【作者】 孟巧红；

【导师】 张嘉宁；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 甘露糖受体(Mannose Receptor,MR)又称CD206,是MR家族成员之一,属C-型凝集素受体,最早发现于大鼠肝枯否细胞,后发现广泛存在于动物体内的多种细胞,主要生物学功能表现在介导内吞清除内外源的多糖,多肽,蛋白,核酸和杀灭病原体,转运和递呈抗原启动免疫应答,是保守的模式识别受体(Pattern Recognition Receptor,PRR)之一。MR的分子量为175kDa,属I-型跨模蛋白,胞外部分分为N-末端的富含半胱氨酸结构域(Cysteine-rich Region,CR), II-型纤连蛋白重复结构域(Fibronectin Type II Repeat Region,FN II),8个C-型凝集素样糖识别结构域(C-Type Lectin like Domain,CTLD1-8),跨膜结构域和胞浆的C-末端序列。MR可通过不同的结构域识别结合多种配体。据报道,表达在淋巴管内皮细胞上的MR是L-选凝蛋白的天然配体,两者的相互作用介导了淋巴细胞的归巢。本实验室的前期工作表明小鼠肝癌细胞高低淋巴道转移株HCa-F和HCa-P的淋巴道转移潜能与其表达的L-选凝蛋白正相关,此外,发现淋巴瘤细胞p388D1的淋巴道转移潜能也与其表达的L-选凝蛋白相关。故认为MR与L-选凝蛋白存在的相互作用可能介导肿瘤的淋巴道转移,转移机制与淋巴细胞的“归巢”相似。实验目的:利用DNA重组技术将甘露糖受体MR的C-型凝集素样糖识别结构域4-7(CT- LD4-7)与分泌性信号肽Sig和人IgG的Fc段构成嵌合基因,插入到真核表达载体pCDNA3.1中,瞬转293T细胞,检测蛋白表达,为以后利用Protein-A亲和层析纯化蛋白,进行黏附实验来验证甘露糖受体MR与L-选凝蛋白相互作用,以及究其相互作用与肿瘤淋巴道转移的相关性提供基础。实验方法:1.酶切鉴定本实验室已有的连接有MR CTLD1-8序列的T载体,记为pMD-18T/ CTLD1-8,并以之为模板自行设计引物进行PCR扩增MR CTLD4-7序列(1701bp),通过TA亚克隆将CTLD4-7基因插入pMD-18T载体中构建载体pMD-18T/ CTLD4-7,转化细菌扩增质粒,酶切鉴定并测序。将pMD-18T/ CTLD4-7的CTLD4-7基因酶切并切胶回收,插入表达载体pX(pX载体的酶切位点上游连有IgG的分泌性信号肽,下游连有人IgG的Fc段)中,扩增并酶切鉴定后命名为pX/ CTLD4-7。单酶切pX/ CTLD4-7和表达载体pCDNA3.1,回收pX/ CTLD4-7的酶切产物片段Sig-CTLD4-7-Fc(2451bp)和pCDNA3.1片段,连接两者,构建的表达载体记作pCDNA3.1/Sig-CTLD 4-7-Fc,酶切鉴定,并用PCR的方法确定连入片段的方向,之后进行测序鉴定。2.培养293T细胞,脂质体法将去内毒素的pCDNA3.1/ Sig-CTLD4-7-Fc表达载体小量瞬转293T细胞,同时作阴性对照和阳性对照,分别在24h,48h,72h时收集培养上清,ELISA检测分泌蛋白的表达量,同时作细胞免疫化学进行表达的定性分析。实验结果:1.质粒转化细菌,扩增后用MluI单酶切和MluI、KpnI双酶切鉴定pMD-18T/ CTLD1-8,产生预期的3420 bp,2800 bp条带和2800bp,2120bp,1300bp条带;AatII,SalI双酶切鉴定pMD-18T/ CTLD4-7,产生预期的2200bp,1695bp,498bp条带,经测序序列正确;AatII,SalI双酶切和NotI单酶切鉴定pX/ CTLD4-7,产生预期的3621bp,1695bp和2865bp,2451bp条带;NotI单酶切鉴定pCDNA3.1/Sig-CTLD4-7-Fc产生5522 bp,2451 bp条带,PCR鉴定方向正确,测序结果正确,表明准确构建出真核表达载体。2. pCDNA3.1/ Sig-CTLD4-7-Fc表达载体瞬转293T细胞后,ELISA实验结果发现,嵌合蛋白在转然后24h时已开始分泌,分泌量在48h及72h升高;免疫细胞化学检测,确定在24h时CTLD4-7-Fc嵌合蛋白已经开始表达,而且在48h及72h持续。结论:1.重组的pcDNA3.1/Sig-CTLD4-7-Fc表达载体构建成功。2.通过脂质体法转染真核细胞293T,可在细胞中成功表达。更多还原

【Abstract】 Mannose receptor (CD206) is one member of the mannose receptor family and belongs to C-type lectin receptor. It was initially founded in rat liver Kupffer cell. Mannose receptor was also founded distributing extensively on many other kinds of tissue cell in vivo. Its major biological functions display in mediating the internalization and clearance of endogenous or exogenous polysaccharide, polypeptide, protein and nucleic acid, eliminating pathogen, conveying and presenting antibody and initiating immune response. It is one of the conservative pattern recognition receptors (PPR).Mannose receptor is a 175 kDa I type trans-membrane receptor consisting of five domains: a cysteine-rich amino terminus; a fibronectin type II repeat region; 8 C-type lectin-like carbohydrate recognition domains (CTLD1-8); a trans-membrane domain and a cytoplasmic domain. Mannose receptor can bind endogenous or exogenous ligands through different domains.It is reported that mannose receptor expressed on human lymphatic endothelium is a noval ligand of L-selectin. The interaction between the two molecules mediates lymph cell homing. The early study work of our laboratory indicates that the lymphatic metastasis capability of mouse high and low metastasis hepatoma carcinoma cell HCa-F and HCa-P is related to their L-selectin