CGT52TGT和GGA57GAA甘露聚糖结合凝集素突变体的构建

目的 构建CGT52TGT和GGA57GAA2种甘露聚糖结合凝集素(MBL)基因突变体。方法 设计2对引物52F／52R和57F／57R(R中引入了点突变)，以含汉族人野生型MBL cDNA的重组质粒pGEM-mbl为模板，采用TaKaRa MutanBEST突变试剂盒进行定点突变，以PER和测序分析进行鉴定。结果 分别用52F／52R和57F／57R为引物对进行PCR，均得到1个约3800bp的DNA片段，自身连接后获得重组质粒。以SP6／T7P为引物对进行PCR，获得约900bp的扩增产物。序列分析表明，2种克隆分别除其第52、57位密码变为TGT、GAA外，其余与野生型MBL cDNA完全相同。结论 构建成功CGT52TGT和GGA57GAA MBL基因突变体，为深入探索MBL基因突变引起调理吞噬缺损的机理和MBL分子的结构-功能关系提供了分子模型。     

MBL突变体在CHO细胞中表达及其产物分析

采用PCR技术,从质粒pMBLm54中获得含GGC54GAC点突变的甘露聚糖结合凝集素(MBL)基因,将其插入真核表达载体构建重组表达载体pcDNA4/HisMax C-MBLm54,DNA测序验证序列正确后将其电转染入CHO细胞。以Zeocin筛选并维持培养转染后CHO细胞,获得2株稳定高效表达目的蛋白的单克隆细胞株。采用Ni~(2+)-NTA agarose层析柱纯化表达产物,获得54Asp突变MBL蛋白,SDS-PAGE和Western blot分析表明,54Asp突变MBL蛋白主要为相对分子质量为60000的成分,而重组野生型MBL蛋白则主要是相对分子质量为200000和>200000的分子。配体结合试验发现,天然MBL和重组野生型MBL能与甘露聚糖结合,而54Asp则否。结果表明,54Asp突变MBL蛋白不能形成正常的MBL结构单位和寡聚体,并进而影响其配体结合活性。     

GGC54GAC MBL突变体的构建

目的：在人甘露聚糖结合凝集素（MBL）基因中引入GGC54GAC点突变。方法：设计上下游引物和诱变引物,以汉族人野生型MBLcDNA为模板,采用megaprimer-PCR技术进行定点突变。将PCR产物克隆至pGEM-T载体,酶切和测序分析。结果：以上游引物和诱变引物进行第一轮PCR,获得一约180bp的DNA片段,以此片段和下游引物行第二轮PCR扩增,得到一长约780bp的产物,将其克隆至pGEM-T载体,酶切了现第54位密码中的BanI酶切位点已被破坏,与计算机酶切图谱分析结果一致。序列分析表明,除第54位密码变为GAC外,余与野生型MBL cDNA完全相同。结论：构建成功了GGC54GAC MBL突变体,为深入探索MBL基因突变引起调理吞噬缺损的机制提供了分子病理模型。     

突变型和野生型MBL基因在COS7细胞中的瞬时表达

采用PCR技术,从质粒pMBLm52、pMBLm54和pMBLm57中分别获取含CGT52TGT、GGC54GAC和GGA57GAA点突变的人甘露聚糖结合凝集素(MBL)突变体全长编码区cDNA序列,将其分别插入质粒pcDNA4/HisMax C中,构建成相应的3种真核表达载体。将各突变型MBL基因的真核表达载体和野生型MBL基因真核表达载体以脂质体法分别转染COS7细胞。72 h后,以双抗体夹心ELISA技术检测各细胞培养上清中目的蛋白的含量分别为0.980μg/ml、0.971μg/ml、0.900μg/ml和1.047μg/ml;用One-way ANOVA分析这些目的蛋白的含量无显著性差异(P>0.05)。因此,MBL结构基因的点突变并不影响MBL蛋白的合成与分泌。     

重组人N端缺失MBL的原核表达及其活性研究

目的 原核表达具生物学活性的重组人N端缺失甘露聚糖结合凝集素(rhMBLΔN)蛋白.方法 应用PCR技术,从含中国人野生型MBL cDNA的质粒pGEM-MBL中扩增rhMBLΔN基因片段,插入pET43.1a载体后,转化E.coli BL21(DE3)感受态菌诱导表达rhMBLΔN蛋白.应用Ni2 -NTA琼脂糖柱纯化目的 蛋白,以SDS-PAGE、Western blot和ELISA进行鉴定.结果 从pGEM-MBL中扩增到长约620bp的DNA片段,构建的重组表达载体经Bam HI和Eco RI酶切后出现约7270bp和620bp的片段,测序结果与预期的完全一致.表达的重组蛋白纯化后,经SDS-PAGE鉴定为Mr97000的蛋白.ELISA证实,纯化蛋白能与抗重组人MBL抗体结合,具备配体结合活性并能与人甘露聚糖结合凝集素相关丝氨酸蛋白酶1、2的N端片段结合.结论 获得了可表达rhMBLΔN的菌株和具活性的rhMBΔN蛋白,为进一步研究提供了条件.     

