, taFvs scFv-immunotoxin conjugate and multivalent scFv complexes, such as the trimer, tetravalent mini antibodies. The exogenous gene expression is a complex process, including the interaction between the gene transcription, translation, post-translational processing, cell metabolism and cell gene and protein, protein and protein. Into the post-genome era, E. coli preferred model for a new topic of research proteomics, gene function, protein networks, reveals the unknown areas of the gene expression for the development of E. coli expression system provides channels. With E. coli expression system constantly-depth study of the mechanism of gene expression and influencing factors will be found, would be more perfect E. coli expression system
In general, the affinity of the antibody capacity and the screened positive correlation only in the high capacity of the antibody library screening to high-affinity antibodies. Ribosome display technology as a new antibody library technology, its greatest superiority is to enable the storage capacity of 1013 ~ 1015, this is because it remove the transformation and cloning steps, completely in vitro, without relying on cells conversion and amplification, thus making the display library of larger capacity, more efficient screening, but also save time and effort, is an entirely in vitro expression and screening of functional proteins. In this study, the improvement of the two fragments overlap extension PCR method efficiently the antibody heavy chain and light chain are randomly connected together, also will serve as the intergenic regions CK gene fragments with 3 'end of the single-chain antibody gene, and then another to set up a pair of primers ribosome display in vitro expression of the T7 promoter and ribosome binding sites (Kozak sequence) into the connection good antibody gene fragments, so that was successfully constructed ribosome display the required template. The end of the spacer region to remove the stop codon after the completion of such protein to form the ribosome - the protein-mRNA complexes, which can be very convenient in vitro screening.
E.coli expression system for the expression of the different active rAbs exist different expression site, including secreted into the extracellular periplasmic space, the outer membrane or the internal and cytoplasm. Use of a promoter such as lac, trp and tac induce antibody expression, these promoters can be lac obstructions regulation and isopropyl-β-galactosidase (IPTG) induction. Cytoplasmic reduced case, the expression can be obtained the high concentrations of antibody, but is easy to accumulate the formation of inclusion bodies, denaturing reagent therefore need to be processed, and then refolding. The refolding efficiency of the different antibodies is also different, especially Fragment scFv. Gou, etc. The method of using nickel chelate chromatography column refolding from inclusion bodies, the successful refolding concentration is greater than 40 mg / L having the activity of IP10-scFv fusion protein. Addition of scFv expression in the pelB leader sequence regulation and under oxidizing conditions, secreted into the periplasm or E. coliα-hemolysin (hyla) system secreted into the medium supernatant. HylA is E. coli outer membrane is formed between the type I protein secretion channel hemolytic toxin secreted by the protein channel. This system has been proved to have secretion, including scFv, including exogenous protein capacity.
The bacterial expression in a bacterial expression system, E. coli (E. coli) is the most widely used, the genetic background is relatively clear, having a safe use, simple operation, substantial expression of the exogenous gene expression system. E. coli expression system to express the non-glycosylated antibody molecules including scFvs, including. Compared with the mammalian cell lines, E. coli expression system is more rapid and easy access to express the antibody, and therefore suitable for large-scale screening and antibody production. Further, using E. coli expression system can obtain a high concentration of polyclonal antibody (RAB), and have reported that the expression level of up to 2 g / L. However, E. coli are not suitable for the expression of glycosylated protein, so to express glycosylated proteins, such as scFv-Fc fusion proteins, the need to select the other expression systems. E. coli expression system for the expression of the different active rAbs exist different expression site, including secreted into the extracellular periplasmic space, the outer membrane or the internal and cytoplasm. Use of a promoter such as lac, trp and tac induce antibody expression, these promoters can be lac obstructions regulation and isopropyl-β-galactosidase (IPTG) induction.
Insect expression system uses one or more baculovirus promoter, exogenous target gene inserted into the recombinant virus promoter, and can copy, while the expression of multiple foreign genes in insect cells or insect body. Insect expression system to express higher levels of glycosylated proteins is to be considered, despite the complete protein glycosylation, insect cells and mammalian cell expression systems have the same N-glycosylation sites, but the mode of action and mammalian different oligosaccharide chains are essentially different. This may be due to the insect cells do not have the processing capacity of mammalian cells to mature oligosaccharides. In addition, different precursor protein cleavage site is also very important, although the mammalian secretion signal in insect cells, precision machining, but with a signal peptide sequence may not be able to secrete into the protein. Upstream with the melittin signal peptide of scFv expressed in insect cells, only the insoluble intracellular precursor protein processing defects of the precursor protein molecules because the baculovirus expression system; the B. subtilis signal peptides and scFv of expression in insect cell lysates to detect less than unprocessed precursor protein secretion also increased.
