Expression And Functional Detection Of Anti-B7-H4-scFv/PE38KDEL Recombinant Immunotoxins

In this study,the anti-B7-H4-scFv gene was ligated with toxin PE38KDEL by SOE-PCR and connector was subcloned into prokaryotic expression vector pET28a to be induced expression.Recombinant toxins obtained through the renaturation of soluble protein,after purification by nickel column to obtain a single target protein,and Western blotting to identify the expression of the protein;after indirect ELISA and Western blot were used to detect the recombinant protein and B7-H4 antigen binding specificity and affinity;MTT assay and subcutaneous xenograft model experiment were used to detect the inhibitory effect of toxin protein on tumor cells in vitro and in vivo.HE staining and immunohistochemical analysis of tumor tissues were performed.Specific experimental steps are as follows:1.Amplification and cloning of anti-B7-H4-scFv-PE38KDEL gene and constru-ction of recombinant expression vector pET28a/anti-B7-H4-scFv-PE38KDELThe anti-B7-H4-scFv gene and toxin PE38KDEL gene were amplified by PCR.Then PE38KDEL gene and anti-B7-H4-scFv gene were ligated by SOE-PCR.After the PCR reaction was over,the target band was taped and recovered.The recovered target gene anti-B7-H4-scFv-PE38KDEL and pUM19-T Vector were cloned according to the kit.Positive clone samples of bacteria were sent to the company for sequencing identification,the correct strain were stored for future use.The plasmid pET28a(+)was extracted according to the Biomiga kit and tested concentration of plasmid.The pET28a(+)plasmid and the anti-B7-H4-scFv-PE38KDEL gene were double-digested with EcoRI and Not I restriction enzymes.The ligation product was transformed into E.coli BL21(DE3)competent cells.The next day,the positive clones that grew out of the plates were picked and sent to the company for sequencing and identification.The target strains of the correct sequencing were stored at-80?.2.Expression,purification and activity Detection of recombinant toxin Anti-B7-H4-scFv-PE38KDEL proteinE.coli BL21(DE3)expressing strains containing the correct pET28a-anti-B7-H4-scFv-PE38KDEL recombinant vector plasmid induced protein expression at different IPTG final concentrations and induction times.SDS-PAGE analysis was performed to detected protein expression under different conditions,and find the optimal expresson conditions.Under the optimized conditions of induced expression,the products were expanded and collected,and the resulting inclusion bodies were precipitated and refolded to obtain more soluble supernatant.The His-tagged product can be bound to a nickel column to purify the protein and elute with different concentrations of imidazole and collect the eluate.The eluted protein solution was dialyzed,concentrated,collected and identified.Indirect fluorescent labeling method was used to detect the binding activity of immunotoxin to human breast cancer cells MCF-7,normal human renal epithelial 293a and human hepatoma HepG2 by flow cytometry.3.Biological function detection of recombinant toxin protein Anti-B7-H4-scFv-PE38KDEL in vitro and in vivoThe biological functions of the recombinant toxin protein were tested in vitro and in vivo,respectively.MTT assay in five concentration gradients(0.01,0.10,1.00,10,100 ug/ml)at 12h,24h,36h and 48h was used to determine the effect of recombinant toxin on the proliferation of A549 cells,HepG2 cells and 293T cells in vitro,respectively.The effect of toxin protein(0.01,1.00,100 ug/ml)on the three kinds of cells of MCF-7 and HepG2,293a cells was detected by trypan blue staining and the survival rate was calculated.The model of mouse tumor xenograft was established in vivo.A certain number of lung cancer A549 cells were injected into mice to establish a model.When the tumor grew up,the mice were randomly divided into the experimental group,the positive control group and the blank control group.The blank group,the positive control group and the experimental group was injected with normal saline,paclitaxel and the recombinant toxin protein and injected continuously for seven times.The body weight and the tumor volumes were recorded during the experiment.The tumor masses were weighed after the nude mice were sacrificed.HE staining and immunohistochemistry analysis was performed on tumor tissue.