IL-24基因克隆与表达及诱导THP-1细胞分泌炎症性细胞因子和肿瘤细胞凋亡作用的研究

发布时间：2019-02-12 14:15

【摘要】： 目的:RT-PCR扩增出人源性成熟肽IL-24基因,构建重组质粒pQE30/ hIL-24,在大肠杆菌(E.coli)中进行诱导表达,纯化获得重组融合蛋白rhIL-24。研究该重组蛋白在体外诱导THP-1人单核细胞白血病细胞表达并产生炎症性细胞因子TNF-α和IL-6的水平及诱导肿瘤细胞凋亡作用,为进一步探索表达蛋白的生物学活性提供试验依据。方法:利用引物设计软件设计hIL-24基因特异性引物,以提取正常成人的外周血单个核细胞的mRNA合成的cDNA为模板,RT- PCR扩增目的基因片段,将其插入pQE30表达载体,构建重组质粒pQE30/hIL-24,并通过酶切分析、PCR及测序鉴定。然后将其转化至宿主菌M15中进行诱导表达,利用SDS-PAGE和Western-Blot进行分析和鉴定表达产物,镍-琼脂糖凝胶亲和层析柱纯化重组蛋白rhIL-24。用不同浓度的rhIL-24刺激人单核细胞白血病细胞THP-1,通过ELISA法检测到经刺激后的THP-1细胞产生的TNF-α和IL-6。再用Annexin V-FITC染色法检测到经rhIL-24处理后THP-1细胞发生凋亡的情况。结果:RT-PCR成功扩增出hIL-24基因片段(474bp),构建了重组质粒pQE30/hIL-24,并在M15菌中高效表达出分子量约为19.2kDa的rhIL-24重组融合蛋白,超声裂解后SDS-PAGE分析显示目的蛋白在菌体细胞内以包涵体的形式表达,经镍-琼脂糖凝胶层析柱纯化获得纯度在95%以上的rhIL-24。rhIL-24能诱导THP-1细胞表达TNF-α和IL-6,并以时间、剂量依赖方式刺激细胞分泌炎症性细胞因子TNF-α和IL-6,当rhIL-24的浓度为28μg/mL时产生的TNF-α和IL-6量最多,分别为278.63±8.03pg/mL和49.48±6.92pg/mL;当rhIL-24刺激THP-1细胞18h产生的TNF-α和IL-6量则达到高峰。另外,rhIL-24以28μg/mL的浓度作用于THP-1细胞18h后发生的凋亡率最高。结论: 1、成功克隆hIL-24基因并构建了pQE30/hIL-24原核表达载体,将其转化E.coli后能高效表达出分子量约19.2kDa的重组融合蛋白。 2、rhIL-24能诱导人单核细胞白血病细胞THP-1表达并分泌炎症性细胞因子TNF-α和IL-6; 3、rhIL-24能诱导人单核细胞白血病细胞THP-1发生凋亡。
[Abstract]:Objective: to amplify the IL-24 gene of human sexual maturation peptide by RT-PCR, construct the recombinant plasmid pQE30/ hIL-24, and express it in Escherichia coli (E.coli), and purify the recombinant fusion protein rhIL-24.. To study the expression of THP-1 human monocytic leukemia cells and the levels of inflammatory cytokines TNF- 伪 and IL-6, and the apoptosis of tumor cells induced by the recombinant protein in vitro. It provides experimental basis for further exploring the biological activity of the expressed protein. Methods: hIL-24 gene specific primers were designed by using primer design software. CDNA synthesized from peripheral blood mononuclear cells (PBMC) of normal adults was extracted as template. The target gene fragment was amplified by RT- PCR and inserted into pQE30 expression vector. The recombinant plasmid pQE30/hIL-24, was constructed and identified by enzyme digestion, PCR and sequencing. The recombinant protein rhIL-24. was purified by nickel agarose gel affinity chromatography. The expression product was analyzed and identified by SDS-PAGE and Western-Blot. THP-1, of human monocytic leukemia cells stimulated with different concentrations of rhIL-24 was used to detect TNF- 伪 and IL-6. produced by stimulated THP-1 cells by ELISA assay. Annexin V-FITC staining was used to detect the apoptosis of THP-1 cells treated with rhIL-24. Results: the hIL-24 gene fragment (474bp) was successfully amplified by RT-PCR, and the recombinant plasmid pQE30/hIL-24, was constructed. The recombinant rhIL-24 fusion protein with a molecular weight of about 19.2kDa was highly expressed in M15 strain. SDS-PAGE analysis after ultrasonic cleavage showed that the target protein was expressed in the form of inclusion body in the cell. Purified by nickel agarose gel chromatography, rhIL-24.rhIL-24 with purity of more than 95% could induce THP-1 cells to express TNF- 伪 and IL-6, for a period of time. When the concentration of rhIL-24 was 28 渭 g/mL, the quantity of TNF- 伪 and IL-6 was the highest, which was 278.63 卤8.03pg/mL and 49.48 卤6.92 PG / mL, respectively. When rhIL-24 stimulated THP-1 cells for 18 h, the production of TNF- 伪 and IL-6 reached its peak. In addition, the apoptosis rate of THP-1 cells treated with 28 渭 g/mL rhIL-24 for 18 h was the highest. Conclusion: 1. HIL-24 gene was cloned successfully and the prokaryotic expression vector of pQE30/hIL-24 was constructed. The recombinant fusion protein with molecular weight of about 19.2kDa could be expressed efficiently by transforming it into E.coli. 2rhIL-24 can induce the expression of THP-1 and the secretion of inflammatory cytokines TNF- 伪 and IL-6; 3rhIL-24 in human monocytic leukemia cells. RhIL-24 can induce THP-1 apoptosis in human monocytic leukemia cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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