GST pull-down和免疫共沉淀联合质谱分析筛查弓形虫ROP18相互作用蛋白的研究

发布时间：2018-06-03 17:42

本文选题：弓形虫 + ROP18　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的筛选神经干细胞内与弓形虫ROP18相互作用的蛋白,为ROP18致病的分子机制研究奠定基础。方法第一部分GST Pull-Down筛选C17.2神经干细胞内与弓形虫ROP18的互作蛋白:(1)运用RT-PCR扩增ROP18基因;运用Optigene TM密码子优化分析平台对弓形虫ROP18编码基因进行优化,合成优化后的全长基因(ROP18u);以优化后的ROP18u基因为模板,PCR扩增去除N端信号肽和前功能区序列的截短ROP18片段(ROP18uc),将此3种基因分别克隆入pGEX-6P-1质粒,构建原核表达载体,所有重组质粒均经PCR、双酶切和测序鉴定正确后,分别转化大肠埃希菌BL21(DE3)诱导表达,Western Blot鉴定重组蛋白,选取可溶性表达最高的菌种。(2)2%N2的DMEM/F12培养液诱导C17.2分化,收集细胞并提取总蛋白。(3)诱导表达ROP18u-GST蛋白和GST标签蛋白,利用体外Pull Down技术分别作用于分化C17.2总蛋白,通过对比获得可能与ROP18u存在相互作用的蛋白,并进行质谱分析。第二部分免疫共沉淀技术筛选c17.2神经干细胞内与弓形虫rop18的互作蛋白:(1)rt-pcr扩增rop18基因,并无缝克隆至慢病毒载体pcdh-cmv-gfp中,经pcr、酶切和测序鉴定正确后,与辅助质粒pmlg/prre、prsv-rev和pmd2.g共转染293t细胞包装pcdh-cmv-gfp-rop18慢病毒,同时包装pcdh-cmv-gfp空载体慢病毒作为对照组。(2)pcdh-cmv-gfp-rop18慢病毒和pcdh-cmv-gfp空载慢病毒分别感染c17.2,加以2%n2的dmem/f12诱导其分化,待绿色荧光大量表达时收集细胞并分别提取总蛋白。(3)合成两段rop18抗原决定簇多肽,分别免疫新西兰兔制备两种兔抗rop18多克隆抗体,并进行elisa和westernblot鉴定。(4)利用rop18多克隆抗体,分别同两种慢病毒感染的分化c17.2总蛋白进行免疫共沉淀,以期通过对比获得在c17.2分化过程中可能与rop18相互作用的蛋白,与gstpull-down的结果相呼应。结果第一部分:(1)经诱导表达后对比发现,优化后的rop18u-gst可溶性蛋白表达量相较于rop18-gst明显上升,经优化剪切后表达的rop18uc-gst蛋白可溶性表达量无显著提升。(2)c17.2经分化后,显微镜下可见胞体上出现明显的形态、数量和长短各不相同的突起,呈树枝分支状。(3)gst标签蛋白pulldownc17.2分化总蛋白前后所得样本经sds-page检测无明显差异;rop18u-gst蛋白与c17.2总蛋白pulldown后的样本相较于pulldown前,sds-page胶多出明显蛋白条带,经质谱分析获得多个可能与rop18存在相互作用的鼠源性蛋白。第二部分:(1)重组慢病毒质粒pcdh-cmv-gfp-rop18经测序鉴定无误,与辅助质粒共转染293t细胞,细胞出现明显cpe且荧光显微镜下观察到明显的绿色荧光,包装后第2天收获并浓缩pcdh-cmv-gfp-rop18慢病毒;同样方法包装空载pCDH-CMV-GFP慢病毒。(2)两种ROP18多克隆抗体,经ELISA检测,效价分别达到1:64000和1:16000;经Western Blot鉴定,能够特异性识别ROP18重组蛋白。(3)两组慢病毒感染的C17.2均出现不同程度的CPE,荧光显微镜下发现明显绿色荧光。(4)ROP18多克隆抗体未能拉下ROP18蛋白,经Western Blot检测,大部分ROP18蛋白仍残留于免疫共沉淀后的上清中。结论(1)密码子优化未能显著提高ROP18重组蛋白的表达量,但提高了可溶性蛋白的含量。(2)GST Pull-Down联合质谱结果表明ROP18与C17.2神经干细胞内的细胞骨架蛋白、组蛋白等多种蛋白间可能存在相互作用。(3)成功包装ROP18慢病毒感染C17.2神经干细胞,引起细胞病变,并稳定表达ROP18。(4)成功制备两种ROP18多克隆抗体,能够特异性识别ROP18重组蛋白,但不适用于免疫共沉淀实验。
[Abstract]:Objective to screen the proteins interacting with Toxoplasma ROP18 in neural stem cells, and to lay the foundation for the molecular mechanism of ROP18. Method first part GST Pull-Down was used to screen the interaction proteins between C17.2 neural stem cells and Toxoplasma ROP18: (1) RT-PCR amplification of ROP18 gene and Optigene TM codon optimization analysis platform for Toxoplasma gondii The ROP18 encoding gene was optimized and the optimized full length gene (ROP18u) was synthesized. The optimized ROP18u base was amplified by PCR to remove the N terminal signal peptide and the truncated ROP18 fragment (ROP18uc) of the sequence of the anterior functional region. The 3 genes were cloned into pGEX-6P-1 plasmids, and the prokaryotic expression vector was constructed. All the recombinant plasmids were cut by PCR, double enzyme and the gene was constructed. After sequencing and identification, BL21 (DE3) induced expression of Escherichia coli was transformed, and Western Blot was used to identify the recombinant protein and select the highest soluble strain. (2) 2%N2 DMEM/F12 culture medium induced C17.2 differentiation, collected the cells and extracted the total protein. (3) the expression of ROP18u-GST protein and GST labelling protein was induced, and Pull Down technology in vitro was used respectively. The C17.2 protein was differentiated and the proteins interacting with ROP18u were obtained by contrast and mass spectrometric analysis. The interaction protein of C17.2 neural stem cells and Toxoplasma rop18 was screened by second part immunoprecipitation techniques: (1) RT-PCR amplification of rop18 gene, no slit cloning to the lentivirus carrier pcdh-cmv-gfp, PCR, enzyme