Screening And Identification Of Target Antigen Recognized By Single Chain Variable Fragment (scFv 4-16) Against Hepatocellular Carcinoma

Background Hepatocellular carcinoma is one of most common cancer worldwide with advanced malignancy,high mortality and poor prognosis.There are about one million deaths of HCC annually in the world.Traditional therapy of resection, chemotherapy and radiotherapy are of limited efficacy and it is short of biomarker for early diagnosis of HCC.Single chain variable fragment is better vector of targeted therapy of HCC owing to its small molecule weight,strong tumor tissue penetration, weak immunogenicity,short half-lives in blood and maintaining the binding and specificity of the whole antibody.Our work team had identified one single chain variable fragment against hepatocellular carcinoma(scFv 4-16) using phage display antibody library technology.After affinity maturation and humanization of single chain variable fragment against HCC,we get humanized scFv with high specificity and affinity against HCC(scFvHDM) which can specifically combine with antigen of HCC tissue and cell.However,the corresponding antigen of single chain variable fragment against hepatocellular carcinoma is unknown,this study is to screen and identify the targeted antigen recognized by single chain variable fragment against hepatocellular carcinoma.The first part:Construction of the cDNA phage expression library of Hepatocellular carcinoma antigensObjective To construction a cDNA phage expression library of hepatocellular carcinoma antigens.Methods The total RNA was extracted from hepatocellular carcinoma cell,and the mRNA was purified.The single-strand and double-strand of cDNA were synthesized through reverse transcription-PCR,cDNA fragment,after removed those smaller than 500bp,were recombined toλTriplEx2 phage vector.The recombined cDNA were packaged in vitro with Gigapackâ…¢Gold packing extract, then a small portion of package phage was used to infect E.coli XL1-blue for titration and determination of the percentage of recombinant clones.PCR method was used to identify the size of inserted cDNA.Result The constructed cDNA phage expression library of hepatocellular carcinoma antigens consisted of 3.4×10⁶ pfu/ml independent clones with a recombination rate of 98.2%.After amplification,the constructed cDNA phage expression library of hepatocellular carcinoma antigens consisted of 3.4×10¹¹ pfu/ml independent clones with a recombination rate of 97.2%.The length of the inserted cDNA fragment ranged from 600bp to 2kb with an average of 1400bp.Conclusion A high-quality cDNA phage expression library of hepatocellular carcinoma antigens has been constructed successfully,which is essential of screening and identifying of tumor specific antigen of hepatocellular carcinoma.The second part:Optimized expression,purity and identification of humanized single chain variable fragment against hepatocellular carcinomaObjective Optimized expression and identification of humanized single chain variable fragment against hepatocellular carcinoma.Methods After induced by IPTG, the soluble scFvs against HCC were expressed in the bacteria periplasm of E.coli HB2151.Using HiTrap Anti-E Tag column,the scFvs were purified by affinity chromatography.SDS-polyacrylamide gel electrophoresis of purified scFvs was analyzed.The consistencies of purified scFvs were measured by Bradford method. The bioactivities of purified scFv were identified by ELISA and immunohistochemistry.Result Alter 12h induced by 1mmol IPTG at 30℃with 0.4M sucrose,the amount of expressed scFv increased greatly.The yield of purified scFv was 325μg/L.The purified scFv was specially binding to tumor antigen of HCC.Conclusions The functional humanized single chain variable fragment against hepatocellular carcinoma was achieved,which is essential of using this single chain variable fragment in hepatocellular carcinoma targeted diagosis and therapy.The third part:screening of the cDNA phage expression library of Hepatocellular carcinoma and analysis of positive cloneObjective To screen and identify the targeted antigen recognized by single chain variable fragment against hepatocellular carcinoma(scFv 4-16).Method:The cDNA phage expression library of hepatocellular carcinoma was screened by single chain variable fragment against hepatocellular carcinoma(scFv 4-16,scFv DM,scFv HDM). The positive clone was sequenced and analyzed by BLAST soft.The positive clone mRNA expression in cell was measured by reverse transcription-PCR.Result:After three times screening,one positive clone(scFv 4-16G) was obtained.The scFv 4-16G sequence was 833bp length,which is similar to keratin 23.The identical ratio is 99%. The mRNA of scFv 4-16G was stongly expressed in hepatocellular carcinoma cell, weakly expressed in gastric cancer cell,but no expressed in normal liver cell. Conclusion:Keratin 23 may be the targeted antigen recognized by single chain variable fragment against hepatocellular carcinoma.