热休克蛋白70与NY-ESO-1多肽复合物的制备以及抗肿瘤效应的体外研究

发布时间：2018-06-09 22:04

本文选题：热休克蛋白70 + NY-ESO-1肽　；参考：《福建医科大学》2011年硕士论文

【摘要】：【目的】将原核表达纯化获得的人热休克蛋白70(heat shock protein70, HSP70)与肿瘤睾丸抗原NY-ESO-1多肽体外连接制备HSP70-NY-ESO-1多肽复合物,检测HSP70-NY-ESO-1多肽复合物负载的树突状细胞(dendritic cells, DC)对NY-ESO-1阳性肿瘤细胞的体外杀伤效率,探讨增强肿瘤免疫治疗的有效策略。【方法】 1.人HSP70的原核表达、鉴定及纯化:RT-PCR技术获得人HSP70全长cDNA,将PCR产物克隆到原核表达载体PET-30a质粒上,酶切与DNA测序鉴定后,将PET-30a-HSP70重组质粒转化至大肠杆菌Rosetta(DE3),IPTG诱导蛋白表达,表达产物行SDS-PAGE和Western-blot鉴定,His-tag层析柱纯化HSP70蛋白。 2.人脐带血树突状细胞的培养及鉴定:联合应用重组人粒细胞/巨噬细胞集落刺激因子(recombination human granulocyte/macrophage colony-stimulating factor, rhGM-CSF)和重组人白细胞介素4(recombination human interleukin-4, rhIL-4)培养人脐带血单个核细胞来源的DC,肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)诱导其成熟。通过倒置显微镜、光学显微镜及透射电镜观察DC形态,流式细胞术鉴定DC表型,混合淋巴细胞反应鉴定DC功能。 3.HSP70-NY-ESO-1多肽复合物负载DC的体外杀伤实验:HSP70与NY-ESO-1多肽体外连接,获得HSP70-NY-ESO-1多肽复合物。分别用HSP70-NY-ESO-1多肽复合物负载的DC、NY-ESO-1多肽负载的DC、HSP70负载的DC以及未经负载的DC刺激淋巴细胞,并以未经DC刺激的淋巴细胞作为对照组,共5组淋巴细胞,乳酸脱氢酶(lactic dehydroge-nase, LDH)释放法检测淋巴细胞对NY-ESO-1阳性脑胶质瘤细胞的体外杀伤效率。【结果】 1.人HSP70的原核表达、鉴定及纯化:RT-PCR扩增出大小约2000bp的目的片段,经酶切和DNA测序证实,HSP70基因成功地克隆到原核表达载体PET-30a质粒上;转入重组质粒的大肠杆菌经IPTG诱导后,SDS-PAGE显示,在分子量为约72KD处有表达量明显增多的蛋白条带,Western-blot证实其为目的条带,纯化后蛋白纯度达95%。 2.人脐带血树突状细胞的培养及鉴定:培养第8d的成熟DC(mature DC, mDC)形态学观察:细胞体积大,形态不规则,有典型的树枝状突起;流式细胞术鉴定:mDC表达HLA-DR(80.65%)、CD86(76.59%)、CD83(74.81%),较不成熟DC(immature DC, imDC)表达明显上升(HLA-DR、CD86与CD83分别为25.20%、17.58%、1.50%);混合淋巴细胞反应证实:mDC体外激发混合淋巴细胞反应能力较强,imDC则较弱。 3.HSP70-NY-ESO-1多肽复合物负载DC的体外杀伤实验:HSP70-NY-ESO-1多肽复合物负载DC组的淋巴细胞对NY-ESO-1阳性脑胶质瘤细胞的杀伤活性明显高于其他四组,差别有显著性意义(P0.001);NY-ESO-1多肽负载的DC组淋巴细胞杀伤活性高于HSP70负载的DC组、未经负载的DC组以及对照组,差别有显著性意义(P0.001);HSP70负载的DC组、未经负载的DC组与对照组相比杀伤率无显著差别(P0.05)。【结论】 1.成功构建HSP70的原核表达载体,并高效表达HSP70蛋白,纯化可获得大量高纯度的HSP70蛋白。 2.联合应用细胞因子rhGM-CSF、rhIL-4和TNF-α可以从人脐带血单个核细胞中培养出足够数量的成熟DC。 3.HSP70-NY-ESO-1抗原肽复合物负载DC能在体外有效激活和扩增肿瘤特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL),HSP70能够协同NY-ESO-1抗原肽对NY-ESO-1阳性脑胶质瘤细胞的杀伤,增强NY-ESO-1抗原肽的抗肿瘤免疫作用。
[Abstract]:[Objective]
HSP70-NY-ESO-1 polypeptide complex was prepared by human heat shock protein 70 (heat shock protein70, HSP70) derived from the prokaryotic expression and purification of human heat shock protein protein70 (HSP70), and the killing efficiency of HSP70-NY-ESO-1 polypeptide complex loaded with dendritic cells (dendritic cells, DC) on the in vitro killing efficiency of NY-ESO-1 positive tumor cells was investigated. An effective strategy for enhancing tumor immunotherapy.
[method]
The prokaryotic expression, identification and purification of 1. people HSP70: RT-PCR technology obtained the full length cDNA of human HSP70, and cloned the PCR product on the prokaryotic expression vector PET-30a plasmid. After the enzyme digestion and DNA sequencing, the recombinant plasmid was transformed to the Escherichia coli Rosetta (DE3), and the egg white expression was induced, and the expression product was identified and identified. HSP70 protein was purified by G chromatography column.
Culture and identification of 2. human umbilical cord blood dendritic cells: combined use of recombinant human granulocyte / macrophage colony stimulating factor (recombination human granulocyte/macrophage colony-stimulating factor, rhGM-CSF) and recombinant human interleukin 4 (recombination human interleukin-4, rhIL-4) to cultivate human umbilical cord blood mononuclear cells DC, tumor necrosis factor alpha (tumor necrosis factor- a, TNF- alpha) induced its maturation. The morphology of DC was observed by inverted microscope, optical microscope and transmission electron microscope. The DC phenotype was identified by flow cytometry and the function of DC was identified by mixed lymphocyte reaction.