digestion After the sequence identification was correct, the pcdh-cmv-gfp-rop18 lentivirus was packaged with the auxiliary plasmid pmlg/prre, prsv-rev and pmd2.g, and the pcdh-cmv-gfp no-load lentivirus was packaged as the control group. (2) pcdh-cmv-gfp-rop18 lentivirus and pcdh-cmv-gfp empty lentivirus infected C17.2 respectively, and 2%n2 dmem/f12 induced their differentiation to be green. When the color fluorescence was expressed in large quantities, the cells were collected and the total protein was extracted respectively. (3) two segments of rop18 antigen determinant polypeptide were synthesized and two Rabbit anti rop18 polyclonal antibodies were prepared from New Zealand rabbits, respectively, and ELISA and Westernblot were identified. (4) rop18 polyclonal antibody was used to immunize the total C17.2 total protein of two kinds of lentivirus infection respectively. Precipitation, in order to obtain the protein that may interact with rop18 during the differentiation of C17.2, echoes with the results of gstpull-down. Results the first part: (1) after the induction of expression, the optimized expression of rop18u-gst soluble protein was significantly higher than that of rop18-gst, and the rop18uc-gst protein expressed after the optimized shear was expressed. There was no significant increase in soluble expression. (2) after the differentiation of C17.2, the apparent morphology was found on the cell body under the microscope, and the number and length were different. (3) there was no significant difference between the samples before and after the GST labeling protein pulldownc17.2 differentiation total protein; rop18u-gst protein and C17.2 total protein pulldown After the samples were compared with the pulldown, the SDS-PAGE glue had more obvious protein bands, and several mouse derived proteins that might interact with rop18 were obtained by mass spectrometry. The second part: (1) the recombinant lentivirus plasmid pcdh-cmv-gfp-rop18 was sequenced and identified, and the 293T cells were co transfected with the auxiliary plasmids, and the cells showed a clear CPE and fluorescence microscope. The obvious green fluorescence was observed, and pcdh-cmv-gfp-rop18 lentivirus was harvested and concentrated at second days after packaging; the same method was used to package the empty pCDH-CMV-GFP lentivirus. (2) two ROP18 polyclonal antibodies were detected by ELISA, and the titers were 1:64000 and 1:16000 respectively. The ROP18 recombinant protein could be identified by Western Blot. (3) two groups of lentiviruses. The infected C17.2 showed different degrees of CPE and found obvious green fluorescence under the fluorescence microscope. (4) ROP18 polyclonal antibody failed to pull down ROP18 protein, and most of ROP18 protein remained in the supernatant after immunoprecipitation after Western Blot. Conclusion (1) optimization of codon did not significantly increase the expression of ROP18 recombinant protein, but improved The content of soluble protein. (2) GST Pull-Down combined mass spectrometry results showed that there might be interaction between the cytoskeleton protein, histone and other proteins in ROP18 and C17.2 neural stem cells. (3) the successful packaging of ROP18 lentivirus infected C17.2 neural stem cells, causing cytopathic disease, and stabilizing the expression of ROP18. (4) to produce two kinds of ROP18. The cloned antibody can specifically recognize ROP18 recombinant protein, but it is not suitable for coprecipitation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R382.5

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