expression level . In lymphoma P388D1, this phenomenon also exist. So, we deduce that the interaction between mannose receptor and L-selectin is involved in tumor lymphatic metastasis.Objective: In this thesis we are to construct a chimeric gene of the mannose receptor C-type lectin-like domain 4-7 fused to rabbit secreting signal peptide gene and the Fc segment gene of human IgG. The chimeric gene named Sig-CTLD4-7-Fc is to be inserted in vector pcDNA3.1, then the secreted chimeric protein is to be transient expressed in 293T cells . It may provide a basis for purifying this chimeric protein by using Protein-A bind , identifying if MR can interact with L-selectin through adhesion experiment . The observation of the two molecules’interaction may give us some hints for further research of the mannose receptor’s function in the process of tumor lymphatic metastasis.Methods:1.We are to testify pMD-18T/ CTLD1-8 vector of our laboratory by enzyme cutting .Then we amplify CTLD4-7 of MR from the vector of pMD-18T/ CTLD1-8 by PCR to inserte it into pMD-18T clone vector and identify sequence after testifing by enzyme cutting.The right MR CTLD4-7 gene is to be inserted into pX vector(pX vector contains secreting signal sequence in upriver position and Fc gene of human IgG in downriver position).Then testify the aim vector by enzyme cutting. It is to be named pX/ CTLD4-7.The right Sig-CTLD4-7-Fc chimeric gene is to be obtained by single NotI enzyme cutting pX/ CTLD4-7 ,then inserted in pcDNA3.1 expression vector. It constructs the recombinant expression vector pcDNA3.1/ Sig-CTLD4-7-Fc.2.Culture 293T cell. Then we transfect the highly-purified plasmid pcDNA3.1/ Sig-CTLD4-7-Fc into 293T cells with TM2000. The transient expression product of 24h, 48h, 72h is to be detected with negtive and positive control by ELISA. At the same time, cell immunochemistry is to be done for qualitative assay of transient expression.Results:1.MluI single enzyme and MluI, KpnI double enzyme cutting pMD-18T/ CTLD1-8 plasmid generate prospective 3420 bp, 2800 bp and 2800bp, 2120bp,1300bp segments respectively. AatII, SalI double enzyme cutting pMD-18T/ CTLD4-7 generate prospective 2200bp,1695bp, 498bp segments, and the CTLD4-7 gene sequence is identified exactly. AatII , SalI double enzyme and NotI single enzyme cutting pX/CTLD4-7 generate prospective 3621bp, 1695bp and 2865bp, 2451bp segments respectively. And NotI single enzyme cutting pCDNA3.1/Sig-CTLD4-7-Fc generate prospective 5522 bp, 2451 bp segments. The direction of Sig-CTLD4-7-Fc in pCDNA3.1/Sig-CTLD4-7-Fc is identified by PCR and the sequence by sequencing. All indicate we construct the eukaryotic expression vector pCDNA3.1/Sig-CTLD4-7-Fc exactly2.After transfecting 293T cell, ELISA experimental result manifests chimeric protein starts to secrete at the time of 24h, and the amount obviously rises at the time of 48h,72h;cell immunochemistry identifys that it strats to express chimeric protein CTLD4-7-Fc at the time of 24h, and it continues to express at the time of 48h,72h.Conclusions:1.We have successfully constructed the recombinant expression vector pCDNA3.1/ Sig-CTLD4-7-Fc.2.The recombinant expression vector can be successfully expressed in the 293T cells by lipid transfecting.更多还原