大鼠补体调节因子DAF功能区的表达纯化及功能鉴定

目的:重组表达促衰变因子(DAF)功能区的4个短的保守重复序列(SCR1-4)。方法:通过克隆大鼠DAF的SCR1-4并插入到原核表达载体pET32a( ),IPTG诱导共表达,根据表达结果选择纯化方式。结果:诱导后表达产物为不溶蛋白,通过纯化包涵体并复性,得到了具有抑制致敏绵羊红细胞发生溶血的可溶性蛋白成分。结论:原核表达SCR1-4易于形成包涵体,稀释复性法适用于DAFSCR1-4包涵体的复性。     

负载HCMV pp65抗原肽的HLA-A*1101-GPI四聚体的制备和鉴定

为体外复性制备负载人巨细胞病毒(HCMV)pp65抗原肽的HLA-A*1101-GPI四聚体,研究优化HLA-A*1101重链与生物素化酶底物肽融合蛋白(HLA-A*1101-BSP)在大肠杆菌中的诱导表达的温度、时间和诱导剂IPTG浓度,并以抗HLA-A*0201抗血清进行免疫印迹鉴定HLA-A*1101-BSP的表达水平。将初步纯化的HLA-A*1101-BSP与β2-微球蛋白(β2m)和HCMV抗原肽pp6516-24(GPISGHVLK,简称GPI)一起利用稀释法进行重折叠复性,获得可溶性HLA-A*1101-GPI单体,经生物素化和纯化后,与Streptavidin-PE结合成四聚体。流式细胞仪分析显示HLA-A*1101-GPI四聚体具有与特异性细胞毒T细胞(CTL)的结合活性,表明成功获得可溶性HLA-A*1101-GPI四聚体,为研究HLA-A*1101限制性CTL的免疫应答打下基础。     

重组人CD40胞外结构域在大肠杆菌中的表达和纯化鉴定

目的构建人CD40胞外结构域（exCD40）的原核表达载体，获得可溶性产物。方法以RT-PCR方法从人白细胞总RNA中扩增编码CD40胞外区的cDNA，构建羧基端融合His6标签的exCD40的原核表达载体，在大肠杆菌中表达，并对表达产物进行复性、纯化和鉴定。结果构建了exCD40的原核表达载体，并在大肠杆菌中获得高表达，Mr为23000，与理论大小相符，表达产物主要存在于包涵体中，经Ni^2＋-NTA柱上复性和纯化获得纯度达95％的可溶性exCD40蛋白，该蛋白能与细胞上的CD40L结合。结论从大肠杆菌中成功获得具有配基结合活性的可溶性exCD40蛋白。     

人EGF受体胞外域在大肠杆菌中表达、复性及抗原性鉴定

目的 构建人EGF受体（EGFR）胞外域的原核表达载体并在大肠杆菌中表达,制备EGFR特异性抗体,对表达产物进行鉴定。方法 以PCR方法从克隆的EGFR胞外区cDNA中扩增编码胞外结构域L1、S1、L2（EGFR-LSL）的DNA片段,并在其3′端加入编码His6标签的序列,与pET-3c连接构建EGFR-LSL原核表达载体,在大肠杆菌中进行表达;同时以纯化的EGFRL2结构域蛋白（EGFR-L2）制备抗血清,以此抗血清鉴定表达产物。结果 EGFR-LSL在大肠杆菌BL21（DE3）中获得高效表达,抗His6抗体免疫印迹分析表明,表达产物全部以包涵体形式存在,通过Ni2^＋．NTA柱上复性获得纯化的可溶性EGFR-LSL蛋白;免疫印迹分析表明,EGFR-LSL与小鼠抗EGFR-L2抗血清具有高特异性反应。结论 从大肠杆菌中获得具有特异抗原性的可溶性EGFR-LSL蛋白。     

