發(fā)布時(shí)間：2018-04-02 01:15

  本文選題：甘露聚糖結(jié)合凝集素　切入點(diǎn)：重組　出處：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 甘露聚糖結(jié)合凝集素(mannan-binding lectin,MBL)是哺乳動(dòng)物C型凝集素超級(jí)家族中膠凝素家族成員,主要由肝細(xì)胞分泌,作為糖蛋白存在于血漿中。通過(guò)其糖識(shí)別域(carbohydrate-recognition domain,CRD)識(shí)別病原體表面糖結(jié)構(gòu),通過(guò)其膠原樣區(qū)(collagen-likeregion,CLR)與2個(gè)MBL相關(guān)絲氨酸蛋白酶(MBL associated serine protease,MASP-1,MASP-2)結(jié)合,以不依賴抗體和C1q的凝集素途徑激活補(bǔ)體,發(fā)揮溶破和間接調(diào)理功能,還能與吞噬細(xì)胞表面膠凝素受體結(jié)合而起直接調(diào)理作用。MBL的識(shí)別譜廣泛,在天然免疫中發(fā)揮重要作用。MBL基因在人群中突變頻率極高,已知3個(gè)MBL結(jié)構(gòu)基因點(diǎn)突變可導(dǎo)致MBL蛋白空間結(jié)構(gòu)改變,與糖基配體和MASPs結(jié)合的能力降低,補(bǔ)體凝集素激活途徑幾乎完全消失,臨床上表現(xiàn)為各種病原體的急慢性反復(fù)感染。 人血漿MBL含量極低,從血漿中大量提取MBL用于科研和臨床,花費(fèi)昂貴。通過(guò)基因工程、細(xì)胞培養(yǎng)等技術(shù),體外大規(guī)模制備MBL,將為進(jìn)一步研究MBL在免疫系統(tǒng)中的作用以及臨床上MBL缺損的治療提供條件。 本課題組曾成功構(gòu)建了兩個(gè)野生型MBL真核表達(dá)載體,PcDNA3.1~+-MBL和PcDNA4/HisMaxC-MBL,并用電穿孔法分別轉(zhuǎn)染入CHO、HEK293和HEPG2細(xì)胞,經(jīng)G418和Zeocin選擇轉(zhuǎn)染子并克隆化培養(yǎng),獲得表達(dá)重組人MBL的細(xì)胞株。通過(guò)RT-PCR和ELISA等方法分析比較,發(fā)現(xiàn)PcDNA3.1~+-MBL在CHO和HEK293細(xì)胞中能夠大量合成RNA,其表達(dá)效率顯著高于其他載體和細(xì)胞的組合。因此,本研究采用本室保存的已轉(zhuǎn)染了PcDNA3.1~+-MBL真核表達(dá)載體的CHO細(xì)胞株進(jìn)行為期2個(gè)月的篩選,通過(guò)ELISA技術(shù)篩選高表達(dá)單克隆細(xì)胞株,然后擴(kuò)大培養(yǎng),分離純化其表達(dá)的蛋白產(chǎn)物,并對(duì)其生物學(xué)特性進(jìn)行深入研究。 一、雙抗夾心ELISA體系的建立 用抗MBL-CRD單克隆抗體捕獲,辣根過(guò)氧化物酶標(biāo)記的抗MBL-CLR多克隆抗體檢測(cè),以丹麥Aarhus大學(xué)Jens Chr.Jensenius教授贈(zèng)送的重組人MBL蛋白作為標(biāo)準(zhǔn)品,來(lái)篩選細(xì)胞培養(yǎng)上清中目的蛋白含量較高的細(xì)胞株,以獲得高表達(dá)重組人MBL蛋白的細(xì)胞克隆。該檢測(cè)體系靈敏度可達(dá)0.1mg/L。 二、篩選穩(wěn)定高效表達(dá)重組人MBL蛋白的細(xì)胞株 本課題組已構(gòu)建了含PcDNA3.1~+-MBL真核表達(dá)載體的CHO細(xì)胞。將其復(fù)蘇后,在96孔板中單克隆化,待細(xì)胞克隆長(zhǎng)至孔底的1/4后,挑取細(xì)胞株置12孔板內(nèi)培養(yǎng)24 h后,以800 mg/L的G418加壓篩選。隨后的1個(gè)月中,每隔3～5天換液一次,維持G418濃度為800 mg/L。然后,通過(guò)雙抗夾心ELISA體系篩選得到6株高表達(dá)的單克隆細(xì)胞株,其上清中目的蛋白濃度均在3 mg/L以上。RT-PCR分析表明,這些轉(zhuǎn)染了MBL基因的CHO細(xì)胞能穩(wěn)定轉(zhuǎn)錄MBLmRNA。 三、重組人MBL蛋白的純化及其生物學(xué)特性研究 利用硫酸銨沉淀法初步純化表達(dá)產(chǎn)物,鼠抗人MBL-CRD mAb親和層析柱進(jìn)一步純化,獲得重組人MBL蛋白。在1L培養(yǎng)上清中可獲得2mg左右目的蛋白。經(jīng)ELISA鑒定,純化后的蛋白產(chǎn)物可與抗MBL單克隆抗體、抗MBL-CRD單克隆抗體、抗MBL-CLR多克隆抗體結(jié)合。通過(guò)非還原SDS-PAGE和WesternBlot分析,重組人MBL分子量多集中在Mr 200 000以上,為三至六聚體形式。結(jié)合實(shí)驗(yàn)證明所純化的重組MBL蛋白能與甘露聚糖及重組MASP蛋白有效結(jié)合;C4d沉積實(shí)驗(yàn)表明重組人MBL蛋白和血漿來(lái)源MBL均能有效介導(dǎo)補(bǔ)體激活,而這種介導(dǎo)作用可被競(jìng)爭(zhēng)性糖類抑制。另外,用C4d沉積試驗(yàn)檢測(cè)保存于不同溫度下6個(gè)月的重組蛋白介導(dǎo)補(bǔ)體激活的功能,發(fā)現(xiàn)其穩(wěn)定性良好,活化補(bǔ)體活性無(wú)明顯降低。
[Abstract]:Mannan binding lectin (mannan-binding lectin MBL) is a mammalian C type lectin superfamily of collectin family members, mainly secreted by liver cells, as a glycoprotein present in plasma. Through the carbohydrate recognition domain (carbohydrate-recognition domain CRD) to identify the pathogen surface carbohydrate structure, through its collagenous region (collagen-likeregion, CLR 2) and MBL (MBL associated serine associated serine protease protease, MASP-1, MASP-2) with the lectin pathway to C1q and antibody dependent complement activation, play 1yse and indirect conditioning, but also with phagocytic cells on the surface of glue hemagglutinin receptor binding and recognition of direct opsonization.MBL broad spectrum play an important effect of.MBL gene mutation in the population of high frequency in innate immunity, 3 known MBL structure gene mutation can cause MBL protein spatial structure change, and the sugar ligand The ability to combine with MASPs is reduced, and the activation pathway of the complement lectin is almost completely disappeared, and the clinical manifestations are acute and chronic infection of various pathogens.
