Screening And Identification Of Single Chain Antibodies To KRAS Protein P.G12S

BACKGROUND:KRAS is one of the most easily activated oncogenes in the RAS family,and 17-25%of tumors have activated KRAS gene mutations.Key regions of oncogenic KRAS gene activation mutations are mainly codon 12,13,59,61,and 63.These mutations are activated in a manner that increases the RAS protein content of the GTP binding state by inactivating endogenous GTP enzyme activity and causing enzyme activity protein resistance.Among cancer patients in China,KRAS gene mutation sites are mainly codon 12 and 13 of exon 2.p.G12D?p.G13D point mutation is the main one,and p.G12S point mutation is also seen.KRAS gene mutations are also different in different types of tumors,such as colorectal adenocarcinoma and non-small cell lung cancer in patients,to some extent their different gene locus mutations are associated with the prognosis of patients.Using genetic engineering to screen specific single-stranded antibodies against mutations at certain sites can provide theoretical basis for the development of personalized targeted therapy strategies.With the continuous development of tumor biotherapy,effective specific targeted therapeutic sites can greatly reduce the toxic side effects of drugs while improving the efficiency of drugs.At present,novel targeted drugs such as cetuximab and some immune checkpoint inhibitors have been applied to target and immunotherapy of many tumors and achieved good results.But for tumors with mutations at specific sites of RAS genes,multiple targeted drugs are difficult to achieve the desired therapeutic effect.Therefore,it is very important to find specific antibodies against specific site mutations for tumor biotherapy.At present,the theory of tumor immunity is that there must be tumor specific antigen in tumor patients,which can induce the body to produce immune response to tumor,and theoretically the body can produce specific antibody against specific antigen.But finding tumor-specific antigens and corresponding antibodies is difficult.Single chain antibody is a recombinant protein that splices the light chain variable region and heavy chain variable region of traditional antibody through a section of nucleotide chain by genetic engineering method.It has the advantages of small molecular weight,low immunogenicity,strong penetration,rapid localization,and easy genetic engineering preparation and modification,and is very suitable for targeted therapy of tumor.In the 1990s,phage display technology laid a theoretical foundation for the establishment of phage antibody library.At the same time make the discovery and preparation of tumor-associated single-chain antibodies possible.To obtain specific antibodies with high affinity depends mainly on two important determinants:the storage capacity of antibody bank and the source of preparation,and the ideal antibody screening should be from the largest possible storage capacity and directly from the human body,but because of the limited conditions,most related studies are more difficult to have both.OBJECTIVE:Human phage monoclonal antibody library was constructed by genetic engineering and phage display technology in order to screen single-stranded antibodies with high affinity and specificity for KRAS p.G12S site mutations,and to provide new protocols for tumor diagnosis and gene targeting therapy.METHODS:1.Collection of 10 normal human peripheral blood,isolation of peripheral blood lymphocytes,extraction of total RNA,reverse transcription as cDNA,expansion of diverse single-stranded antibody heavy and light chain variable region fragments by soe-pcr and construction of single-stranded antibodies,double-enzyme of phage physique,linking with post-cut single-stranded antibodies,and then electroconversion of the junction product into escherichia coli.TG1 to construct a primary single-stranded antibody library;2.Using the synthesized p.G12S-containing polypeptide as a target,a single-stranded antibody with high affinity specifically resistant to this site mutation was selected by multi-wheel washing and its structural sequence was analyzed;3.The affinity and specificity of the obtained single-stranded antibody,as well as the basic expression of the protein,were identified by methods such as elisa et.RESULTS:Based on the techniques of genetic engineering and molecular cloning,the primary phage monoclonal antibody library with a capacity of about 1×10¹³was successfully constructed.Using recombinant protein containing p.G12S as target antigen and phage display method,we successfully constructed a single-stranded antibody library with high affinity for p.G12S site mutation by 2 rounds of washing.By enzyme digestion,nucleic acid sequencing and other methods,we determined whether the light chain sequence of the obtained single-stranded antibody gene had high homology with the human IgG antibody light-weight chain variable region sequence?>90%?.Conclusion:To construct the human-derived primary single-chain antibody library with10¹³storage capacity;Screening single-stranded antibodies with high affinity and specificity for mutation of KRAS protein p.G12S sites.