血管抑制素和内皮抑制素在大肠杆菌中的融合表达与鉴定

血管抑制素（AS）和内皮抑制素（ES）是两种具有较强活性的内源性血管抑制剂，联合应用具有协同作用。本研究通过大肠杆菌表达AS-ES融合蛋白，观察其对血管生成的抑制作用。首先用RT-PCR方法分别获得AS和ES基因，通过基因拼接获得融合基因，构建了含有该融合基因的原核表达质粒——pET-42（b）／AS-ES。表达菌株经IPTG诱导后目的蛋白以包涵体形式表达，表达量为14％，分子量约65KD。Western blotting检测表明表达产物可分别与AS和ES抗体产生特异的免疫反应。经复性、肝素亲和层析柱纯化的表达产物对鸡胚尿囊膜血管有明显抑制作用。本研究获得了AS、ES在大肠杆菌中的融合表达．目的蛋白具有特异的免疫反应性和血管抑制活性。     

CD83重组蛋白的原核表达、复性纯化及多克隆抗体制备北大核心CSCD

目的:利用原核表达系统表达、纯化CD83蛋白,制备CD83多克隆抗体。方法:构建CD83胞外结构域的原核表达质粒,利用TOP10F’菌株表达,使用IPTG诱导并寻找最佳条件;采用0.3%SKL将包涵体溶解变性,透析复性后通过GST亲和层析纯化GST-CD83蛋白;通过QuickAntibody免疫佐剂免疫SD大鼠制备CD83多克隆抗体,ELISA分析抗体的反应活性和特异性。结果:成功构建重组质粒pGEX-2T-CD83(Met1-Arg133),获得最佳诱导条件:0.2 mmol/L IPTG、25℃、O/N。成功获得CD83重组蛋白,免疫大鼠后获得了特异性及活性较高且效价为1∶104~1∶105的多克隆抗体。结论:获得的CD83重组蛋白及多克隆抗体,为后期深入开展CD83蛋白及CD83抗体的免疫抑制特性研究提供支持。     

小鼠FcγRⅢ原核表达、纯化及鉴定

目的: 制备纯化重组蛋白mFcγRⅢ,并鉴定其生物学活性.方法: 从克隆载体mRⅢ-T中扩增mFcγRⅢ胞外区,构建原核表达载体pETmRⅢ,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,SDS-PAGE和Western blot对重组表达蛋白进行鉴定;表达产物通过包涵体纯化后用稀释方法复性;ELISA检测复性蛋白活性.结果: 成功构建了重组表达质粒pETmRⅢ,纯化和复性了重组蛋白mFcγRⅢ;复性的重组蛋白mFcγRⅢ具有较强的体外活性.结论: 原核重组蛋白mFcγRⅢ复性后具有较强的体外结合配体特性,为探索重组蛋白治疗自身免疫病奠定了基础.     

Nogo-66融合蛋白的原核表达及鉴定

目的原核表达轴突再生抑制蛋白Nogo-66,并对其进行鉴定。方法采用聚合酶链反应方法从含Nogo-66编码序列的质粒DNA中扩增出Nogo-66基因开放阅读框,构建原核表达载体,转化大肠杆菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,裂解细菌抽提蛋白,表达产物进行聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western免疫印迹鉴定。结果测序鉴定Nogo-66开放阅读框成功插入pET-46-EK/LIC质粒;重组质粒在大肠杆菌中成功表达大鼠Nogo-66蛋白,SDS-PAGE测定其相对分子质量(Mr)约7000,WesternBlot结果证实表达产物融合有聚组氨酸标签。结论应用原核表达系统成功表达了Nogo-66蛋白,为下一步制备Nogo-66蛋白疫苗及疫苗功能研究打下了基础。     

