【作者】 连继勤；

【导师】 张玉静；

【作者基本信息】 中国人民解放军军需大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 层粘连蛋白(Laminin, LN)是各种动物胚胎及成体组织基底膜的主要结构组分之一，对基底膜基质的组装起关键作用。它可介导细胞粘着于胶原进而铺展，并促进细胞生长。LN通过与其受体结合能够调节细胞的粘附、扩散、迁移和增殖，在炎症的发生及肿瘤的转移中发挥着重要作用。层粘连蛋白G域样(LG)组件位于层粘连蛋白α链的C末端，由180-200个氨基酸残基组成，存在于许多细胞外蛋白和受体蛋白中，一般以串联排列的形式存在。层粘连蛋白LG组件含有许多作用位点，这些位点对层粘连蛋白与其细胞受体及配体间的相互作用至关重要，对它们的研究将有助于揭示层粘连蛋白在细胞的分化、生长、运动及肿瘤的转移、浸润中所起的作用。本实验通过克隆人层粘连蛋白α4链LG4-5组件(hLNα4LG4-5)基因，分别利用原核BL21(DE3)表达菌、真核293细胞及Pichia酵母菌对LG4-5组件进行了表达研究，以期得到具有生物活性的LG4-5组件蛋白，为进一步研究该蛋白的结构及功能打下基础。 hLNα4LG4-5组件cDNA序列的克隆及序列测定 用Trizol法提取人胎盘组织总RNA。利用DNASIS软件设计一对LG4-5组件的特异性引物GTCAGAATTCCCACCTTTCCAACAGCCCTAGAG和GTCACTCGAGCTAGGCTGCTGGACAGGAGTTGAT，通过RT-PCR方法扩增得到一与人LNα4链LG4-5组件基因(1098bp)大小相符的特异性片段。将该片段纯化回收后与pMD-18T载体连接，连接产物转化入JM109感受态菌中，用碱裂解法从阳性转化菌中提取质粒，并分别用PCR与双酶切对重组质粒进行鉴定，将鉴定正确的质粒进行序列测定。将测序结果与GENBANK中人LNα4链基因序列进行同源性比较，结果表明大小为1098bp的扩增片段与LN4α的同源性为99.8％，共有2处发生了突变，分别为399位的T变为C和519位的T变为C，但编码氨基酸并没有发生改变，仍为Asp与Ser。测序结果证实已成功克隆得到人LNα4链LG4-5组件的cDNA基因片段。 hLNα4 LG4-5组件在原核细胞中的表达及抗体制备 对测序正确的克隆载体进行双酶切，回收LG4-5组件片段并将之定向克隆至原核表达载体pET-28a中，构建LG4-5组件的原核表达载体pET-28a-LG4-5。将重组载体转化至BL21(DE3)宿主菌中，并用IPTG进行诱导表达，结果得到了以包涵体形式存在的LG4-5组件的原核表达产物。对表达条件进行优化，然后制备LG4-5组件表达蛋白的包涵体，纯化后作为抗原免疫实验用兔，间接ELISA检测证实所制多抗血清效价为1∶10240。以所制多抗血清为一抗，通过Western印迹检测到了hLNα4LG3-4组件的特异性原核表达产物，说明抗体具有较好的特异性。 hLNα4 LG4-5组件在真核293细胞中的表达研究 通过设计新的引物及PCR扩增，在LG4-5组件基因片段的两端加入NheⅠ和XhoⅠ酶切位点，并将它定向克隆至真核表达载体pCEP-Pu／AC7中，构建了LG4-5组件的真核表达载体pCEP-Pu／AC7-LG4-5。分别利用磷酸钙法和脂质体法将重组质粒pCEP-Pu／AC7-LG4-5转染至293细胞系中，用G418和嘌呤霉素抗性筛选得到稳定的转染细胞株。收集转染细胞，利用Rl’- PCR方法在转录水平上检测到了LG4一5组件基因在293细胞内表达的mRNA。收集转染细胞的培养上清，浓缩后分别用ELIsA、SDS一PAGE和Westem印迹法检测到了目的基因在上清中的特异性表达产物，表明转染细胞可稳定表达LG4一5组件蛋白并能将之分泌到培养上清中。 址“No 4 LG4一5组件酵母表达载体的构建及在尸ichia酵母菌中的表达研究设计新的引物，通过PCR扩增在LG4一5组件基因片段的两端加入EcoRI和Notl酶切位点，并将它定向克隆至酵母表达载体PPIcz oA中，构建了LG4一组件的单拷贝酵母表达载体PPIcz“A一LG4一5.以单拷贝重组质粒为基础，利用载体中同尾酶刀口脚Hl和几才11酶切位点，体外构建了LG4一5组件的2拷贝和4拷贝酵母表达载体。用电击法将构建的重组质粒转化至Pichia酵母感受态菌中，用Zcocin抗性筛选阳性克隆，并得到21株Mut+型转化菌和3株Mut-型转化菌。提取阳性菌落的基因组DNA，并利用PcR法检测到了LG4一5组件基因片段，说明它能够与酵母基因组进行重组并整合。对转化的酵母菌进行诱导表达，利用Rl’- PCR方法在转录水平上检测到了LG4一5组件基因在酵母细胞内表达的mRNA。对表达上清用间接ELIsA、SDS一PAGE和Westem印迹检测均呈阳性，表明LG4一5组件可在酵母菌中完成翻译过程并被分泌至培养基中。对表达的目的蛋白进行定量测定表明，每升培养基可分泌表达目的蛋白25.46毫克，表达获得成功。更多还原

