《胶原样凝集素》PPT课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,胶原样凝集素COLLECTIN,郑州大学医学院免疫学与微生物学教研室,李付广,一、凝集素（LECTIN）的分类,C-型凝集素: 需要Ca,2+,维持活性的凝集素,S-型凝集素: 巯基依赖或-半乳糖结合的凝集素,二、胶原样凝集素历史上的重要发现,1906 conglutinin; first animal lectin to be associated with the immune system,1989-1991 mannan-binding lectin,1999 collectin liver 1(CL-L1),2001 collectin placenta 1(CL-P1),2003 collectin kidney 1(CL-K1),三、胶原样凝集素的一般特性,四、胶原样凝集素的结构,（一）一级结构特点,1. N端富含半胱氨酸区段：7-28氨基酸，其中1-3半胱氨酸,2. 胶原样区：53-177氨基酸(-Gly-Xaa-Yaa-),n,重复序列, Xaa、Yaa多为脯aa或羟脯aa,3. 颈区：24-28氨基酸，-helice coiled-coils,4. 糖识别区（CRDs）：与相应的糖配体结合,(二)三级结构,1.同源三聚体的形成(亚单位),相同的三条肽链形成类似郁金香花朵,2.亚单位进一步聚集成寡聚体,单体-CL-43, CL-L1,四聚体-SP-D, Conglutinin, CL-46(十字形结构),六聚体-SP-A, MBL (一束郁金香花),(三)糖结合特异性,胶原样凝集素的凝集素样活性存在于CRDs, 由三个氨基酸基序决定:,1.甘露糖结合基序: Glu-Pro-Asn,2.半乳糖结合基序: Gln-Pro-Asp,所有胶原凝样集素(SP-A除外)均具有甘露糖特异基序Glu-Pro-Asn, SP-A第三位的氨基酸由Arg(狗)或Ala替换Asn.,系统树,(五)结合微生物种类,五、胶原样凝集素在宿主防御方面的生物学功能,（一）凝集作用,通过桥联，可使病原微生物凝集成大块，加强呼吸道黏膜纤毛清除作用，阻止病原吸附到细胞表面，抑制其定居和侵入，促进吞噬作用。,（二）补体激活作用,1. MBL产生,病原微生物感染早期 刺激激活 巨噬细胞和中性粒细胞 TNF-,IL-1,IL-6 急性期反应 肝细胞 MBL, CRP,2.MBL途径(lectin pathway),MBL与病原微生物表面的mannose, fucose, GlcNAc结合启动的补体激活级联反应,(三)调理作用,1.collectin的受体,见下表,2.collectin直接作为调理素,3.激活补体成分,C4b, C3b, iC3b,4.吞噬作用的激活,(四)抑制微生物的生长,SP-A,SP-D直接引起微生物细胞壁通透性增强,导致微生物生长抑制或死亡,(五)调节炎症反应,调节前炎症因子的合成分泌,(六)调节特异性免疫,DCs抗原提呈, DCs成熟, T细胞增殖,(七)调节过敏反应,SP-A,SP-D抑制IgE与过敏原结合, 抑制过敏早期组织胺释放, 抑制晚期支气管炎症中淋巴细胞增殖,(八)对细胞凋亡的影响,SP-A抑制型肺泡细胞凋亡, SP-A、SP-D,、MBL刺激凋亡细胞清除,Human CL-L1的研究,1999年Ohtani k.等克隆和鉴定出一个编码新的胶原凝集素基因，称肝脏胶原凝集素1（collectin liver 1， CL-L1），该基因定位于第八号染色体的长臂上，其基因结构由6个外显子（EX1EX6）和5个内含子（IN1IN5）组成，cDNA含有831bp插入片段，编码277个氨基酸残基。推测的氨基酸序列具有典型的胶原凝集素结构：N端富含半胱氨酸区、胶原样区、颈部和糖识别区。,CL-L1与其他胶原凝集素相比，具有独特之处，MBL、SP-A和SP-D基因定位于第10号染色体，而CL-L1基因定位于第8号染色体，C末端具有4个重复的赖氨酸结构，CRD和颈区由一个外显子编码，胶原样区有5个外显子编码。胶原凝集素绝大多数为分泌型，存在于体液和分泌液中，只有CL-L1为可溶性胞浆内蛋白，因此推测CL-L1可能具有独特的功能。,Comparison of three constructs,Constructs,Max.O.D.,Solubility,IPTG induction,Leakage,2GXY+NECK+CRD,0.8,no(PBS),yes,yes,NECK+CRD kkkk,1,part(TBS),no(PBS),no,yes,2GXY+NECK+CRDkkkk,1,no,no,yes,Neck+CRDkkkk,M 1 2 3 4,25kDa,17kDa,M marker,1 before induction,induction 1 hr,induction 3 hrs,induction 5 hrs,Growth of standard E. coli expression culture(200 ml),(1)Inoculate 20 ml of SOB (containing ampicillin 100,g/ml, chloramphenicol 35,g/ml) in 100 ml flask. Grow the cultures overnight at 37,C.,(2)Inoculate 200 ml of prewarmed SOB media with 10 ml of the overnight cultures and grow at 37,C with vigorous shaking until an OD600 of 0.6 is reached.,(3)Take a 1 ml sample immediately before induction.,(4)Induce expression by adding IPTG to a final concentration 1 mM.,(5)Incubate the cultures for 3 hrs. Collect a second 1 ml sample.,(6)Harvest the cells by centrifugation at 3500rpm for 25 min.,(7)Freeze the cells at -70.,Preparation of cleared E. coli lysate under denaturing conditions,(1)Thaw the cell pellet for 15 min on ice and resuspend in lysis buffer at 5 ml per gram wet weight.,(2)Stir cells for 15-60 min at room temperature.,(3)Centrifuge lysate at 10000,g for 20-30 min at room temperature to pellet the cellular debris. Save supernatant, and filter the supernatant with 0.45,m filter.,(4)Add 10,l 2SDS-PAGE sample buffer to 10,l supernatant and store at -20,C for SDS-PAGE analysis.,Purification of Neck+CRD KKKK of hCL-L1 with Ni-NTA column,(1)Equilibrate column with 5 column volumes of lysis buffer .,(2)Apply lysate to column. Collect the pass-through for SDS-PAGE analysis(recycle once).,(3)Wash with 10 column volumes of wash buffer until A280 is below 0.01.,(4)Elute protein with elution buffer. Collect the single protein peak completely.,Dialysis of recombinant protein,buffer 1-8M urea,TBS(pH7.6)/300ml; buffer 2- TBS(pH7.6)/3000ml,(1)Add elution into membrane tube (MWCO15000), put the membrane in 300 ml buffer 1, stirring slowly.,(2)Add 100 ml buffer 2 at 6 hour intervals, total 8 times.,(3)At last put membrane in 2000 ml buffer 2 for dialysis overnight.,1 2 3 4,M,25kDa,16.5kDa,M marker,1,3 before induction,2,4 induction 3hrs,CBB staining,1 2 3 4 5 3 4 5,M,M marker,1 pre-sample,pass,elution,sup,ppt,25kDa,16.5kDa,CBB staining W.B,免疫兔子获得免疫血清,谢谢!,