基于链亲和素四聚体的特性体外组装寡聚体重组MBL蛋白

发布时间：2018-07-01 20:44

本文选题：甘露聚糖结合凝集素 + Strep-tagⅡ标签肽　；参考：《南方医科大学》2008年硕士论文

【摘要】： 天然免疫是机体识别病原微生物,抵御病原体感染的第一道防线。甘露聚糖结合凝集素(mannan-binding lectin,MBL)是天然免疫系统中的关键分子,为肝细胞分泌的血浆蛋白,属C型凝集素超家族中胶凝素(collectins)家族成员。MBL籍其模式识别作用选择性识别多种病原体的糖结构,然后通过激活补体凝集素途径而发挥溶破和间接调理功能,并能与吞噬细胞胶凝素受体结合而启动调理吞噬,还可介导MBL依赖的细胞介导的细胞毒作用。而且,MBL也是一个重要的粘膜表面防御分子。成熟MBL肽链自N端至C端依次有4个结构域:富含Cys的N端区、胶原样区(collagen-like region,CLR)、颈区和C端糖识别域(carbohydraterecognitiondomain,CRD)。完整MBL分子是同质三肽链亚单位的寡聚体,多至六聚体。只有高寡聚体MBL分子才具有生物学活性。CRD是MBL分子的识别功能区,能选择性识别多种病原体的糖结构;而CLR是其效应功能区,MBL结合MBL相关丝氨酸蛋白酶(MBL associated serine proteases,MASP1、MASP2)和胶凝素受体的功能定位于此区。在CRD识别、结合病原微生物的糖结构后,可活化MASP酶原而激活补体凝集素途径,并能与吞噬细胞胶凝素受体结合启动调理吞噬,从而发挥天然抗感染免疫效应。获取足量的有生物学活性的MBL蛋白是开展MBL研究的前提。但人血清中MBL含量甚微(约1 mg/L),提取困难且费用高昂,同时由于MBL蛋白结构很复杂,真核表达的产量很低,而原核表达系统缺乏真核细胞所特有的翻译后加工系统,无法组装形成寡聚体的蛋白,这些严重制约了对MBL分子的研究。因此,本课题拟通过原核表达系统表达MBL的单肽链并在其N端加一段标签肽(即rhMBL△N-strep-tagⅡ),利用胶原蛋白的3条肽链自动相互缠绕成α-螺旋的性质而装配形成胶原样蛋白的三级结构即亚单位,然后籍标签肽与链亲和素之间的相互作用而形成寡聚体形式的MBL分子。通过与血浆来源的天然MBL比较,分析其结合配体、MASP并启动补体凝集素途径及结合吞噬细胞胶凝素受体的活性,评价其生物学活性和免疫学活性。一、重组人N端缺失MBL与strep-tagⅡ标签融合蛋白表达及鉴定使用Primer premier5.0软件,设计并合成引物,并在上游引物酶切位点后插入Strep-tagⅡ标签肽基因序列,以含有汉族人野生型MBL全长编码区cDNA的质粒pGEM-MBL为模板,PCR扩增出长度约640 bp的人N端缺失MBL-Strep-tagⅡ融合蛋白(MBL△N-strep-tagⅡ)基因片段,将其克隆至pET43.1a原核表达载体中,构建的重组表达载体经BamHⅠ和EcoRⅠ酶切后出现约7525 bp和640 bp的片段,经测序鉴定序列正确后,导入大肠杆菌BL21(DE3)中,以IPTG诱导重组pET43.1a-MBL△N-strep-tagⅡ质粒表达MBL△N-strep-tagⅡ融合蛋白。可溶性表达的MBL△N-strep-tagⅡ融合蛋白经Ni~+-NTA agarose层析柱纯化,获得了纯化的融合蛋白。纯化蛋白经SDS-PAGE电泳出现Mr约为97 000的条带,Western blot分析显示,纯化的目的蛋白能与鼠抗人MBL-CRD抗体结合;ELISA表明,目的蛋白既能与糖基配体结合又能与2个MASP结合,同时也显示与链亲和素有良好的结合能力。二、基于链亲和素四聚体特性体外组装寡聚体MBL蛋白本室曾原核表达MBL的CRD和CLR蛋白,都因其胶原样蛋白的3条肽链相互缠绕成α-螺旋而自装配成具有生物学活性的三级结构,而rhMBL△N-strep-tagⅡ单链蛋白中的包含有CLR和CRD两部分,所以rhMBL△N-strep-tagⅡ单链蛋白在缓冲液中反复透析,自动装配成了三聚体即亚单位。还原和非还原SDS-PAGE和Western blot结果显示,在约Mr 50 000的蛋白条带为重组MBL的亚单位。ELISA结果表明亚单位蛋白对甘露聚糖的结合能力与单链蛋白比较有所提高,但其对链亲和素的结合能力却比单链蛋白明显降低。因此,改善结合缓冲液后,加入链亲和素进行组装。SDS-PAGE结果出现Mr97 000、Mr125 000和Mr220 000三处条带,表明获得了与链亲和素结合的二、三、四聚体重组MBL蛋白。三、组装寡聚体MBL蛋白功能初步鉴定本实验的目的分析组装寡聚体MBL蛋白是否具有生物学活性和免疫学功能。组装寡聚体MBL蛋白与酵母菌细胞壁甘露聚糖结合,使之产生可见的凝集现象,但效价比天然MBL蛋白和哺乳细胞表达的重组MBL蛋白低。配体结合试验发现,组装寡聚体MBL蛋白能与配体甘露聚糖和2种MASP(即MASP1、MASP2)结合,但结合能力低于天然的MBL蛋白。补体C4d沉积试验揭示,组装寡聚体MBL蛋白与包被的甘露聚糖结合,启动补体凝集素途径,但这种活性比天然MBL蛋白低。此外,该组装寡聚体MBL蛋白能与THP1细胞结合,提示其可结合于吞噬细胞的胶凝素受体。本实验基于链亲和素四聚体的特性,通过体外组装,制备了寡聚体重组MBL蛋白;有关实验表明,该组装寡聚体MBL蛋白具备了天然MBL蛋白的基本生物学活性与功能。诚然,由于技术的限制,未能得到大量的高纯度组装寡聚体MBL蛋白,暂无法进一步深入研究其免疫学功能。但是,本课题的初步实验结果,对于获取困难的结构复杂的多聚体蛋白,似乎有一定的指导意义。若能寻找到比链亲和素-有关标签肽有着更好亲和力和安全性的小分子-蛋白质结合对,则可为体外大量制备寡聚体蛋白提供科学依据并供相关药物的开发研究以借鉴。
