| Objective: To investigate the role of CD40×HER2 Single chain Diabody(CD40×HER2 Sc Db)by gene recombination and prokaryotic expression of prokaryotic cells and detect the function of tumor-specific immunity and HER2 molecular targeting.Methods Immunohistochemistry was used to screen the immunized library anti-CD40 phage display library.High affinity anti-CD40 Sc Fv clone was obtained by phage ELISA.The heavy chain variable region(VH)and the light chain variable(VL)sequence of CD40 Sc Fv were amplified by PCR.The sequence of the VL and VH were connected with the HER2 Sc Fv gene fragment by Overlap Extension PCR in two steps.DNA electrophoresis and sequencing were used to detect the correctness of the splicing sequence.The CD40×HER2 Sc Db sequence and the p ET28 a prokaryotic expression vector were digested by Nde I and Sal I and constructe the p ET28a-CD40×HER2 Sc Db prokaryotic expression vector.The recombinant plasmids were transformed into Rosetta(DE3)competent cells,the protein expression was induced by IPTG,thallus was splited by ultrasonic lysis,and purified by Ni-NTA agarose.The physical and chemical properties of CD40×HER2 Sc Db were analyzed and the molecular structure was predicted.The effect of CD40×HER2 Sc Db on the expression of CD80/CD86 and MHC-II on dendritic cells was detected by flow cytometry.Relying on the differnet intervention,experiment groups were separated as follows: Normal Saline group(NS),tumor antigen loading group(Ag),Ag+CD40×HER2 Sc Db group and Ag+TNF-? group.The effect of CD40×HER2 Sc Db on the expression of IL-12 from dendritic cells and IFN-? from T lymphocytes were detected by ELISA.In vitro culture of mouse breast cancer cell lines 4T1,CCK8 was used to detect CD40×HER2 Sc Db-induced tumor-specific T lymphocytes on 4T1 proliferation inhibition.The tumor growth model of BALB/c mice was constructed.Relying on the differnet intervention,experiment groups were separated as follows: Normal Saline group(NS),HER2 Sc Fv group;tumor CD40×HER2 Sc Db1(750?g/kg)group;CD40×HER2 Sc Db2(1.5mg/kg)group and CD40 m Ab(5mg/kg)group.The tumor growth curve was recorded and analyzed.The expression of IL-12 and IFN-? in tumor tissue was detected by ELISA.The expression of Caspase-3 in tumor cells was detected by immunohistochemistry(IHC).The lmphocyte infiltration was detected by HE staining.The mouse Tumor cell T6-17 was cultured in vitro.The HER2 target function of CD40×HER2 Sc Db was detected by immunofluorescence.CCK8 was used to detect the cytotoxicity of CD40×HER2 Sc Db to T6-17 cells.Relying on the differnet intervention,experiment groups were separated as follows: Normal Saline group(NS),CD40 Sc Fv(2.5?g/m L)group;HER2 Sc Fv(2.5?g/m L)group;CD40×HER2Sc Db1(2.5?g/m L)group;CD40×HER2Sc Db2(5?g/m L)group and Trastuzumab(15?g/m L)group.The tumor model of BALB/c-Nude mice was constructed and relying on the differnet intervention,experiment groups were separated as follows: NS group,CD40 Sc Fv group(150?g/kg);HER2 Sc Fv group(150?g/kg);CD40×HER2 Sc Db1 group(150?g/kg);CD40×HER2 Sc Db2(300?g/kg)group and Trastuzumab(1mg/kg)group.The volume of subcutaneous tumor was observed.The tumor tissue was embedded in paraffin and sliced.The morphological changes of T6-17 tumor cells were observed by HE staining.Results The phylogenetic analysis showed that the sequence diversity of phage display library clone had a convergence effect.Sequencing of the third round of screening clones 100% matched clones accounted for 3.2% of the total clones and screened for the acquisition of eight potential positive clones.The affinity of the gradient phage ELISA to detect the potential clones showed that the affinity of the 8 # clone with CD40 and its ratio to BSA affinity > 3,which was consisted with the criteria for positive clonal screening.The VH and VL fragments of CD40 Sc Fv were amplified by PCR.The results of DNA electrophoresis and sequencing