The Screening Of The Single Chain Antibody Variable Of Protein TP53’s Mutation At Locus R175H

As a kind of tumor suppressor protein,P53 protein mainly regulates cell division by controlling cell cycle and DNA replication during tumor growth.It will lose its function of inhibiting cancer and become a cancer-promoting factor to promote the occurrence and development of tumor when P53 protein mutates or aggregates.It was found that more than50% of the cancer tissues had mutants of P53 protein,and in most cancers,P53 mutants were found at 175,245,248,249,273 and 282 amino acid positions in the detection of cancer tissue samples in patients.Mutations of P53 mutants R248 Q,R248W and R175 H show aggregation in a variety of tumor tissues,forming stable protein aggregates that activate other gene expression programs and promote carcinogenesis and drug resistance.Unlike WT-P53,which is rapidly degraded by the ubiquitin-proteasome system,P53 mutant proteins,such as P53.R175 H,are highly stable and tend to form higher-order aggregates.Targeting P53 mutant protein has become a potential therapeutic opportunity for cancer in view of the widespread existence of P53 mutant protein in human cancer and its key role in disease progression.In fact,molecular targeted therapy mainly depends on drugs or other substances targeting specific molecules(molecular targets)to block the growth and spread of cancer cells.The discovery of ideal targets is very important for the successful development of molecular targeted therapy for cancer.One of the bases of cancer occurrence is determined by changes in genetic characteristics,which lead to mutations or changes in proteins and receptors.In addition,it promotes cell survival and proliferation.These specific genetic changes that distinguish cancer cells from normal cells can be used as molecular targets in the development of molecular targeted drugs.Destroying the specific mechanism by which cancer cells develop for survival and growth is a reasonable way to selectively kill cancer cells with minimal impact on normal cells.Mutant P53 is one of the best treatable targets because more than half of human cancers have P53 mutations in this regard.However,most normal cells do not have mutations in the P53 gene.Taking advantage of the above characteristics,cancer targeted therapy strategies for P53 mutants are also produced.Targeting depletion of P53 mutant proteins can be used as one of the potential strategies with the least impact on wild-type P53 proteins in vivo.Recombinant monoclonal antibodies have high specificity and affinity to cell surface or intracellular antigens.As a consequence,they have a wide range of clinical applications in the diagnosis and treatment of diseases.Although its targeting effect is significant,it limits the role of recombinant monoclonal antibodies in the treatment of human diseases because of its wide range of effects.The emergence of scFv overcomes these shortcomings.A small molecular weight scFv consisting mainly of antibody heavy chain and light chain variable regions connected by junctions(containing 15 to 20 amino acids)that maintain the specific antigen binding characteristics of natural antibodies.The scFv has the characteristics of improved tissue permeability,faster blood clearance,significantly reduced kidney uptake and less activated monoclonal antibody-mediated immune response because of its smaller molecular weight and volume compared with the complete monoclonal antibody.These properties greatly promote pharmacokinetics and drug delivery system and reduce immunogenicity.ScFv can be easily produced in a variety of hosts,including plants,yeast and microbial expression systems,especially E.coli.It can be produced by conventional hybridoma technology,but the level of antibody affinity obtained from this method is usually not enough to be effectively used in clinic.As a consequence,screening large combinatorial antibody libraries by using yeast,bacteriophage,ribosome and m RNA provides a powerful platform for effectively enriching specific and high affinity clones.OBJECTS:The mutant protein at P53.R175 H site and the protein expressed by P53 wild type were designed and synthesized.Besides,the scFv with high affinity and specificity against P53.R175 H site mutation was screened out through the constructed human phage scFv library with 1X1013 capacity.It provided a new idea for the diagnosis and treatment of related tumors.METHODS:The designed sequence entrusted a third party to synthesize the mutated antigen protein at P53.R175 H site and the antigen protein expressed by P53 wild type.Taking this antigen as the target antigen,scFv with high affinity against P53.R175 H site mutation was screened by multiple liquid phase screening.In addition,these scFv did not bind to wild-type P53 protein and had high specificity.The selected scFv was verified by ELISA,and the affinity and specificity of the scFv were identified.RESULTS: P53 wild type protein and mutant protein at P53.R175 H site were synthesized.Taking these two proteins as target antigens,scFv with high affinity to P53.R175 H mutant protein was successfully screened by multi-round liquid phase screening,and these scFv showed low affinity with P53 wild type protein.Moreover,the selected scFv was digested with restriction endonuclease and sequenced to determine whether it has high homology with the light and heavy chain variable region sequence of human IgG antibody.Conclusion:The mutant antigen protein at P53.R175 H site and P53 wild type antigen protein were synthesized successfully.In addition,the scFv against the mutant protein at P53.R175 H site with high affinity and specificity was screened.