本文關(guān)鍵詞：野生型和突變型MBL的體外表達(dá)及其特性研究　出處：《浙江大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 重組甘露聚糖結(jié)合凝集素 GGC54GAC突變體 真核表達(dá)載體 CHO細(xì)胞

【摘要】：研究背景： 甘露糖結(jié)合凝集素(mannose-binding lectin, MBL)是天然免疫系統(tǒng)中的關(guān)鍵分子。它是由肝臟合成的C型凝集素超家族成員,主要存在于人體的血清中。MBL主要的生物學(xué)功能是識(shí)別并結(jié)合糖基配體以及凝集素途徑激活補(bǔ)體。人血清MBL濃度范圍為0-8.24μg/ml,一般認(rèn)為MBL濃度大于1μg/ml可發(fā)揮正常的生理功能。而MBL濃度低于0.5μg/ml則為MBL缺損。目前已知6個(gè)基因位點(diǎn)突變可引起MBL濃度降低。其中編碼膠原樣區(qū)的外顯子1區(qū)的54,57,52位密碼子突變(分別稱(chēng)為B、C、D型)：CGT52TGT、GGC54GAC和GGA57GAA對(duì)血清中MBL濃度影響較大。此外,啟動(dòng)子區(qū)5’端的-550(H/L),-221(X/Y)和5’端非轉(zhuǎn)錄區(qū)+4(P/Q)位點(diǎn)突變對(duì)血清中MBL濃度也有一定影響。B型突變?cè)趤喼藜案呒铀魅巳褐斜容^普遍,基因頻率約為25%,C型突變?cè)谌龉侵奕巳罕容^普遍,基因頻率50%-60%,而D型突變頻率偏低,主要見(jiàn)于歐洲人群,基因頻率僅有14%,且對(duì)MBL濃度影響較小。目前研究發(fā)現(xiàn)中國(guó)人群MBL基因突變以B型突變最常見(jiàn),而C和D型突變較為罕見(jiàn)。MBL的亞單位由3個(gè)多肽鏈組成,每條鏈都含有一個(gè)凝集素區(qū)(又稱(chēng)為糖類(lèi)識(shí)別區(qū)CRDs)。CRDs可以識(shí)別病原體上的甘露糖、N-乙酰葡糖胺、葡萄糖和巖藻糖等碳水化合物。MBL與病毒表面多種糖基高效結(jié)合組成了機(jī)體抗病毒的第一道防線(xiàn)且參與感染進(jìn)程的調(diào)控。目前對(duì)A型流感病毒、HIV、絲狀病毒：Ebola病毒和Marburg病毒都有相關(guān)研究,表明MBL在阻斷病毒感染、抑制病毒擴(kuò)散方面發(fā)揮重要作用。研究表明MBL缺陷個(gè)體對(duì)HIV和HCMV易感性增加,疾病進(jìn)展過(guò)程加快,繼發(fā)隱孢子蟲(chóng)病等二重感染率也顯著增加。國(guó)內(nèi)的研究資料也表明,低MBL水平兒童易患HCMV感染,且癥狀較重并存在反復(fù)感染傾向。但目前尚未見(jiàn)將MBL應(yīng)用于HCMV感染研究的報(bào)道。因此本研究構(gòu)建了在中國(guó)人群基因突變頻率最高的GGC54GAC突變型(即B型)和野生型(A型)MBL真核表達(dá)載體,轉(zhuǎn)染入CHO細(xì)胞,獲得MBL的體外表達(dá),研究重組MBL的特性,并與天然MBL比較,探索MBL基因突變導(dǎo)致MBL免疫缺陷的發(fā)病機(jī)制,為下一步野生型和GGC54GAC突變型MBL在體外對(duì)HCMV抑制作用和機(jī)制的研究奠定基礎(chǔ),并為MBL缺損患者替代治療的研究奠定一定基礎(chǔ)。 目的： 構(gòu)建野生型和GGC54GAC突變型MBL的真核表達(dá)載體,獲得重組野生型和突變型MBL,并研究獲得的重組MBL的特性,為下一步MBL阻斷HCMV感染的作用和機(jī)制的研究奠定基礎(chǔ)。 方法： 構(gòu)建野生型和突變型MBL表達(dá)質(zhì)粒：PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54,通過(guò)陽(yáng)離子脂質(zhì)體法轉(zhuǎn)染方法轉(zhuǎn)染CHO細(xì)胞,G418篩選轉(zhuǎn)染子,在轉(zhuǎn)染后24小時(shí),48小時(shí),1周通過(guò)熒光顯微鏡觀察MBL基因在CHO細(xì)胞的表達(dá),獲得穩(wěn)定轉(zhuǎn)染子MBL-CHO和CHO-MBLm54。Trizol法提取MBL的mRNA,以熒光定量PCR方法分析野生型和突變型MBL基因在CHO細(xì)胞的表達(dá)。大量培養(yǎng)MBL-CHO和CHO-MBLm54,第3天收集培養(yǎng)上清,ELISA法測(cè)定重組野生型和突變型MBL的糖基結(jié)合活性。超濾管濃縮,培養(yǎng)上清預(yù)處理,通過(guò)mannan-Sepharose 4B親和層析獲得純化的野生型MBL。Bradford法測(cè)定野生型MBL的濃度,聚丙酰胺凝膠電泳和考馬斯亮藍(lán)染色分析野生型MBL的分子量和純度。 結(jié)果： 1.真核表達(dá)質(zhì)粒PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54經(jīng)雙向凝膠電泳和測(cè)序驗(yàn)證構(gòu)建成功。 2.真核表達(dá)質(zhì)粒PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54經(jīng)陽(yáng)離子脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染CHO細(xì)胞,熒光顯微鏡觀察及熒光定量PCR驗(yàn)證野生型及突變型MBL基因在CHO細(xì)胞獲得成功表達(dá)。 3. ELISA法顯示重組野生型MBL具備甘露聚糖結(jié)合活性,而突變型MBL則無(wú)甘露聚糖結(jié)合活性 4. Bradford法測(cè)定培養(yǎng)上清中野生型MBL的濃度為559.82 ng/ml。 5.聚丙酰胺凝膠電泳分析野生型MBL的分子量,單條肽鏈分子量為32KDa,非還原條件下MBL主要為分子量大于170KDa的寡聚體及多聚體。 結(jié)論： 1.構(gòu)建了野生型和GGC54GAC突變型MBL基因的真核表達(dá)載體,其在CHO細(xì)胞成功表達(dá)。 2. GGC54GAC結(jié)構(gòu)基因突變導(dǎo)致寡聚體及多聚體結(jié)構(gòu)的MBL表達(dá)不佳。 3.獲得了高純度的寡聚體及多聚體結(jié)構(gòu)的野生型重組MBL,具備甘露聚糖結(jié)合活性,其在培養(yǎng)上清中的濃度可滿(mǎn)足下一步實(shí)驗(yàn)需求。 本課題屬“甘露聚糖結(jié)合凝集素阻斷HCMV侵入靶細(xì)胞機(jī)制的研究”課題的部分內(nèi)容,為后續(xù)研究奠定了基礎(chǔ),同時(shí)也為MBL缺損患者替代治療的研究奠定了一定的基礎(chǔ)。
[Abstract]:Research background:
Mannose binding lectin (mannose-binding lectin MBL) is a key molecule of the innate immune system. It is composed of C type synthetic liver lectin superfamily, mainly in the main body of the biological function of.MBL in serum is to recognize and bind sugar ligand and coagulation in angiotensin pathway complement activation of human serum MBL concentration range. 0-8.24 g/ml, general MBL concentration is more than 1 g/ml can play a normal physiological function. But the concentration of MBL is less than 0.5 g/ml for the MBL defect. At present 6 gene loci known mutation can cause a decrease of MBL concentration. The collagenous region encoding exon 54,57,52 codon 1 mutation sub region (referred respectively B, C, D):CGT52TGT, GGC54GAC and GGA57GAA has great influence on serum MBL concentration. In addition, the promoter region of 5 'end of -550 (H/L), -221 (X/Y) and 5' untranslated region of +4 (P/Q) mutation on serum MBL concentration Have a certain effect of.B mutation is common in Asian and Caucasian population, the gene frequency was about 25%, C mutation in sub Saharan African population is relatively common, the frequency of 50%-60% gene, and D mutation frequency is low, mainly in the European population, the gene frequency is only 14%, and had little effect on the concentration of MBL. The present study found that people Chinese MBL gene mutations in the B mutation is the most common form of subunit C and D mutations were rare.MBL is composed of 3 polypeptide chains, each chain contains a lectin region (also known as carbohydrate recognition region CRDs).CRDs can identify pathogens on N- mannose, N-acetylglucosamine, galactose and glucose carbohydrates such as.MBL and virus surface of various sugar based efficient combination of control components of the first line of defense and participate in the process of antiviral infection. The influenza A virus, HIV, filamentous virus: Ebola virus and Marburg virus Have the relevant research, showed that MBL in blocking viral infection, plays an important role in inhibiting the spread of the virus. The results indicate that the MBL defect of the individual susceptibility to HIV and HCMV increase, accelerate the progress of the disease, the double infection secondary to Cryptosporidium disease rate significantly increased. The domestic research material also shows that low levels of MBL children susceptible to HCMV infection, and severe symptoms and repeated infection tendency. But there is no research on the application of MBL infection HCMV reported. Therefore this study constructed GGC54GAC the highest frequency of mutations in the population China gene mutation type (B type) and wild type (A type) MBL eukaryotic expression vector was transfected into CHO MBL cells, in vitro expression, characterization of recombinant MBL, and compared with natural MBL, to explore the pathogenesis of MBL mutations in the MBL gene cause immunodeficiency, the next step for the wild type and GGC54GAC mutant MBL in vitro inhibition of HCMV It lays the foundation for the study of use and mechanism, and lays a foundation for the study of replacement therapy for MBL defect patients.
Objective:
We constructed the eukaryotic expression vector of wild-type and GGC54GAC mutant MBL, obtained the recombinant wild-type and mutant MBL, and studied the characteristics of the recombinant MBL. It laid the foundation for further research on the role and mechanism of MBL blocking HCMV infection.
Method錛，

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