本文選題：Zmp1　切入點：巨噬細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.誘導(dǎo)表達Zmp1,純化獲得Zmp1重組蛋白;用純化到的重組蛋白Zmp1免疫新西蘭大白兔,制備Zmp1多克隆抗體.2.用Zmp1蛋白定量作用雷帕霉素處理過的RAW264.7細(xì)胞,觀察Zmp1蛋白對巨噬細(xì)胞RAW264.7自噬的影響。3.構(gòu)建Zmp1真核表達載體,轉(zhuǎn)染RAW264.7細(xì)胞,驗證Zmp1的表達,雷帕霉素處理轉(zhuǎn)染Zmp1的RAW264.7細(xì)胞,觀察真核表達Zmp1對巨噬細(xì)胞RAW264.7自噬的影響。方法:1.將原核表達載體p ET32a(+)-zmp1轉(zhuǎn)化至大腸桿菌BL21(DE3)中,經(jīng)IPTG誘導(dǎo),用SDS-PAGE和Western blot鑒定其表達;用金屬螯合磁珠純化,超濾管濃集,獲得純化蛋白;用BCA蛋白定量試劑盒定量純化蛋白。2.用純化后的Zmp1重組蛋白作為抗原免疫新西蘭大白兔,制備Zmp1多克隆抗體,用Western blot檢測抗體的特異性和ELISA檢測抗體效價。3.培養(yǎng)RAW264.7細(xì)胞,用不同濃度雷帕霉素誘導(dǎo)小鼠巨噬細(xì)胞RAW264.7,用同一濃度雷帕霉素誘導(dǎo)RAW264.7不同時長,Western blot檢測自噬相關(guān)蛋白LC3的表達,觀察雷帕霉素作用量和時間與誘導(dǎo)自噬強度的關(guān)系。選取雷帕霉素最適作用量和時間,誘導(dǎo)RAW264.7細(xì)胞,用不同劑量Zmp1蛋白作用巨噬細(xì)胞,透射電鏡下觀察自噬體形成的變化,Western blot檢測自噬相關(guān)蛋白LC3表達,實時熒光定量PCR(RT-q PCR)檢測自噬相關(guān)基因Atg5,Atg8,Atg12的m RNA表達水平。4.設(shè)計引物,采用PCR從BCG基因組中克隆zmp1基因,將其連接到真核表達載體pEGFP-C1上,構(gòu)建重組真核表達載體pEGFP-C1-zmp1,經(jīng)酶切、測序鑒定重組載體構(gòu)建成功后,用LipofectamineTM 3000 Reagent將質(zhì)粒轉(zhuǎn)染至RAW264.7中,在倒置熒光顯微鏡下觀察細(xì)胞轉(zhuǎn)染情況,實時熒光定量PCR檢測轉(zhuǎn)染細(xì)胞中zmp1基因m RNA的表達水平,Western blot檢測Zmp1表達。5.雷帕霉素作用轉(zhuǎn)染pEGFP-C1-zmp1后的RAW264.7細(xì)胞,透射電鏡下觀察自噬體形成的變化,Western blot檢測自噬相關(guān)蛋白LC3表達,q RT-PCR檢測自噬相關(guān)基因Atg5,Atg8,Atg12的m RNA表達水平。結(jié)果:1.SDS-PAGE顯示,IPTG誘導(dǎo)出大小約為95 KD的蛋白,與Zmp1重組融合蛋白大小相符,Western blot結(jié)果在95 k D處可見陽性條帶。經(jīng)磁珠純化和超濾管超濾濃集的重組融合蛋白,電泳后在95 k D處可見純化且濃集的蛋白條帶,測得重組蛋白含量為3.59 mg/m L。2.ELISA檢測抗血清效價大于1∶102400,Western blot結(jié)果證實制備的多克隆抗體與Zmp1原重組蛋白特異結(jié)合。3.通過Western blot檢測自噬相關(guān)蛋白LC3的表達,100 ng/μL雷帕霉素作用RAW264.7細(xì)胞16 h后,LC3Ⅱ表達水平較對照組明顯升高,差異有統(tǒng)計學(xué)意義(P0.05)。透射電鏡下觀察自噬體形成,Zmp1蛋白作用組較雷帕霉素組自噬體數(shù)量明顯減少;Western blot檢測自噬相關(guān)蛋白LC3表達,Zmp1作用組較雷帕霉素組LC3Ⅱ表達水平降低,差異有統(tǒng)計學(xué)意義(P0.05),且隨著Zmp1蛋白劑量增大,LC3Ⅱ表達水平有逐漸降低的趨勢;RT-q PCR檢測自噬相關(guān)基因Atg5,Atg8,Atg12的m RNA表達水平,Zmp1作用組較雷帕霉素組表達水平低,且差異有統(tǒng)計學(xué)意義(P0.05)。4.PCR克隆出zmp1基因,重組真核表達質(zhì)粒pEGFP-C1-zmp1酶切后,得到與理論值大小相符合的條帶,DNA雙向測序結(jié)果與Gen Bank中錄注的序列相同。轉(zhuǎn)染至RAW264.7后,熒光顯微鏡下可見綠色熒光蛋白的表達,RT-q PCR和Western blot鑒定重組真核表達載體pEGFP-C1-zmp1在RAW264.7細(xì)胞中能表達出Zmp1 m RNA和蛋白,Western blot結(jié)果在98 k D處可見陽性條帶。5.將pEGFP-C1-zmp1轉(zhuǎn)染至RAW264.7 24 h后,雷帕霉素誘導(dǎo)自噬,收集細(xì)胞,透射電鏡下觀察自噬體形成的變化,Zmp1質(zhì)粒轉(zhuǎn)染組較雷帕霉素組自噬體數(shù)量明顯減少;Western blot檢測自噬相關(guān)蛋白LC3表達,Zmp1質(zhì)粒轉(zhuǎn)染組較雷帕霉素組LC3Ⅱ表達水平降低,差異有統(tǒng)計學(xué)意義(P0.05);RT-q PCR檢測自噬相關(guān)基因Atg5,Atg8,Atg12的m RNA表達水平,Zmp1作用組較雷帕霉素組表達水平低,且差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.成功表達純化濃集Zmp1蛋白,免疫新西蘭大白兔制備出Zmp1多克隆抗體。2.成功構(gòu)建pEGFP-C1-zmp1真核表達載體,轉(zhuǎn)染RAW264.7可表達Zmp1蛋白。3.巨噬細(xì)胞內(nèi)和細(xì)胞外表達的Zmp1蛋白均可能抑制小鼠巨噬細(xì)胞RAW264.7巨噬自噬過程。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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