HLA-A*0206-BSP和-A*0207-BSP融合基因的构建与表达

采用RT-PCR技术从HLA-A*0206和-A*0207阳性个体的PBMC中分别克隆出HLA-A*0206和-A*0207基因的全长cDNA序列,构建HLA-A*0206和-A*0207克隆载体。再利用PCR技术从构建的克隆载体中扩增HLA-A*0206和-A*0207的α链(重链)胞外段序列,分别经双酶切置换本室保存的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使HLA-A*0206和-A*0207与BirA酶底物肽(BirA substrate peptide,BSP)序列融合,构建HLA-A*0206-BSP和-A*0207-BSP融合基因的表达载体,经限制性酶切和DNA测序证实。然后将该表达载体转化E.coliBL21(DE3)后获得表达产物,通过体外稀释复性,初步纯化的表达产物通过ELISA和Western blot检测证明能够与β2微球蛋白(HLA I类分子轻链)及HLA-A2限制性抗原肽(HBV core 18-27)折叠形成具有HLA I类分子天然构象的抗原肽/HLA-A2复合物单体。为进一步构建HLA-A*0206和-A*0207四聚体,探讨相应HLA-A2亚型的功能特点提供了物质基础。     

pS2融合蛋白的原核表达及其性质鉴定

目的：获得ｐＳ２融合蛋白。方法：ＲＴ－ＰＣＲ法从乳腺癌组织中扩增ｐＳ２片段，定向克隆到ＰＭＳ－３１ｂ载体上，重组质粒ＰＭＳ－ｐＳ２转化温度敏感型细菌ＰＯＰ２１３６，使之高效表达融合蛋白ＭＳ２－ｐＳ２。结果：免疫组织化学试验（ＩＨＣ）结果表明：纯化的融合蛋白ＭＳ２－ｐＳ２能与抗ｐＳ２进口单抗特异反应。酶联免疫吸附试验（ＥＬＩＳＡ）及Ｗｅｓｔｅｒｎ ｂｌｏｔｉｎｇ试验表明；ＭＳ２－ｐＳ２能与ｐＳ２Ｃ端     

High Level Expression of HLA-A＊0203-BSP Fusion Protein in Escherichia coli and Construction of Soluble HLA-A＊0203 Monomer and Tetramer Loaded with Epstein-Barr Virus Peptide

Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.     

Identification of a novel HLA-A allele, HLA-A*9216

The new human leukocyte antigen (HLA)-A*9216 differs from A*02010101 by a nonsynonymous nucleotide change at condon 76 (GTG>CTG).     

Identification and nucleotide sequence of HLA-A*02015

A novel HLA-A*02 allele, A*02015, is described that possess a unique non-coding substitution of G to A at position 113 in exon 3.     

Identification and nucleotide sequence of HLA-A*03013

We have identified a further HLA-A*03 allele, A*03013, in three unrelated Caucasoid individuals. The allele was initially detected by the polymerase chain reaction using sequence-specific primers (PCR-SSP) and was shown to differ from HLA-A*03011 by a single non-coding substitution of G to T at position 167 in exon 2 by DNA sequencing. The A*03013 bearing haplotype in one of the three individuals, was HLA-A*03013, B*07, Cw*0702, DRB1*1101, DRB3*0202, DQA1*05, DQB1*0301. The estimated phenotype and gene frequencies for A*03013 was <0.02% and <0.00007, respectively.     

可生物素化HLA-A2-生物素化序列融合基因的构建与表达

本文采用RT PCR技术从人外周血PBMC中克隆HLA A2基因的胞外区 ,拼接上依赖BirA酶的可生物素化序列后 ,装入pET 42b (+)高效表达载体进行诱导表达。并对表达产物进行纯化与复性。结果成功地扩增出HLA A2基因并测序证实 ;构建了融合表达载体pET HLA A2 ,并获表达与纯化。为研究在原核系统中表达、纯化与复性及进一步体外构建MHC I类分子四聚体 ,探讨免疫识别机制奠定了基础。     

A novel HLA-A*33 (A*3309) allele in a Caucasian family that differs at protein position 66 from HLA-A*3308

A novel human leucocyte antigen (HLA)-A33 (HLA-A*3309) allele has been identified in a Caucasian family from Middle Europe using single allele-specific sequencing strategy. This allele is identical to the HLA-B*3308 allele except for one point mutation in exon 2 at codon 66 (AAA-->AAT), resulting in an amino acid change from lysine (K) to asparagine (N).     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.