showed that the VH and VL of CD40 Sc Fv were 345 bp and 324 bp,respectively,which were in agreement with the expected size.The length of CD40×HER2 Sc Db gene was 1464 bp,consistent with expectations.The optimal protein induction effect was obtained by using 0.5 mmol/L IPTG.The protein was denatured at about 55 Kd.The size of the protein was similar to expected size.Western Blot detection of His-tag was also seen at about 55 kd The concentration of protein was 2.7 mg/ml by BCA detection.The expression ratio of CD80,CD86 and MHC-II on the DC surface of Ag+CD40×HER2 Sc Db group were 93.5±3.1% 86.00±3.2% 92.3±2.8%,respectively.Compared with Ag+TNF-? group,no significant difference founded(P>0.05).Compared with NS group and Ag group,the difference was statistically significant(P<0.05).The concentration of IL-12 in the supernatant of DC cells in Ag+CD40×HER2 Sc Db group was 659.90±61.28 pg/m L,which have no significantly different from that of Ag+TNF-? group(P>0.05)Group and Ag group,the difference was statistically significant(P<0.05).The inhibition rate of T+lymphocytes on the proliferation of 4T1 cells was 75.65±2.53% in Ag+CD40×HER2 Sc Db group,and there was no significant difference compared with Ag+TNF-? group(P>0.05)Compared with the difference was statistically significant(P<0.05).The levels of IL-12 and IFN-? in 4T1 tumor tissues of CD40×HER2 Sc Db2 group were 448.85±51.10 pg/m L and 273.44±44.60 pg/m L,respectively.Compared with NS group and HER2 Sc Fv group,the difference was statistically significant(P<0.05),and there was no significant difference compared with CD40 m Ab group(P> 0.05).The changes of tumor volume of 4T1 in different groups showed that CD40×HER2 Sc Db2 group(1.5mg/kg)and CD40 m Ab(5mg/kg)could significantly inhibit the proliferation of 4T1 tumor in vivo.There was no significant difference between the two groups(P>0.05).The expression of Cleaved-Caspase-3 in tumor tissue was detected by immunohistochemistry.The expression of Cleaved-Caspase-3 in CD40×HER2 Sc Db group was significantly up-regulated.The inhibition of CD40×HER2 Sc Db on T6-17 cells showed that the inhibitory rate of CD40×HER2 Sc Db(5?g/m L)on T6-17 was 86.89 ± 6.35%,which was similar to that of trastuzumab(15?g / m L)Was 85.56 ± 1.73%,the difference was not statistically significant(P>0.05).Western blot was used to detect the expression of Akt,p-Akt,ERK1/2 and p-ERK1/2 in different intervention groups.The results showed that CD40×HER2 Sc Db,HER2 Sc Fv and trastuzumab could inhibit Akt,The expression of Akt,ERK1/2 and p-ERK1/2 was statistically significant(P<0.05)compared with NS group.The results of HE staining showed that the cells in the NS group were normal,the nuclei were intact,the nucleolus was positive and the cell growth was active.The tumor cells of CD40×HER2 Sc Db and trastuzumab group lost normal morphology,nuclear pyknosis Fragmentation is obvious.Conclusion The anti-CD40 high affinity Sc Fv clone was successfully obtained by screening the immunized library against CD40 phage display.The full length of CD40×HER2 Sc Db gene sequence can be successfully obtained by overlapping PCR.CD40×HER2 Sc Db protein can be obtained by IPTG induction,and the functional protein can be obtained by protein renaturation.CD40×HER2 Sc Db can induce tumor cell apoptosis by activating tumor cells by promoting tumor cell specificity,activating tumor-specific immunity,in vitro killing of tumor,and by promoting tumor-specific CTL in tumor tissue nest set,by activating Caspase-3 in tumor cells Activation of tumor-specific immune responses by upregulation of activated immune cytokines within the tissue.CD40×HER2 Sc Db can inhibit the phosphorylation of Akt and ERK1/2 by binding to HER2 on T6-17 surface,inhibit the proliferation of T6-17 in vitro,and inhibit the proliferation of T6-17 cells by HER2 targeting in vivo. |
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