[Abstract]:Natural immunity is the first line of defense to identify pathogenic microorganisms and resist pathogens infection. Mannan-binding lectin (MBL) is a key molecule in the natural immune system. It is a plasma protein secreted by the liver cells, and is a.MBL pattern recognition of the collectins family members of the C agglutinin superfamily. By selectively identifying the sugar structure of a variety of pathogens, then using the activation of the complement lectin pathway to play dissolving and indirect conditioning functions, and to combine with the phagocytic agglutinin receptor to initiate phagocytosis, and mediate the cytotoxic action mediated by MBL dependent cells. Moreover, MBL is also an important membrane surface defense molecule.
The mature MBL peptide chain has 4 domains from the N end to the C end: the N endpoint rich in Cys, the collagen like region (collagen-like region, CLR), the neck region and the C terminal sugar recognition domain (carbohydraterecognitiondomain, CRD). The whole MBL molecule is the oligomer of the homogeneous three peptide chain, from the six polymer. Only the high oligomer has the biological activity. It is the functional area of the MBL molecule, which can selectively identify the sugar structure of various pathogens, and CLR is its functional area. MBL combines the function of MBL associated serine protease (MBL associated serine proteases, MASP1, MASP2) and gelatin receptor in this area. Activating the complement lectin pathway and binding to the phagocyte receptor can activate the phagocytic phagocytosis, thereby playing a natural anti infection immune effect.
Obtaining a full amount of biological active MBL protein is the prerequisite for the development of MBL research. But the content of MBL in human serum is very small (about 1 mg/L), which is difficult and expensive. At the same time, the production of eukaryotic expression is very low because of the complex structure of MBL protein, and the prokaryotic expression system lacks the posttranslational processing system peculiar to the eukaryotic cells and can not be assembled. The protein of oligomers seriously restricts the study of MBL molecules. Therefore, this topic is intended to express MBL's single peptide chain through the prokaryotic expression system and add a tagged peptide (rhMBL Delta N-strep-tag II) at its N end, and use 3 peptide chains of collagen to automatically intertwine each other into alpha spire to form a three grade junction of the original protein. It is a subunit, and then forms oligomer form MBL molecules with the interaction between tagged peptide and streptavidin. By comparing with natural MBL from plasma sources, the binding ligands, MASP and the activity of binding agglutinin receptor with phagocytic cells are activated, and their biological and immunological activities are evaluated.
