Screening Of Binding-protein Of RON From Phage Library Baseed On 10Fn3 Scaffold

BackgroundMacrophage stimulating protein（MSP） recepteur d,origine naetais（RON） is the receptor tyrosine kinase, a members of the family of tyrosine kinase receptor（RTKs）, usually is expressed in the epithelial cells, mononuclear macrophage, granulocyte, lungs, bone marrow and other normal cells.Study shows that the abnormal expression of RON is relevant to malignant transformation of a variety of cell, invasion of the tumor.So inhibiting abnormal expression of RON has important clinical significance in the treatment of cancer.With the development of Cell biology,Some reserch have prepared monoclone antibodies of against RON. However, the shortcoming such as the big quality of rat monoclonal antibody molecules making it too difficult to work into the organization and the source of rat easily producing immune rejection limits its clinical application.Along with the rapid development of phage display technology, the preparation of humanized antibody using the technology not only overcomes the drawback of different source of rat monoclonal antibody, but also retained its the advantage of strong homogeneity, strong specificity.Although the development of phage antibody library provided a new way for the preparation of humanized anti-platelet antibody, these antibodies preparated by the technology have the big molecular and the relatively complex structure, sometimes they must be modificated（glycosylation, joining the disulfide bond, etc.） to activate their functions.Therefore, it has become a new method for the preparation of new antibodies that using small molecule protein based on the skeleton protein similar the function with antibody constructs phage random peptide library, then gets high affinity small molecular peptides combined with target molecules by phage display technology.There are many skeleton protein in nature, its secondary structure has important protein interaction activity sites, such as cyclic（loop） structure, the α-helix or β- fold, etc. the binding sites of protein with the structure skeleton exposed outside and are similar function with complementary decision domain antibody, making the skeleton protein has the possibility to construct random peptide library.The accounts of 10 FN3（The tenth human fibronectin type III domain） domains proteins are higer to be in about 2% of all human proteins and it is found in the bacteriophage. The tenth 10FN3 domain in human fibronectin was first proposed in 1998 as a scaffold to construct random peptide, because it combines several molecular features favorable for therapeutic use. First, comparable to those of other scaffolds,10FN3 domains have immunoglobulin（Ig）-like structure domains and exist a β-fold with noncontiguous loops that allow a large binding interface in the N terminal and C terminal. Moreover, the hydrophobic core of the Ig-like fold provides a stable framework structure, providing an unusually high thermostability to 10FN3（Tm₈8℃）. Importantly, compare with variable domains of Igs, as a monomer, lacks the post-translational modifications of stable cysteine residues, high-level expression in bacteria, 10FN3 is stable.In this experiment, we selected the 10 Fn3 as the supporter, and randomized amino acids of the two loops BC and FG loop structures.Then we builded restrictive random peptide library based on the changes, and screened the high affinity small molecule polypeptide combined with RON by phage display technology. ObjectiveConstructed restrictive random polypeptide library by phage display technology and selected the high affinity protein with RON receptor tyrosine kinase extracellular region from the phage random polypeptide library and cells combined with. Mathods1. Fishing the gene of RON extracellular region from breast cancer cells MDA–MB-231 by RT-PCR technology.2. Building the RON extracellular cloning vector and expression vector.3. Adopting the method of transient transfection, transfected expression vector into the 293 t cells, then harvested the supernatant and purified the protein expression by RON extracellular gene.4. By overlap extension PCR, gained genes of phage random ploypeptide library based on skeleton 10Fn3 protein, and then connected with expression vector p Cantab5 E by enzyme digestion site Sfi and ⅠNot Ⅰ.5. Electrotransformated recombination plasmid into E. coli XL- Blue, established primary phage random ploypeptide library.The bacterium solution was directly prepared for PCR amplification,positive clone to be sequenced in company,respectively authenticated the injection rate and the diversity of the phage random polypeptide library.6. By packaging diffrent antigen concentration（10 mu g/ml, 5 mu g/ml, 2.5 mu g/ml, 1.25 mu g/ml, 0.63 mu g/ml, 0.31 mu g/ml and 50 mu g/ml, 25 mu g/ml, 12.5 mu g/ml, 6.25 mu g/ml, 3.13 mu g/ml, 1.56 mu g/ml）,we compared the enrichment degree impaction of antigen package concentration for the result of screening,.Then we used diffrent washing environment（ 1 x, 4 x, 8 x, 12 x, 16 x, 20 x PBST and 1 x, 2 x, 4 x,10 x, 8 x, 14 x PBST） to compare the affect of harshly washing enrichmen for screening results.7. Screening high affinity protein molecules combind with RON extracellular region by ELISA. Results1. We have successfully constructed eukaryotic expression vector of RON extracellular region.2. We have successfully purified the protein of antigen RON extracellular region.3. We have successfully builded phage random polypeptide library that the database is 1.92 x 107, and successfully screened high affinity protein molecules with RON extracellular region.4. By compared the screening condition in this experiment, we reached a decision that the influence of washing conditions on the enrichmen degree is impotent than the affect of antigen packaging concentration. Conclusion1. Builded phage random polypeptide library which database is 1.92 x 107, established a phage random polypeptide library technology platform, Selected the protein molecules combined with RON,which lay the foundation for the screening of small molecule protein.2. In this experiment, through the comparison of the influence on the enrichment efficiency of antigen package concentration with the washing conditions, we draw a conclution that in the process of screening small molecular combined with RON, influence of PBST washing concentration that is the influence of washing conditions is stricter than RON antigen package concentration.