人MBL糖识别域在大肠杆菌中的表达、纯化及鉴定

目的原核表达重组人甘露聚糖结合凝集素（MBL）糖识别域（CRD）。方法采用PCR技术,从含汉族人MBL全长编码区cDNA的重组质粒pGEM-mbl中扩增出CRD包括颈区的基因片段,将其插入原核表达载体pGEX-4T1中,经PCR、限制性酶切和测序确证后,以IPTG诱导其在大肠杆菌中表达。包涵体经变性、复性后以GSTtrap亲和层析柱纯化,融合蛋白经凝血酶切割后回收目的蛋白。分别以Western blot和ELISA分析表达产物的免疫学和糖结合活性。结果PCR扩增得到长约450bp的目的基因片段,插入pGEX-4T1载体所获重组表达载体pGEX4T1-CRD的酶切图谱和序列与预期的一致。重组子经诱导表达,表达产物主要以包涵体形式存在。以GSTtrap亲和层析柱纯化获得约Mr43000的GST-CRD融合蛋白,经凝血酶切割后得到约Mr17000的CRD蛋白。纯化的GST-CRD蛋白能与抗人CRD单克隆抗体特异性结合并具糖结合活性。结论获得了具生物学活性的人MBL CRD蛋白,为进一步探索MBL分子CRD的效应功能提供了实验材料。     

人MBL-CLR表达载体的构建及其在大肠杆菌中的表达

目的：在大肠杆菌中表达人甘露聚糖结合凝集素（MBL）胶原样区（CLR）蛋白。方法：应用PCR技术,从含中国人野生型MBLcDNA的质粒pGEM-MBL中扩增CLR基因片段,将其克隆至pET32a原核表达载体后,转化E.coliBL21（DE3）感受态菌株诱导表达CLR蛋白。通过Ni^2＋-NTA琼脂糖柱层析纯化目的蛋白,以SDS-PAGE、Western blot和ELISA法进行鉴定。结果：从重组质粒pGEM-MBL中扩增到长约180bp的DNA片段。构建的重组表达载体经BamHⅠ和HindⅢ酶切后出现约5900bp和180bp的片段,测序结果同预期的结果完全一致。重组质料在大肠杆菌中诱导表达后,纯化的蛋白经SDS-PAGE电泳出现Mr约为30000、60000和120000的3条带。Western blot分析表明,3条蛋白带均可与抗His抗体起反应,3条蛋白带对应于融合蛋白的单体和寡聚体。ELISA检测证实,纯化的蛋白能与鼠抗重组人MBL蛋白抗体结合。结论：获得可表达人MBL-CLR的大肠杆菌菌株和重组的人MBL-CLR-Trx融合蛋白,为MBL分子结构、功能关系的进一步研究提供了条件。     

37Glu突变型MBL蛋白在CHO细胞中的表达及其生物学特性研究

目的:探索甘露聚糖结合凝集素(MBL)基因GGA57GAA点突变引起免疫缺损的机制.方法:采用PCR从质粒pMBLm57中扩增含GGA57GAA点突变的MBL基因,构建重组真核表达载体,电转染CHO细胞,以Zeocin筛选并克隆化培养,用mAb-Sepharose 4B和Ni2 -NTA agarose层析纯化目的蛋白,以SDS-PAGE、Western blot、ELISA、配体结合试验、C4d沉积试验等鉴定目的蛋白.结果:重组真核表达载体pcDNA4/HisMax C-MBLm57转染CHO细胞后获得5株稳定表达目的蛋白的单克隆细胞株;37Glu突变型MBL蛋白主要以双链(Mr约60000)存在,不能形成高聚体;其配体结合能力低下,与MASP-2N端蛋白的结合能力也降低,不能通过凝集素途径激活补体系统.结论:GGA57GAA点突变产生结构有缺陷的MBL蛋白,进而影响其识别功能和效应功能.     