Expression and identification of fusion protein of recombinant human N terminal deletion MBL and Strep-tag II tag
Using Primer premier5.0 software, primers were designed and synthesized, and the Strep-tag II tagged peptide gene sequence was inserted after the loci of the upstream primers. The plasmid pGEM-MBL containing the cDNA of the wild type MBL full-length encoding region of the Han people was used as a template. The N terminal deletion MBL-Strep-tag II fusion protein (MBL Delta N-strep-tag II) of the human N terminal deletion (MBL Delta N-strep-tag II) based on the length of 640 BP was amplified by PCR The recombinant expression vector was cloned into the pET43.1a prokaryotic expression vector, and the recombinant expression vector was cut into about 7525 BP and 640 BP fragments after being cut through BamH I and EcoR I enzyme. After the sequence identification sequence was correct, the recombinant expression vector was introduced into Escherichia coli BL21 (DE3), and the recombinant pET43.1a-MBL Delta N-strep-tag II plasmid was induced by IPTG to express MBL Delta N-strep-tag II fusion egg. The soluble expression of MBL Delta N-strep-tag II fusion protein was purified by Ni~+-NTA agarose chromatography column, and purified fusion protein was obtained. The purified protein showed a band of about 97000 Mr by SDS-PAGE electrophoresis. The Western blot analysis showed that the purified target protein could combine with the mouse anti human MBL-CRD antibody; ELISA showed that the target protein could not only be with the glycosyl group. Ligand binding can also bind to the 2 MASP, and also shows good binding ability to streptavidin.
Two, oligomeric MBL protein was assembled in vitro based on the characteristics of streptavidin four dimer.
The CRD and CLR protein of MBL, which had been prokaryotic expression of the protein, were intertwined into alpha helix of the 3 peptide chain of its original protein, and self assembled into a three stage structure with biological activity. The rhMBL Delta N-strep-tag II single strand protein contained CLR and CRD two parts, so rhMBL Delta N-strep-tag II single chain protein was dialytic repeatedly in buffer solution. The redox and non reductive SDS-PAGE and Western blot results showed that the subunit.ELISA results in the protein strip of about Mr 50000 showed that the binding ability of the subunit protein to the mannan was higher than that of the single strand protein, but the binding ability to the streptavidin was better than that of the single chain protein. Therefore, after improving the combination of the buffer solution, the.SDS-PAGE results of Mr97 000, Mr125 000 and Mr220 000 three bands were added to the assembly of the streptavidin, indicating that the recombinant MBL protein of two, three, four polymer combined with streptavidin was obtained.
Three, preliminary identification of the function of the assembled oligomer MBL protein
The purpose of this study was to analyze whether the oligomer MBL protein had biological and immunological functions. The assembly of oligomer MBL protein was combined with the yeast cell wall mannan to produce visible agglutination, but the potency was lower than the recombinant MBL protein expressed by natural MBL protein and mammalian cells. The oligomeric oligomerization was found by ligand binding test. The body MBL protein can bind to the ligand mannan and 2 kinds of MASP (MASP1, MASP2), but the binding ability is lower than the natural MBL protein. The complement C4d deposition test reveals that the assembly of oligomer MBL protein is combined with the coated mannan and initiates the complement lectin pathway, but this activity is lower than that of the natural MBL protein. In addition, the oligomer MBL protein can be compared to T. The binding of HP1 cells suggests that it can bind to the gelling receptor of phagocytes.
Based on the characteristics of the streptavidin four polymer, the oligomer recombinant MBL protein was prepared by in vitro assembly. The experiment showed that the oligomer MBL protein had the basic biological activity and function of the natural MBL protein. However, the preliminary experimental results of this project seem to be of certain guiding significance for obtaining difficult structure complex polymer proteins. If we can find a small molecule protein binding pair with better affinity and safety of the tagged peptide related to the tagging peptide, a large number of oligomers can be prepared in vitro. The polymer protein provides scientific basis for the development and research of related drugs for reference.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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