Low plasma MBL content in plasma from the extraction of large amount of MBL used in research and clinical, expensive. Through genetic engineering, cell culture technique in vitro, large-scale preparation of MBL, will provide the conditions for the further research of MBL role in the immune system and clinical treatment of MBL deficiency.
The research group has successfully constructed two wild-type MBL eukaryotic expression vector, PcDNA3.1~+-MBL and PcDNA4/HisMaxC-MBL, and by electroporation were respectively transfected into CHO, HEK293 and HEPG2 cells by G418 and Zeocin clones were selected and cultured, expression of recombinant human MBL cell lines by RT-PCR and ELISA methods. Comparative analysis found that PcDNA3.1~+-MBL RNA and HEK293 CHO in the synthesis of a large number of cells, the expression rate was significantly higher than that of other combinations of vectors and cells. Therefore, this study used the room saved PcDNA3.1~+-MBL transfected with CHO eukaryotic expression cell line carrier were screened for a period of 2 months, through the ELISA technology to screen high expression monoclonal cell lines, and then expand the culture, the expression of protein separation purification, and further study of its biological characteristics.
The establishment of a dual anti sandwich ELISA system
Capture using anti MBL-CRD monoclonal antibody, horseradish peroxidase labeled anti MBL-CLR polyclonal antibody detection by Chr.Jensenius Aarhus Jens, Professor of University of Denmark presented the recombinant human MBL protein as a standard, to screen cell culture to high protein content in the supernatant of cell line, in order to obtain high cell clones expressing recombinant human MBL protein. The detection the system sensitivity can reach 0.1mg/L.
Two, screening cell lines for stable and efficient expression of recombinant human MBL protein
This research has constructed the eukaryotic expression vector of PcDNA3.1~+-MBL CHO cells. The recovery after the monoclonal 96 well plate, to be long to the bottom of the hole cell clones after 1/4 cells were cultured in 12 well plates after 24 h to 800 mg/L G418 pressure screening. 1 months later every 3~5 days, was changed once, to maintain the concentration of G418 was 800 mg/L. and then by double antibody sandwich ELISA system for screening 6 strains with high expression of the monoclonal cell lines, the supernatant protein concentration was above 3 mg/L.RT-PCR analysis showed that these MBL transfected CHO cells stably transcription of MBLmRNA.
Three, purification of recombinant human MBL protein and Study on its biological characteristics
By using ammonium sulfate precipitation method preliminary purification, further purification of mouse anti human MBL-CRD mAb affinity chromatography, the recombinant human MBL protein. The 1L supernatant can obtain about 2mg. The target protein was identified by ELISA, protein purified with anti MBL monoclonal antibody, anti MBL-CRD monoclonal antibody, polyclonal anti MBL-CLR antibody binding. Analysis of SDS-PAGE and WesternBlot by non reductive, recombinant human MBL molecular weight concentrated in Mr more than 200000, three to six dimer form. Combined with the experiment results showed that the purified recombinant MBL protein can effectively combine with mannan and recombinant MASP protein; C4d deposition experiments showed that the recombinant human MBL protein and plasma source MBL can effectively mediate complement activation, and this effect may be mediated by competitive inhibition of saccharide. In addition, C4d deposition test stored in different temperature 6 months of recombinant protein mediated by complement activation It was found that the stability was good and the activated complement activity was not significantly reduced.

【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
【分類號(hào)】：R392

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