The in vitro killing experiment of the 3.HSP70-NY-ESO-1 polypeptide complex loaded with DC: HSP70 and NY-ESO-1 polypeptide were connected in vitro, and the HSP70-NY-ESO-1 polypeptide complex was obtained. The DC of the HSP70-NY-ESO-1 polypeptide complex, DC of the NY-ESO-1 polypeptide loaded, DC on HSP70 load, and the unloaded DC to stimulate the lymphocyte. As a control group, a total of 5 groups of lymphocytes and lactic dehydroge-nase (LDH) release assay were used to detect the killing efficiency of lymphocytes against NY-ESO-1 positive glioma cells in vitro.
[results]
1. human HSP70 prokaryotic expression, identification and purification: RT-PCR amplification of the size of 2000bp of the target fragment, confirmed by enzyme digestion and DNA sequencing, the HSP70 gene was successfully cloned to the prokaryotic expression vector PET-30a plasmid, and the recombinant plasmid was induced by IPTG and SDS-PAGE showed a significant increase in the molecular weight of about 72KD. The protein bands and Western-blot confirmed that they were the target bands, and the purity of the purified protein reached 95%.
Culture and identification of 2. human umbilical cord blood dendritic cells: mature DC (mature DC, mDC) morphological observation of culture 8D: large cells, irregular morphology, typical dendritic protuberances, flow cytometry identification: mDC expression HLA-DR (80.65%), CD86 (76.59%), CD83 (74.81%), and less mature DC (immature DC) increased significantly CD83 was 25.20%, 17.58%, 1.50%, respectively. Mixed lymphocyte reaction confirmed that mDC had strong ability to stimulate mixed lymphocyte reaction in vitro, while imDC was weak.
3.HSP70-NY-ESO-1 polypeptide complex loaded DC in vitro killing experiment: HSP70-NY-ESO-1 polypeptide complex loaded DC group lymphocytes to NY-ESO-1 positive brain glioma cells were significantly higher than the other four groups, the difference was significant (P0.001); NY-ESO-1 polypeptide loaded DC group lymphocyte killing activity was higher than the HSP70 load DC Groups, unloaded DC group and control group, the difference was significant (P0.001), HSP70 loaded DC group, unloaded DC group compared with the control group, there was no significant difference in killing rate (P0.05).
[Conclusion]
1. the prokaryotic expression vector of HSP70 was successfully constructed, and HSP70 protein was efficiently expressed, and a large quantity of high purity HSP70 protein could be obtained.
2. the combined use of cytokines rhGM-CSF, rhIL-4 and TNF- a can produce enough mature DC. from human umbilical cord blood mononuclear cells.
The 3.HSP70-NY-ESO-1 antigen peptide complex can effectively activate and amplify the tumor specific cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) in vitro. HSP70 can cooperate with NY-ESO-1 antigen peptide to kill NY-ESO-1 positive glioma cells and enhance the anti-tumor immunity of NY-ESO-1 antiproto peptide.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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