發(fā)布時(shí)間：2018-07-01 20:44

  本文選題：甘露聚糖結(jié)合凝集素 + Strep-tagⅡ標(biāo)簽肽��； 參考：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 天然免疫是機(jī)體識(shí)別病原微生物,抵御病原體感染的第一道防線。甘露聚糖結(jié)合凝集素(mannan-binding lectin,MBL)是天然免疫系統(tǒng)中的關(guān)鍵分子,為肝細(xì)胞分泌的血漿蛋白,屬C型凝集素超家族中膠凝素(collectins)家族成員。MBL籍其模式識(shí)別作用選擇性識(shí)別多種病原體的糖結(jié)構(gòu),然后通過(guò)激活補(bǔ)體凝集素途徑而發(fā)揮溶破和間接調(diào)理功能,并能與吞噬細(xì)胞膠凝素受體結(jié)合而啟動(dòng)調(diào)理吞噬,還可介導(dǎo)MBL依賴(lài)的細(xì)胞介導(dǎo)的細(xì)胞毒作用。而且,MBL也是一個(gè)重要的粘膜表面防御分子。 成熟MBL肽鏈自N端至C端依次有4個(gè)結(jié)構(gòu)域:富含Cys的N端區(qū)、膠原樣區(qū)(collagen-like region,CLR)、頸區(qū)和C端糖識(shí)別域(carbohydraterecognitiondomain,CRD)。完整MBL分子是同質(zhì)三肽鏈亞單位的寡聚體,多至六聚體。只有高寡聚體MBL分子才具有生物學(xué)活性。CRD是MBL分子的識(shí)別功能區(qū),能選擇性識(shí)別多種病原體的糖結(jié)構(gòu);而CLR是其效應(yīng)功能區(qū),MBL結(jié)合MBL相關(guān)絲氨酸蛋白酶(MBL associated serine proteases,MASP1、MASP2)和膠凝素受體的功能定位于此區(qū)。在CRD識(shí)別、結(jié)合病原微生物的糖結(jié)構(gòu)后,可活化MASP酶原而激活補(bǔ)體凝集素途徑,并能與吞噬細(xì)胞膠凝素受體結(jié)合啟動(dòng)調(diào)理吞噬,從而發(fā)揮天然抗感染免疫效應(yīng)。 獲取足量的有生物學(xué)活性的MBL蛋白是開(kāi)展MBL研究的前提。但人血清中MBL含量甚微(約1 mg/L),提取困難且費(fèi)用高昂,同時(shí)由于MBL蛋白結(jié)構(gòu)很復(fù)雜,真核表達(dá)的產(chǎn)量很低,而原核表達(dá)系統(tǒng)缺乏真核細(xì)胞所特有的翻譯后加工系統(tǒng),無(wú)法組裝形成寡聚體的蛋白,這些嚴(yán)重制約了對(duì)MBL分子的研究。因此,本課題擬通過(guò)原核表達(dá)系統(tǒng)表達(dá)MBL的單肽鏈并在其N(xiāo)端加一段標(biāo)簽肽(即rhMBL△N-strep-tagⅡ),利用膠原蛋白的3條肽鏈自動(dòng)相互纏繞成α-螺旋的性質(zhì)而裝配形成膠原樣蛋白的三級(jí)結(jié)構(gòu)即亞單位,然后籍標(biāo)簽肽與鏈親和素之間的相互作用而形成寡聚體形式的MBL分子。通過(guò)與血漿來(lái)源的天然MBL比較,分析其結(jié)合配體、MASP并啟動(dòng)補(bǔ)體凝集素途徑及結(jié)合吞噬細(xì)胞膠凝素受體的活性,評(píng)價(jià)其生物學(xué)活性和免疫學(xué)活性。 一、重組人N端缺失MBL與strep-tagⅡ標(biāo)簽融合蛋白表達(dá)及鑒定 使用Primer premier5.0軟件,設(shè)計(jì)并合成引物,并在上游引物酶切位點(diǎn)后插入Strep-tagⅡ標(biāo)簽肽基因序列,以含有漢族人野生型MBL全長(zhǎng)編碼區(qū)cDNA的質(zhì)粒pGEM-MBL為模板,PCR擴(kuò)增出長(zhǎng)度約640 bp的人N端缺失MBL-Strep-tagⅡ融合蛋白(MBL△N-strep-tagⅡ)基因片段,將其克隆至pET43.1a原核表達(dá)載體中,構(gòu)建的重組表達(dá)載體經(jīng)BamHⅠ和EcoRⅠ酶切后出現(xiàn)約7525 bp和640 bp的片段,經(jīng)測(cè)序鑒定序列正確后,導(dǎo)入大腸桿菌BL21(DE3)中,以IPTG誘導(dǎo)重組pET43.1a-MBL△N-strep-tagⅡ質(zhì)粒表達(dá)MBL△N-strep-tagⅡ融合蛋白�？