MBL高表达细胞株筛选及其产物生物学特性研究

发布时间：2018-04-02 01:15

本文选题：甘露聚糖结合凝集素　切入点：重组　出处：《南方医科大学》2008年硕士论文

【摘要】： 甘露聚糖结合凝集素(mannan-binding lectin,MBL)是哺乳动物C型凝集素超级家族中胶凝素家族成员,主要由肝细胞分泌,作为糖蛋白存在于血浆中。通过其糖识别域(carbohydrate-recognition domain,CRD)识别病原体表面糖结构,通过其胶原样区(collagen-likeregion,CLR)与2个MBL相关丝氨酸蛋白酶(MBL associated serine protease,MASP-1,MASP-2)结合,以不依赖抗体和C1q的凝集素途径激活补体,发挥溶破和间接调理功能,还能与吞噬细胞表面胶凝素受体结合而起直接调理作用。MBL的识别谱广泛,在天然免疫中发挥重要作用。MBL基因在人群中突变频率极高,已知3个MBL结构基因点突变可导致MBL蛋白空间结构改变,与糖基配体和MASPs结合的能力降低,补体凝集素激活途径几乎完全消失,临床上表现为各种病原体的急慢性反复感染。人血浆MBL含量极低,从血浆中大量提取MBL用于科研和临床,花费昂贵。通过基因工程、细胞培养等技术,体外大规模制备MBL,将为进一步研究MBL在免疫系统中的作用以及临床上MBL缺损的治疗提供条件。本课题组曾成功构建了两个野生型MBL真核表达载体,PcDNA3.1~+-MBL和PcDNA4/HisMaxC-MBL,并用电穿孔法分别转染入CHO、HEK293和HEPG2细胞,经G418和Zeocin选择转染子并克隆化培养,获得表达重组人MBL的细胞株。通过RT-PCR和ELISA等方法分析比较,发现PcDNA3.1~+-MBL在CHO和HEK293细胞中能够大量合成RNA,其表达效率显著高于其他载体和细胞的组合。因此,本研究采用本室保存的已转染了PcDNA3.1~+-MBL真核表达载体的CHO细胞株进行为期2个月的筛选,通过ELISA技术筛选高表达单克隆细胞株,然后扩大培养,分离纯化其表达的蛋白产物,并对其生物学特性进行深入研究。一、双抗夹心ELISA体系的建立用抗MBL-CRD单克隆抗体捕获,辣根过氧化物酶标记的抗MBL-CLR多克隆抗体检测,以丹麦Aarhus大学Jens Chr.Jensenius教授赠送的重组人MBL蛋白作为标准品,来筛选细胞培养上清中目的蛋白含量较高的细胞株,以获得高表达重组人MBL蛋白的细胞克隆。该检测体系灵敏度可达0.1mg/L。二、筛选稳定高效表达重组人MBL蛋白的细胞株本课题组已构建了含PcDNA3.1~+-MBL真核表达载体的CHO细胞。将其复苏后,在96孔板中单克隆化,待细胞克隆长至孔底的1/4后,挑取细胞株置12孔板内培养24 h后,以800 mg/L的G418加压筛选。随后的1个月中,每隔3～5天换液一次,维持G418浓度为800 mg/L。然后,通过双抗夹心ELISA体系筛选得到6株高表达的单克隆细胞株,其上清中目的蛋白浓度均在3 mg/L以上。RT-PCR分析表明,这些转染了MBL基因的CHO细胞能稳定转录MBLmRNA。三、重组人MBL蛋白的纯化及其生物学特性研究利用硫酸铵沉淀法初步纯化表达产物,鼠抗人MBL-CRD mAb亲和层析柱进一步纯化,获得重组人MBL蛋白。在1L培养上清中可获得2mg左右目的蛋白。经ELISA鉴定,纯化后的蛋白产物可与抗MBL单克隆抗体、抗MBL-CRD单克隆抗体、抗MBL-CLR多克隆抗体结合。通过非还原SDS-PAGE和WesternBlot分析,重组人MBL分子量多集中在Mr 200 000以上,为三至六聚体形式。结合实验证明所纯化的重组MBL蛋白能与甘露聚糖及重组MASP蛋白有效结合;C4d沉积实验表明重组人MBL蛋白和血浆来源MBL均能有效介导补体激活,而这种介导作用可被竞争性糖类抑制。另外,用C4d沉积试验检测保存于不同温度下6个月的重组蛋白介导补体激活的功能,发现其稳定性良好,活化补体活性无明显降低。
[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.

【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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