Preparation Of Monoclonal Antibody Against Human TK1 And Preliminary Establishment Of Chemiluminescence Detection

Objective: The present studies suggest that thymidine kinase 1(TK1)can be used as a sensitive and effective biomarker for evaluating tumor cell proliferation.It is used for early screening,treatment monitoring and prognosis of cancer patients.In the last decade,several laboratories have prepared different anti-TK1 antibodies for the identification and monitoring of tumor diseases,but the sensitivity is weak.The main purpose of this study to obtain a higher quality TK1 antibody and applied to initial establishment of chemiluminescence detection method,to provide material and technical support for tumor science and clinical application.Methods: 1.The target gene was amplified by PCR,double digested with Xho I / Eco RI restriction endonuclease,and linked to the expression vector p ET32 a,and then transferred into competent cells to prepare BL21-p ET32a-TK1 recombinant strain.The plasmids were extracted and identified by restriction endonuclease digestion.2.The TK1 protein was expressed by IPTG-induced BL21-p ET32a-TK1 recombinant bacteria and optimized from IPTG concentration,culture temperature and time.The expression level of recombinant protein TK1 was tested by SDS-PAGE,and determined the optimal induction conditions.The expressed product was purified by nickel ion affinity chromatography,used SDS-PAGE to detect the purity of the target protein,and the antigenicity of the target protein was detected by western blot.3.Used the obtained TK1 recombinant protein to immunize the mice,took the spleen cells of the best immune effect mice to fuse with SP2/0.Screened monoclonal cell lines that secreting specific antibodies stably.A large number of monoclonal antibodies were prepared by mouse intraperitoneal inducing method.The monoclonal antibody was purified by n-octanoic acid-ammonium sulfate precipitation and affinity chromatography.The purified monoclonal antibody was identified by purity,subtype and specificity.4.Screened the matching antibody,ensured the optimal reaction system and the linear range of the detection method,and established the chemiluminescence detection method initially.Results: 1.Obtained the BL21-p ET32a-TK1 recombinant strain.2.At 37 ?,the concentration of IPTG was 0.2m M,and the expression level of recombinant protein TK1 was the highest and soluble at 6h.When the condition of nickel ion affinity chromatography gradient elution was at 80 m M imidazole,the content of recombinant protein TK1 was the highest purity,and the protein was proved to have antigenicity by western blot.3.Through indicrect-ELISA,selected eight strains Ig G1 subtype and specific anti-TK1 monoclonal cell lines,SDS-PAGE showed that the antibody purity was good.4.A pair of antibody 4G3 and an enzyme-labeled antibody 5E3,which can be used for the initial establishment of the TK1 chemiluminescence detection method,were obtained.The best detection system is: 4G3 coated with 200 ng per hole,enzyme-labeled antibody 5E3 diluted 2000 times.In this detection method,the linear range is 100-5000 pg / m L.Conclusion: Eight TK1 monoclonal antibodies were successfully prepared by gene engineering technology and hybridoma technique,and a pair of pairing antibodies were found.The method of chemiluminescence detection was established by using the pair of pairing antibodies.