扇苄员磉_(dá)的MBL△N-strep-tagⅡ融合蛋白經(jīng)Ni~+-NTA agarose層析柱純化,獲得了純化的融合蛋白。純化蛋白經(jīng)SDS-PAGE電泳出現(xiàn)Mr約為97 000的條帶,Western blot分析顯示,純化的目的蛋白能與鼠抗人MBL-CRD抗體結(jié)合;ELISA表明,目的蛋白既能與糖基配體結(jié)合又能與2個(gè)MASP結(jié)合,同時(shí)也顯示與鏈親和素有良好的結(jié)合能力。 二、基于鏈親和素四聚體特性體外組裝寡聚體MBL蛋白 本室曾原核表達(dá)MBL的CRD和CLR蛋白,都因其膠原樣蛋白的3條肽鏈相互纏繞成α-螺旋而自裝配成具有生物學(xué)活性的三級(jí)結(jié)構(gòu),而rhMBL△N-strep-tagⅡ單鏈蛋白中的包含有CLR和CRD兩部分,所以rhMBL△N-strep-tagⅡ單鏈蛋白在緩沖液中反復(fù)透析,自動(dòng)裝配成了三聚體即亞單位。還原和非還原SDS-PAGE和Western blot結(jié)果顯示,在約Mr 50 000的蛋白條帶為重組MBL的亞單位。ELISA結(jié)果表明亞單位蛋白對(duì)甘露聚糖的結(jié)合能力與單鏈蛋白比較有所提高,但其對(duì)鏈親和素的結(jié)合能力卻比單鏈蛋白明顯降低。因此,改善結(jié)合緩沖液后,加入鏈親和素進(jìn)行組裝。SDS-PAGE結(jié)果出現(xiàn)Mr97 000、Mr125 000和Mr220 000三處條帶,表明獲得了與鏈親和素結(jié)合的二、三、四聚體重組MBL蛋白。 三、組裝寡聚體MBL蛋白功能初步鑒定 本實(shí)驗(yàn)的目的分析組裝寡聚體MBL蛋白是否具有生物學(xué)活性和免疫學(xué)功能。組裝寡聚體MBL蛋白與酵母菌細(xì)胞壁甘露聚糖結(jié)合,使之產(chǎn)生可見(jiàn)的凝集現(xiàn)象,但效價(jià)比天然MBL蛋白和哺乳細(xì)胞表達(dá)的重組MBL蛋白低。配體結(jié)合試驗(yàn)發(fā)現(xiàn),組裝寡聚體MBL蛋白能與配體甘露聚糖和2種MASP(即MASP1、MASP2)結(jié)合,但結(jié)合能力低于天然的MBL蛋白。補(bǔ)體C4d沉積試驗(yàn)揭示,組裝寡聚體MBL蛋白與包被的甘露聚糖結(jié)合,啟動(dòng)補(bǔ)體凝集素途徑,但這種活性比天然MBL蛋白低。此外,該組裝寡聚體MBL蛋白能與THP1細(xì)胞結(jié)合,提示其可結(jié)合于吞噬細(xì)胞的膠凝素受體。 本實(shí)驗(yàn)基于鏈親和素四聚體的特性,通過(guò)體外組裝,制備了寡聚體重組MBL蛋白;有關(guān)實(shí)驗(yàn)表明,該組裝寡聚體MBL蛋白具備了天然MBL蛋白的基本生物學(xué)活性與功能。誠(chéng)然,由于技術(shù)的限制,未能得到大量的高純度組裝寡聚體MBL蛋白,暫無(wú)法進(jìn)一步深入研究其免疫學(xué)功能。但是,本課題的初步實(shí)驗(yàn)結(jié)果,對(duì)于獲取困難的結(jié)構(gòu)復(fù)雜的多聚體蛋白,似乎有一定的指導(dǎo)意義。若能尋找到比鏈親和素-有關(guān)標(biāo)簽肽有著更好親和力和安全性的小分子-蛋白質(zhì)結(jié)合對(duì),則可為體外大量制備寡聚體蛋白提供科學(xué)依據(jù)并供相關(guān)藥物的開(kāi)發(fā)研究以借鑒。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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