野生型和突变型MBL的体外表达及其特性研究

发布时间：2018-01-01 00:34

  本文关键词：野生型和突变型MBL的体外表达及其特性研究　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 重组甘露聚糖结合凝集素 GGC54GAC突变体 真核表达载体 CHO细胞

【摘要】：研究背景： 甘露糖结合凝集素(mannose-binding lectin, MBL)是天然免疫系统中的关键分子。它是由肝脏合成的C型凝集素超家族成员,主要存在于人体的血清中。MBL主要的生物学功能是识别并结合糖基配体以及凝集素途径激活补体。人血清MBL浓度范围为0-8.24μg/ml,一般认为MBL浓度大于1μg/ml可发挥正常的生理功能。而MBL浓度低于0.5μg/ml则为MBL缺损。目前已知6个基因位点突变可引起MBL浓度降低。其中编码胶原样区的外显子1区的54,57,52位密码子突变(分别称为B、C、D型)：CGT52TGT、GGC54GAC和GGA57GAA对血清中MBL浓度影响较大。此外,启动子区5’端的-550(H/L),-221(X/Y)和5’端非转录区+4(P/Q)位点突变对血清中MBL浓度也有一定影响。B型突变在亚洲及高加索人群中比较普遍,基因频率约为25%,C型突变在撒哈拉非洲人群比较普遍,基因频率50%-60%,而D型突变频率偏低,主要见于欧洲人群,基因频率仅有14%,且对MBL浓度影响较小。目前研究发现中国人群MBL基因突变以B型突变最常见,而C和D型突变较为罕见。MBL的亚单位由3个多肽链组成,每条链都含有一个凝集素区(又称为糖类识别区CRDs)。CRDs可以识别病原体上的甘露糖、N-乙酰葡糖胺、葡萄糖和岩藻糖等碳水化合物。MBL与病毒表面多种糖基高效结合组成了机体抗病毒的第一道防线且参与感染进程的调控。目前对A型流感病毒、HIV、丝状病毒：Ebola病毒和Marburg病毒都有相关研究,表明MBL在阻断病毒感染、抑制病毒扩散方面发挥重要作用。研究表明MBL缺陷个体对HIV和HCMV易感性增加,疾病进展过程加快,继发隐孢子虫病等二重感染率也显著增加。国内的研究资料也表明,低MBL水平儿童易患HCMV感染,且症状较重并存在反复感染倾向。但目前尚未见将MBL应用于HCMV感染研究的报道。因此本研究构建了在中国人群基因突变频率最高的GGC54GAC突变型(即B型)和野生型(A型)MBL真核表达载体,转染入CHO细胞,获得MBL的体外表达,研究重组MBL的特性,并与天然MBL比较,探索MBL基因突变导致MBL免疫缺陷的发病机制,为下一步野生型和GGC54GAC突变型MBL在体外对HCMV抑制作用和机制的研究奠定基础,并为MBL缺损患者替代治疗的研究奠定一定基础。 目的： 构建野生型和GGC54GAC突变型MBL的真核表达载体,获得重组野生型和突变型MBL,并研究获得的重组MBL的特性,为下一步MBL阻断HCMV感染的作用和机制的研究奠定基础。 方法： 构建野生型和突变型MBL表达质粒：PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54,通过阳离子脂质体法转染方法转染CHO细胞,G418筛选转染子,在转染后24小时,48小时,1周通过荧光显微镜观察MBL基因在CHO细胞的表达,获得稳定转染子MBL-CHO和CHO-MBLm54。Trizol法提取MBL的mRNA,以荧光定量PCR方法分析野生型和突变型MBL基因在CHO细胞的表达。大量培养MBL-CHO和CHO-MBLm54,第3天收集培养上清,ELISA法测定重组野生型和突变型MBL的糖基结合活性。超滤管浓缩,培养上清预处理,通过mannan-Sepharose 4B亲和层析获得纯化的野生型MBL。Bradford法测定野生型MBL的浓度,聚丙酰胺凝胶电泳和考马斯亮蓝染色分析野生型MBL的分子量和纯度。 结果： 1.真核表达质粒PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54经双向凝胶电泳和测序验证构建成功。 2.真核表达质粒PIRES2-AcGFP-MBL和PIRES2-AcGFP-MBLm54经阳离子脂质体转染法转染CHO细胞,荧光显微镜观察及荧光定量PCR验证野生型及突变型MBL基因在CHO细胞获得成功表达。 3. ELISA法显示重组野生型MBL具备甘露聚糖结合活性,而突变型MBL则无甘露聚糖结合活性 4. Bradford法测定培养上清中野生型MBL的浓度为559.82 ng/ml。 5.聚丙酰胺凝胶电泳分析野生型MBL的分子量,单条肽链分子量为32KDa,非还原条件下MBL主要为分子量大于170KDa的寡聚体及多聚体。 结论： 1.构建了野生型和GGC54GAC突变型MBL基因的真核表达载体,其在CHO细胞成功表达。 2. GGC54GAC结构基因突变导致寡聚体及多聚体结构的MBL表达不佳。 3.获得了高纯度的寡聚体及多聚体结构的野生型重组MBL,具备甘露聚糖结合活性,其在培养上清中的浓度可满足下一步实验需求。 本课题属“甘露聚糖结合凝集素阻断HCMV侵入靶细胞机制的研究”课题的部分内容,为后续研究奠定了基础,同时也为MBL缺损患者替代治疗的研究奠定了一定的基础。
[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.
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