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@#[摘 要] 目的：构建靶向CD70分子的重组免疫毒素，通过表达、纯化制备PE38与抗CD70纳米抗体重组蛋白，体外抗肿瘤实验探究重组蛋白是否对高表达CD70分子的阳性肿瘤细胞具有杀伤活性。方法：通过基因工程手段，将CD70纳米抗体Nb 2B3基因片段通过一个连接子与pET21a-PE38基因片段相连，获得重组表达载体pET21a-Nb 2B3-PE38并转入BL21（DE3）感受态细胞中进行表达、纯化与鉴定。用间接ELISA及FACS法检测Nb 2B3-PE38与CD70分子的结合活性，MTT法检测Nb 2B3-PE38对高表达CD70分子的肾透明细胞癌786-O细胞的体外杀伤活性，Annexin Ⅴ-FITC/PI双染法检测Nb 2B3-PE38对786-O细胞凋亡的影响。结果：成功构建抗CD70纳米抗体重组免疫毒素Nb 2B3-PE38，纯化获得纯度>90%的重组蛋白，SDS-PAGE及WB检测结果表明目的蛋白正确表达，分子量为56 000。纯化后的Nb 2B3-PE38能与重组CD70抗原及786-O细胞表面的CD70分子特异性结合；25 µg/mL Nb 2B3-PE38即对786-O细胞产生极显著的杀伤作用（P<0.001），并且促进786-O细胞的细胞凋亡（P<0.01），其杀伤效应强于阳性对照顺铂（P<0.01）。结论：成功制备了特异性靶向CD70分子的免疫毒素Nb 2B3-PE38，其能够有效杀伤786-O细胞并诱导细胞凋亡且效果强于顺铂。
ABSTRACT
Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody (scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography (Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease di-gestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.
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Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.
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Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Sehistosorna japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The V_H and V_L genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F' induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Westem-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.
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Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector pIRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165-PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.
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Objective:To construct a prokaryotic expression plasmid of PQE30-IL3-Linker-PE38KDEL and identify its recombinant protein expression.Methods:The IL3 and PE38KDEL gene were amplified by polymerase chain reaction(PCR) and cloned into the prokaryotic expression plasmid PQE30-Linker constructed after being sequenced.The recombinant vector confirmed by restriction endonucleases digestion,coenobium PCR,and DNA sequence analysis was transformed into E.coli SG13009.The expression of the protein was induced by IPTG.Relative molecular weight of the expression product was detected by SDS-PAGE.Finally,the fusion protein was examined by Western blot.Results:The results of restriction endonuclease digestion,coenobium PCR and DNA sequence analysis showed that the prokaryotic expression vector PQE30-IL3-Linker-PE38KDEL was constructed successfully.With induction of IPTG,the relative molecular weight of the expression product was identical to the expected value.The expressed 6?His-IL3-PE38KDEL fusion protein were identified at relative molecular mass of 57KD by Western blot with anti-His monoclonal antibody,showing the fusion protein expressed correctly.Conclusion:The fusion protein IL3-PE38KDEL is successfully constructed,which lays a solid foundation for the further research of protein purification and function.
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Objective:To prepare a new type of anti-leukemia immunotoxin with killing activity.Methods:The method of cytotoxicity was used to study the activity of the immunotoxin after the induction of IPTG. Results:The expressed fusion proteins were detected mostly as inclusion bodies at high level.The result showed IL3-PE38KDEL had liable activity of toxicity. Conclusion:The fusion protein IL3-PE38KDEL has good biological activity,which paves way for the further study on its treatment of leukemia.
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Objective To construct a new recombinant immunotoxin expression vector fused with a murine interleukin18(IL18) gene and a truncated pseudomonas exotoxin (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli (E. coli). Method Murine IL-18 (mIL-18) cDNA was cloned from murine liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was ligased with a PE38 gene carried by PRKL expression vector through T4 DNA ligase and constructed into fusion protein expression plasmid PRKL-IL18-PE38. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and the molecular weight and specificity were determined by SDS-PAGE and Western-blotting. Result The new recombinant immunotoxin expression vector was constructed successfully. DNA sequencing revealed that the mIL-18 and PE38 gene were consistent with NCBI Gene Bank. The IL-18-PE38 fusion protein was expressed in E.coli BL21, and Western-blotting analysis indicated that the molecular weight of the expression product is about 56 kDa, and could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cells and may have some potential value in the treatment of Th1 cell-mediated autoimmune diseases.