The Antitumor Effect Of ScFv Antibody Against 7 Transmembrane Receptor Of Trichinella Spiralis On A549 Cells

At present,many researches from China and abroad have confirmed that the infection of Trichinella spiralis can inhibit the growth of tumor,but the specific anti-tumor mechanism still can not be fully explained.In our laboratory,through the screening of Trichinella Spiralis c DNA library,a variety of tumor-related antigen of Trichinella spiralis were obtained,and the mouse hyperimmune serum and monoclonal antibody prepared with the cross antigen has achieved great anti-tumor effects in nude mice model of experimental treatment among them,Our lab prepared hyper-immune serum of 7TR,a A549 related antigen of Trichinella Spiralis,for the experiment and the tumor inhibition rate was 64.77% in the treatment of nude mice bearing A549.However,the mouse antibody has not achieved satisfactory effect in clinical treatment.Both mice polyclonal antibody and mice monoclonal antibody can give rise to strong human anti-mouse antibody response.In addition,mouse monoclonal antibodies are difficult to be produced on large scale for their high cost.What is more,there are also some problems such as the limited therapeutic effect,rejection response and complex technology for the application of chimeric antibody and CDR antibody.Huston J S et al.And Bird R et al.prepared sc Fv by DNA recombination technique.Sc Fv has complete antigen binding site,its molecular weight is only about 1/6 of the complete antibody.Its ability to penetrate into tissue is strong and its metabolism in blood is very fast,researchers can carry out the whole screening process in vitro.What is more important,sc Fv can be expressed in engineering bacteria.Also it can be easily genetically modified and connected to antitumor effect molecules for the constrction of tumor-targeting fusion proteins.It has broad application prospects in tumor imaging and tumor targeting therapy.In recent years,researchers have used single chain antibody directly in the animal experiment of solidtumor therapy,and achieved good therapeutic effect.It also demonstrates the potential of new applications of sc Fv drugs.In this study,firstly,the prokaryotic expression of 7TR protein was performed,and then screen Tomlinson(I+J)single chain antibody library.The positive antibody secreting strain was confirmed by the test of ELISA.Then the sc Fv was secreted by postive purified.IFA was used to examine the binding activity of positive antibody to A549 cells.Indirect immunofluorescence assay was used to examine the binding activity of positive antibody to A549 cells.CCK-8 was used to detect the inhibitory activity of different concentrations of antibody on A549 cells.In the end,we evaluated the therapeutic effect of anti-7TR protein sc Fv against A549 in nude mice bearing A549 tumor and analyze the potential mechanism that the sc Fv inhibit the proliferation of A549.Prokaryotic expression,purification and renaturation of 7TR protein from Trichinella spiralis According to the published 7TR gene of Trichinella spiralis on NCBI website,the specific primers were designed with Trichinella spiralis c DNA as template.The target gene was amplificated by PCR,and the gene was connected into p ET-32 a vector.Then the recombinant plasmid was transformed into E.coli BL21(DE3).Then induced by IPTG.The expression protein was refolded on the DEAE chromatographic column,and the refolded protein was purified by Cu2+ affinity chromatography column.Finally,the recombinant 7TR protein was purified and refolded.Screening of Tomlinson(I+J)human phage antibody library After four rounds of phage antibody enrichment against 7TR protein,2000 E.coli HB2151 colonies were randomly selected from infected colonies,induced by IPTG and with ELISA test,when the sample OD490sample≥3OD490negative,the sample can be determined as positive.After three rounds of parallel test and the removal of antibodies against p ET-32 a tags and milk powder,9 positive clones were obtained:1A2、2B1、2B8、4C7、5D1、6D2、7H5、7H9、8G5Validation of A549 binding activity of anti-7TR positive antibody 8G5 was selected and purified,and then its A549 binding activity was detected by IFA.Theantibodies had good A549 binding activity.Inhibition of A549 cell growth by single chain Fv antibody in vitro and in vivo Take CCK-8 for the detection of anti-7TR protein sc Fv against A549 lung cancer cells.The results showed that when the initial concentration of human single chain antibody was 45μg/100μl,the inhibition rate of lung cancer cells was 22.39%when the cells were treated with sc Fv for 48 H.In animal experiments,the results showed when the concentration of sc Fv was 100μg/100μl,each mouse of each injection volume is 100μl,the tumor growth inhibition rate of anti-7TR sc Fv was59.10%.With immunohistochemical analysis of expression of cancer cell proliferation and apoptosis of tumor related genes PCNA,VEGF,Bcl-2 after the treatment of anti-7TR sc Fv,the results show that,the expression of PCNA,VEGF was significantly decreased,the expression of Bcl-2 had no significant change.In addition,with the result of HE stain,after the treatment of sc Fv antibody,no histopathological changes were caused in hearts,livers,spleens,lungs and kidneys of nude mices.This study laid the foundation for the humanization of mouse antibody against the tumor-related antigens of Trichinella spiralis,and provided the experimental basis for the clinical application of anti-tumor biological agents.