MBL与人单核细胞系THP1/CD14细胞结合及其特性研究

采用Cell-ELISA和流式细胞术,发现人单核细胞系THP1/CD14在生理条件下可结合甘露聚糖结合凝集素(MBL),这种结合具有Ca2+依赖性,能被甘露糖、N-乙酰葡糖胺、D-葡萄糖、半乳糖、蔗糖、海藻糖等及重组人MBL-CRD蛋白和MBL-CLR蛋白所部分抑制,但C1q或抗人C1qR单克隆抗体对之无影响。还发现,炎性状态下的THP1/CD14细胞与MBL的结合增强。结果表明,THP1/CD14细胞表达Ca2+依赖的、糖敏感的MBL受体或结合蛋白,包括对CLR特异和CRD特异的两种受体,均与C1q受体无关;炎性刺激可上调THP1/CD14细胞MBL受体或结合蛋白的表达。     

中国人MBL cDNA的克隆与序列分析

从中国汉族人胎肝组织提取ＲＮＡ,以ＲＴ－ＰＣＲ方法获得了编码含号顺序的全长甘露聚糖结合凝集素肽链的ｃＤＮＡ片段,将其与ｐＧＥＭ－Ｔ载体连接,转化大肠杆菌ＴＧ１,     

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.     

慢性丙型肝炎患者血浆内毒素和血清MBL变化及临床意义

目的:探讨了慢性丙型肝炎患者血浆内毒素和血清甘露聚糖结合凝集素(MBL)水平的变化及意义.方法:应用鲎试验和酶联法对31例慢性丙型肝炎患者进行了血浆内毒素和血清MBL测定,并与35名正常健康人作比较.结果:慢性丙型肝炎患者血浆内毒素和血清MBL水平均非常显著地高于正常人水平(P<0.01),且内毒素水平与MBL呈正相关...     

CD40突变体融合蛋白真核表达载体的构建及表达

目的:构建CD40突变体(muCD40)融合蛋白的真核表达载体pIRES2-EGFP/muCD40Ig,并在中国仓鼠卵巢细胞(CHO)中表达,以获得muCD40Ig融合蛋白,为检测体内可溶性muCD40分子建立实验基础。方法:从L929/muCD40基因转染细胞中通过RT-PCR扩增出人muCD40胞外段基因序列,从人脾脏细胞中扩增出人IgG1恒定区基因,分别插入真核表达载体pIRES2-EGFP,采用Superfectin转染CHO细胞,G418筛选出能稳定分泌muCD40Ig蛋白的基因转染细胞并亚克隆。筛选出的阳性克隆经无血清培养后,收集上清进行Western blot检测;并收集细胞进行RT-PCR鉴定,同时用流式细胞仪分析muCD40Ig蛋白与CD40L的结合能力。结果:成功构建了人pIRES2-EGFP/muCD40Ig重组真核表达载体,PCR及Western blot结果显示该CHO基因转染细胞能够稳定分泌人muCD40Ig蛋白;流式检测muCD40Ig融合蛋白能够与CD40L分子结合。结论:获得了能稳定分泌人muCD40Ig的CHO基因转染细胞株,muCD40Ig融合蛋白具有与CD40L结合的能力。     

Serum MASP-1 in complex with MBL activates endothelial cells

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca²⁺ signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL–MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response.     

Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene

We describe a rapid and simple method for genotyping the three known structural mutations within exon 1 of the mannan-binding lectin (MBL) gene. A PCR-amplifiable synthetic DNA (Universal Heteroduplex Generator) was annealed to genomic PCR product from exon 1 to generate unique DNA heteroduplexes for each mutation. Heteroduplexes were then resolved by non-denaturing polyacrylamide gel electrophoresis. The technique was initially validated with previously typed samples and then applied to previously untyped samples with the results confirmed by DNA sequencing. © 1997 Wiley-Liss, Inc.     

人巨细胞病毒PP150蛋白C端多肽与gp52蛋白片段的融合表达

目的：将人巨细胞病毒PP150蛋白C端多肽与gp52蛋白片段融合,构建表达该融合蛋白的工程菌。方法：合成PP150蛋白C端25个氨基酸的基因片段,并选有细菌偏爱的密码子。用PCR方法扩增gp52蛋白基因片段。将这2个基因片段分别克隆至同一质粒pET28a( )内的NcoI/BamHI和BamHI/EcoRI位点,使两者串联在一起,并且翻译框架一致,可表达1个融合蛋白。将重组质粒转化大肠杆菌,用酶切及SDS－PAGE等方法筛选表达融合蛋白的工程菌。结果：构建成功了高效表达PP150C端多肽与pg52蛋白片段的融合蛋白工程菌,表达量为35％左右。初步纯化获得了表达的融合蛋白。结论:初步纯化的融合蛋白能与已知的抗gp52-IgM阳性血清和抗PP150－IgM阳性血清反应,说明融合蛋白C端的gp52蛋白保持较好的抗原性,融合蛋白N端的PP150C端多肽也显示有抗原性。