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Ca2+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-Asn2.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, CRP2.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, iC3b4.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,Neck+CRDkkkk,M 1 2 3 4,25kDa,17kDa,M marker 1 before inductioninduction 1 hrinduction 3 hrsinduction 5 hrs,Growth of standard E. coli expression culture(200 ml),(1)Inoculate 20 ml of SOB (containing ampicillin 100g/ml, chloramphenicol 35g/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 10000g 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-samplepasselutionsupppt,25kDa,16.5kDa,CBB staining W.B,免疫兔子获得免疫血清,谢谢!,