Study On The Construction And Expression Of Ovarian Cancer-related Fusion Protein IP10-CDR3-scFv

Objective:This study aimed to link interferon-? inducible protein 10(IP10)and T cell receptor complementary determination region 3(CDR3)with a single chain variable fragment(scFv)that targets mesothelin to construct fusion protein IP10-CDR3 scfv through genetic engineering technology and then to detect the expression of fusion protein in human ovarian cancer cell line SKOV3.Prokaryotic expression system was used to induce the expression of fusion protein IP10-CDR3-scFv,and the expressed protein was isolated,purified and identified,so as to achieve a large number of preparation of the target protein,and verify its binding ability to SKOV3 cells in vitro,which lays a foundation for further research.Methods:1.IP10 and CDR3 were linked with scFv to cnstruct IP10-CDR3-scFv by artificial synthesis.Then,the constructed IP10-CDR3 scfv was cloned into the eukaryotic expression vector pcDNA3.1(+)by HindIII and EcoRI digestion.The accuracy of the recombinant expression vector was verified by restriction enzyme digestion and sequencing.2.PcDNA3.1-IP10-CDR3-scFv plasmid was transfected into ovarian cancer SKOV3 cells.The mRNA expression level of IP10 and CDR3 was detected by real-time quantitative PCR and the expression of fusion protein was detected by Western blot.3.The prokaryotic expression vector pET28a-IP10-CDR3-scFv of IP10-CDR3-scFv protein was constructed,and the recombinant vector was identified by restriction enzyme digestion and sequencing.Then the recombinant plasmid was transfected into E.coli BL21(DE3),and the fusion protein was induced to express by IPTG.The conditions of IPTG induction were optimized,and the solubility of the expressed protein was analyzed by SDS-PAGE.After purification and renaturation,the fusion protein was identified by Western Blot.4.After the FITC-labeled fusion protein was treated with SKOV3,the binding ability of the fusion protein to SKOV3 cells was detected by flow cytometry.Results:1.The pcDNA3.1-IP10-CDR3 scfv plasmid was digested by HindIII/EcoRI and then analyzed by 1% agarose gel electrophoresis.The results showed that the position of the target band was the same as expected,and the result of DNA sequencing was consistent with the sequence of the target gene,indicating that the constructed recombinant plasmid was correct.2.IP10-CDR3-scFv plasmid was transfected into SKOV3 cells for 48 hours,the transcription level of IP10 and CDR3 and the expression level of fusion protein with His tag were detected:(1)the results of real-time quantitative PCR showed that the transcriptional levels of IP10 and CDR3 in pcDNA3.1-IP10-CDR3-scFv transfection group were significantly higher than those in non-transfection group and pcDNA3.1(+)transfection group.(2)the results of Western blot test showed that the fusion protein was expressed in pcDNA3.1-IP10-CDR3-scFv transfection group,and the molecular weight was about 45 kD.3.The fusion protein was successfully induced by prokaryotic expression system,and was identified by Western blot after purified :(1)prokaryotic expression vector pET28a-IP10-CDR3-scFv was constructed with confirmed by enzyme digestion and sequencing.(2)the recombinant plasmid was transfected into BL21(DE3)expression strain,and the fused protein was induced by IPTG.The results of SDS-PAGE analysis showed that the fusion protein was expressed at 45 kD compared with the uninduced group.(3)the fusion protein was induced by temperature(37 ? and 15 ?)and IPTG concentration(final concentration was 0.2mM and 1.0mM).SDS-PAGE analysis showed that the expression of fusion protein was the highest in IPTG induced by 0.2mM at 37 ?,and the target protein expressed in the form of inclusion body.(4)under the condition of 37 ? 0.2mM IPTG induction,the high purity fusion protein was obtained after washed and then purified on Ni column.The protein induced by IPTG was proved to be IP10-CDR3-scFv fusion protein with Western blot.4.The results of flow cytometry showed that compared with the SKOV3 group(0.20 �0.01%),the binding rate of FITC-labeled fusion protein to SKOV3 cells in the SKOV3+IP10-CDR3-scFv group was 6.54�0.08%,and the difference was statistically significant(p < 0.05).Conclusion:In this study,the recombinant IP10-CDR3-scFv expression vector was successfully constructed,and the fusion protein P10-CDR3-scFv can be expressed in ovarian cancer cell line SKOV3.A large number of fusion proteins were prepared by prokaryotic induction and expression,Western blot results showed that the induced and purified protein was IP10-CDR3-scFv fusion protein,and the protein could bind to SKOV3 cells.