ZMP1调节RAW264.7自噬的初步研究

发布时间：2018-03-14 21:07

本文选题：Zmp1　切入点：巨噬细胞　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.诱导表达Zmp1,纯化获得Zmp1重组蛋白;用纯化到的重组蛋白Zmp1免疫新西兰大白兔,制备Zmp1多克隆抗体.2.用Zmp1蛋白定量作用雷帕霉素处理过的RAW264.7细胞,观察Zmp1蛋白对巨噬细胞RAW264.7自噬的影响。3.构建Zmp1真核表达载体,转染RAW264.7细胞,验证Zmp1的表达,雷帕霉素处理转染Zmp1的RAW264.7细胞,观察真核表达Zmp1对巨噬细胞RAW264.7自噬的影响。方法:1.将原核表达载体p ET32a(+)-zmp1转化至大肠杆菌BL21(DE3)中,经IPTG诱导,用SDS-PAGE和Western blot鉴定其表达;用金属螯合磁珠纯化,超滤管浓集,获得纯化蛋白;用BCA蛋白定量试剂盒定量纯化蛋白。2.用纯化后的Zmp1重组蛋白作为抗原免疫新西兰大白兔,制备Zmp1多克隆抗体,用Western blot检测抗体的特异性和ELISA检测抗体效价。3.培养RAW264.7细胞,用不同浓度雷帕霉素诱导小鼠巨噬细胞RAW264.7,用同一浓度雷帕霉素诱导RAW264.7不同时长,Western blot检测自噬相关蛋白LC3的表达,观察雷帕霉素作用量和时间与诱导自噬强度的关系。选取雷帕霉素最适作用量和时间,诱导RAW264.7细胞,用不同剂量Zmp1蛋白作用巨噬细胞,透射电镜下观察自噬体形成的变化,Western blot检测自噬相关蛋白LC3表达,实时荧光定量PCR(RT-q PCR)检测自噬相关基因Atg5,Atg8,Atg12的m RNA表达水平。4.设计引物,采用PCR从BCG基因组中克隆zmp1基因,将其连接到真核表达载体pEGFP-C1上,构建重组真核表达载体pEGFP-C1-zmp1,经酶切、测序鉴定重组载体构建成功后,用LipofectamineTM 3000 Reagent将质粒转染至RAW264.7中,在倒置荧光显微镜下观察细胞转染情况,实时荧光定量PCR检测转染细胞中zmp1基因m RNA的表达水平,Western blot检测Zmp1表达。5.雷帕霉素作用转染pEGFP-C1-zmp1后的RAW264.7细胞,透射电镜下观察自噬体形成的变化,Western blot检测自噬相关蛋白LC3表达,q RT-PCR检测自噬相关基因Atg5,Atg8,Atg12的m RNA表达水平。结果:1.SDS-PAGE显示,IPTG诱导出大小约为95 KD的蛋白,与Zmp1重组融合蛋白大小相符,Western blot结果在95 k D处可见阳性条带。经磁珠纯化和超滤管超滤浓集的重组融合蛋白,电泳后在95 k D处可见纯化且浓集的蛋白条带,测得重组蛋白含量为3.59 mg/m L。2.ELISA检测抗血清效价大于1∶102400,Western blot结果证实制备的多克隆抗体与Zmp1原重组蛋白特异结合。3.通过Western blot检测自噬相关蛋白LC3的表达,100 ng/μL雷帕霉素作用RAW264.7细胞16 h后,LC3Ⅱ表达水平较对照组明显升高,差异有统计学意义(P0.05)。透射电镜下观察自噬体形成,Zmp1蛋白作用组较雷帕霉素组自噬体数量明显减少;Western blot检测自噬相关蛋白LC3表达,Zmp1作用组较雷帕霉素组LC3Ⅱ表达水平降低,差异有统计学意义(P0.05),且随着Zmp1蛋白剂量增大,LC3Ⅱ表达水平有逐渐降低的趋势;RT-q PCR检测自噬相关基因Atg5,Atg8,Atg12的m RNA表达水平,Zmp1作用组较雷帕霉素组表达水平低,且差异有统计学意义(P0.05)。4.PCR克隆出zmp1基因,重组真核表达质粒pEGFP-C1-zmp1酶切后,得到与理论值大小相符合的条带,DNA双向测序结果与Gen Bank中录注的序列相同。转染至RAW264.7后,荧光显微镜下可见绿色荧光蛋白的表达,RT-q PCR和Western blot鉴定重组真核表达载体pEGFP-C1-zmp1在RAW264.7细胞中能表达出Zmp1 m RNA和蛋白,Western blot结果在98 k D处可见阳性条带。5.将pEGFP-C1-zmp1转染至RAW264.7 24 h后,雷帕霉素诱导自噬,收集细胞,透射电镜下观察自噬体形成的变化,Zmp1质粒转染组较雷帕霉素组自噬体数量明显减少;Western blot检测自噬相关蛋白LC3表达,Zmp1质粒转染组较雷帕霉素组LC3Ⅱ表达水平降低,差异有统计学意义(P0.05);RT-q PCR检测自噬相关基因Atg5,Atg8,Atg12的m RNA表达水平,Zmp1作用组较雷帕霉素组表达水平低,且差异有统计学意义(P0.05)。结论:1.成功表达纯化浓集Zmp1蛋白,免疫新西兰大白兔制备出Zmp1多克隆抗体。2.成功构建pEGFP-C1-zmp1真核表达载体,转染RAW264.7可表达Zmp1蛋白。3.巨噬细胞内和细胞外表达的Zmp1蛋白均可能抑制小鼠巨噬细胞RAW264.7巨噬自噬过程。
[Abstract]:Objective: 1. expression of Zmp1 induced by Zmp1, the purified recombinant protein; with purified recombinant protein Zmp1 to New Zealand white rabbits were immunized to prepare Zmp1 polyclonal antibody of.2. Zmp1 protein quantitative effect of rapamycin treated RAW264.7 cells, Zmp1 protein was observed on macrophage RAW264.7 effects of autophagy.3. Zmp1 eukaryotic expression vector was constructed and transfected into RAW264.7 cells, expression of Zmp1 was confirmed, RAW264.7 cells transfected with Zmp1 rapamycin treatment, observe the effect of eukaryotic expression of Zmp1 on macrophage autophagy in RAW264.7. Methods: 1. the prokaryotic expression vector p ET32a (+) -zmp1 was transformed into Escherichia coli BL21 (DE3), induced by IPTG, the expression of SDS-PAGE and Western for blot identification; purified by metal chelating beads, ultrafiltration concentration, purified protein; BCA protein assay kit quantitative purified protein.2. as antigen to immunize New Zealand white with purified recombinant Zmp1 protein Rabbit, preparation of Zmp1 polyclonal antibody, RAW264.7 cells cultured with.3. specific ELISA and Western antibody titer detection blot detection antibody, RAW264.7 mouse macrophages with different concentrations of rapamycin induced by the same concentration of rapamycin induced RAW264.7 and Western expression, blot detection of autophagy related protein LC3, to observe the relationship between the effect of rapamycin on amount and time with the strength of the selection of rapamycin induced autophagy. The most suitable for the amount and time in RAW264.7 cells induced by different dose of Zmp1 protein in macrophages, changes of autophagy bodies were observed under transmission electron microscope, the expression of Western blot detection of autophagy related protein LC3, real-time fluorescence quantitative PCR (RT-q PCR) to detect autophagy related genes Atg5, Atg8, m, RNA the expression of Atg12.4. primers were designed to clone the zmp1 gene from the BCG