【Abstract】 The laminins (LN) are a family of modular proteins that provide an integral part of the structural scaffolding of basement membranes in almost every animal tissue. In addition to interacting with cell surface receptors such as integrins and alpha-dystroglycan, they function as structural components and are essential for morphogenesis. By virtue of their receptor interactions, they initiate intracellular signaling events that regulate cellular organization and differentiation. These interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ in various laminins. Laminin G domain-like (LG) modules of approximate 180-200 residues are found in a number of extracellular and receptor proteins and often present in tandem arrays. LG modules lie in C-termini of Laminin alpha chain and contain many sites, which are important for the interactions of laminins with their receptors and ligands. Studies on LG modules will help to investigate the functions of laminin in cell differentiation, growth and movement, and also in tumor metastasis and infiltration.Cloning and sequencing ofhLNα4LG4-5 module cDNA sequence Total RNA was prepared from placenta by Trizol reagent, and a specific cDNA fragment of hLNα 4LG4-5 module was obtained by RT-PCR. Primers used were GTCAGAATTCCCACC TTTCCAACAGCCCTAGAG for the 5’-end and GTCACTCGAGCTAGGCTGCTGGA CAGGAGTTGAT for the 3’-end and they were designed with DNASIS software. The cDNA fragment of LG4-5 was ligated with pMD18-T vector and transfered into competent cells (JM109). Plasmid was isolated from recombinant clones by alkaline lysis and sequenced confirmed by restriction digests and PCR amplification. Sequence comparing between LG4-5 module and hLN a 4 of GENBANK showed that the homogeneity was 99.8% (1098bp) and mutations occurred in 2 sites (T to C at 399 and T to C at 519), but the amino acids remained the same. So the cDNA sequence of hLN a 4LG4-5 module has been cloned and recombinant pMD-18T-LG4-5 vector was constructed successfully.Prokaryotic Expression of hLNα4LG4-5 module and antibody preparation pMD-18T-LG4-5 and pET-28a plasmids were digested with EcoR I and Xho I respectively, and the prokaryotic expression vectors pET-28a-LG4-5 was constructed by recombination of these restriction fragments. Recombinant pET-28a-LG4-5 plasmid was transferred into BL21(DE3) competent cells and the LG4-5 module protein was expressed as inclusion bodies after induced by IPTG. Inclusion bodies of LG4-5 were prepared and purified from BL21(DE3) expression strain and they were used to immunize rabbits as antigen. Hightiter (1:10240) polyclonal antiserum of LG4-5 module was isolated from the immunized rabbits. The protein of hLNα4 LG3-4 module expressed by E.coli BL21(DE3) was detected by Western blot in which the polyclonal antiserum of LG4-5 module was used as the first antibody.Expression ofhLNα4 LG4-5 module in 293 cell Nhe I and Xho I restriction sites were introduced to the LG4-5 module cDNA gene through new primers and PCR amplification. Eukaryotic expression vector pCEP-Pu/AC7-LG4-5 was constructed by subclone at Nhe I and Xho I sites. 293 cells were transfected with recombinant pCEP-Pu/AC7-LG4-5 plasmid by means of liposome and calcium phosphate respectively. Stable transfectants were obtained by selecting with G418 and puromycin. 293 cells and supernatant were collected and concentrated from stable transfectants respectively. RT-PCR amplification, ELISA assay and Western Blot showed the LG4-5 module was expressed in transfected 293 cells and was secreted into the medium successfully.Expression ofhLN α 4 LG4-5 module in Pichia yeast The yeast expression vector pPICZ α A-LG4-5 containing one copy of LG4-5 gene was constructed with the same method as pCEP-Pu/AC7-LG4-5. And the recombinants containing 2 and 4 copies of LG4-5 gene were constructed through Bgl II and BamH I restriction sites, which have the same ends after digestion. The recombinant plasmids containing LG4-5 module gene was transfered into Pichia yeast compete更多还原