The single-chain antibody (single-chain variable fragment, scFv) by an appropriate period of oligonucleotides (Linker) antibody light chain and heavy chain variable region gene at the DNA level will be linking in vivo performance of a singlepeptide chain, with the minimum structural unit of antibody activity, and connected with a variety of effector molecules to construct the ideal components of the new function of antibody molecules, is one of the hotspots in the field of genetically engineered antibodies. Today, scFv gradual replacement of the monoclonal antibody (mAb) to become a valuable molecules for the treatment and detection of diseases such as cancer, inflammation, autoimmune diseases, hepatitis B and AIDS, biological pathogens, send
Health insect and microbial contamination prevention and detection has great application prospects. Kim et al. Exceed the superparamagnetic iron oxide nanoparticles (SPI-ONs) and scFv composed of abf-SPIONs, the treatment of carcinoembryonic anti-(CEA) has a potential role. Shanta and other applications for the amyloid precursor protein (APP) of scFv to inhibit the activity of β-secretase enzyme of Alzheimer's disease cells to solve the difficulties of the mAb in the treatment process. scFv expression in different expression systems, including the prokaryotic expression system (such as E. coli, Bacillus subtilis) and eukaryotic expression systems (such as Saccharomyces cerevisiae, Pichia pastoris, insect cells, plant cells and animal cells). Affect scFv expression yield and activity of many factors, such as the size of the scFv was, solubility, stability and amino acid sequence. Therefore, according to the requirements of different types of scFv expression and the purity and quantity of the final product required in the expression of scFv to choose the best expression system
Construction of human liver cancer natural sink capacity, diversity good ribosome display single-chain antibody library, to lay a good experimental basis for the development of therapeutic human antibodies. Methods: The collection of 40 cases of fresh human peripheral blood 2mL (including healthy adults and patients with hepatocellular carcinoma), lymphocyte separation and total RNA was extracted by the design of appropriate optimization of primers by RT-PCR amplification of human antibody heavy chain variable region gene (variable region of heavy chain, VH) and light chain variable region gene (variable region of light chain VL) and intergenic regions of the light chain constant region gene (consistregion of the light the chain, CK). Adoption of improved overlap extension PCR (SOE PCR) technology The VH and VL spliced connection peptide Linker (Gly4Ser) 3. After the introduction of building a ribosome display single-chain antibody (scFv) library template the necessary components, including a T7 promoter, a ribosome binding site (Kozak sequence) and the translation initiation codon, as well as the intergenic regions of the CK gene. Template gene fragments Connection T-vector was transformed into E.coli DH5a E. coli colonies identified by PCR analysis and sequencing, constructed of single-chain antibody library. Results: ① Construction of the storage capacity of 2.65 × 1013 human hepatocarcinoma single chain antibody library; ② sequence of positive recombinants by colony PCR and sequencing analysis, we found that the antibody library has a rich diversity. Conclusion: The use of improved overlap extension PCR (SOE-PCR) method, building a high storage capacity, diversity, good human-derived liver cancer ribosome display single-chain antibody library, improve the efficiency of the library building. Antibody library was constructed successfully, and lay a solid experimental basis for the follow-up screening antibodies, antibody modification. At present, liver cancer is second only to gastric cancer, the third most common malignant tumor of the esophagus, the initial symptoms are not obvious, its high relapse rate after treatment is one of the worst prognosis of malignant tumors. Therefore, we constructed ribosome display single-chain antibody library derived from liver cancer patients and normal peripheral blood lymphocytes of the heavy chain, light chain variable region gene, and identification for the further screening of hepatocellular carcinoma-specific antigen single-chain The antibody has laid the experimental basis for the further development of anticancer drugs such as immunotoxins, immunoliposomes, as well as tumor vaccine development have very significant practical significance.