genome by PCR, connected to the eukaryotic expression vector pEGFP-C1 to construct. Construction of recombinant eukaryotic expression vector pEGFP-C1-zmp1 by restriction enzyme digestion, sequencing the recombinant vector constructed successfully, using LipofectamineTM 3000 Reagent plasmid was transfected into RAW264.7 cells, observe the transfection under the inverted fluorescence microscope, the expression level of zmp1 gene in m real time fluorescence quantitative PCR detection of transfected cells RNA the expression of Western, blot detection of Zmp1.5. of rapamycin the role of pEGFP-C1-zmp1 after transfection of RAW264.7 cells, changes of autophagosome formation were observed under transmission electron microscope, the expression of Western blot detection of autophagy related protein LC3, Q RT-PCR to detect the autophagy related genes Atg5, Atg8, the expression level of M RNA Atg12. Results: 1.SDS-PAGE showed that IPTG induced the size of approximately 95 KD protein, consistent with protein size Zmp1 Western blot recombinant fusion, results showed positive bands in 95 K D. After purification beads and ultrafiltration ultrafiltration concentration of recombinant fusion protein after electrophoresis. In 95 K D protein purification and concentration of the visible band, the measured recombinant protein content was 3.59 mg/m L.2.ELISA serum titer of more than 1: 102400, Western blot confirmed the expression of polyclonal antibody and Zmp1 recombinant protein specifically binds to.3. by Western blot detection of autophagy related protein LC3, ng/ 100 L rapamycin RAW264.7 cells after 16 h, the expression level of LC3 was significantly higher than the control group, the difference was statistically significant (P0.05). The formation of autophagosomes were observed under transmission electron microscope, Zmp1 protein group than in rapamycin group significantly reduced the number of autophagosomes; the expression of Western blot detection of autophagy related protein LC3, Zmp1 expression group than in rapamycin group LC3 II levels decreased, the difference was statistically significant (P0.05), and with the increasing doses of Zmp1 protein, LC3 II expression level decreased; RT-q PCR detection of autophagy related gene Atg5, Atg 8, m RNA Atg12 the expression level of Zmp1 group than in rapamycin group the expression level is low, and the difference was statistically significant (P0.05.4.PCR) zmp1 gene was cloned and the recombinant eukaryotic expression plasmid pEGFP-C1-zmp1 was digested by strip size consistent with the theoretical value, the sequence of DNA bidirectional sequencing result and Gen Bank. Note the same. After RAW264.7 transfection, expression of green fluorescent protein under fluorescence microscope, pEGFP-C1-zmp1 vector can express Zmp1 m and RNA protein in RAW264.7 cells of eukaryotic expression RT-q PCR and Western blot blot results in the identification of recombinant Western, 98 K D showed positive bands was transfected with pEGFP-C1-zmp1.5. RAW264.7 24 h after rapamycin induced autophagy, collecting cells and changes of autophagosome formation were observed under transmission electron microscope, Zmp1 plasmid transfection group than in rapamycin group significantly reduced the number of autophagosomes; Western blot detected the autophagy related protein White LC3 expression, Zmp1 expression plasmid was transfected into LC3 II group than in rapamycin group decreased, the difference was statistically significant (P0.05); RT-q PCR detection of autophagy related genes Atg5, Atg8, m RNA Atg12 the expression level of Zmp1 group than in rapamycin group low expression levels, and the difference was statistically significant (P0.05) conclusion. 1.: the successful expression and purification of Zmp1 protein concentration, New Zealand white rabbits were immunized to prepare Zmp1 polyclonal antibody.2. pEGFP-C1-zmp1 eukaryotic expression vector was successfully constructed and transfected into RAW264.7 Zmp1 protein expression in.3. macrophages and extracellular expression of Zmp1 protein may inhibit RAW264.7 mouse macrophage macrophage autophagy.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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