单核细胞趋化蛋白—1在大肠杆菌中的表达

将人单核细胞趋化蛋白－１（ＭＣＰ－１）的ｃＤＮＡ插入融合蛋白表达载体ｐＧＥＸ－４Ｔ－１中，构建成质粒ｐＧＥＸ／ＭＣＰ转化大肠杆菌ＪＭ１０９，经ＩＰＴＧ诱导后合成ＧＳＴ－ＭＣＰ－１融合蛋白。用１２％ＳＣＳ－ＰＡＧＥ检测在３０ｋＤ左右有新生蛋白条带出现，表达量约占菌体总蛋白的３１．７％，趋化实验证明，该产物具备明确的单核细胞趋化活性。     

抗人CD4单链抗体的表达及其分离纯化

本研究将构建的抗人ＣＤ３单链抗体基因，克隆到融合蛋白表达载体ｐＧＥＸ－４Ｔ－１，用ＩＰＴＧ诱导表达ＧＳＴ－ＳｃＦｖ融合蛋白，并以ＳＤＳ－ＰＡＧＥ分析，在Ｍｒ为５２０００左右出现的一条新生蛋白带，表达量约占菌体总蛋白的４０％。经初步纯化和复性后，用ＧＳＴ亲和色谱纯化，再经凝血酶水解获得抗人ＣＤ３ＳｖＦｖ，竞争结合抑制实验证明，该表达产物具有ＣＤ３亲和活性。     

抗人CD3单链抗体的表达及其分离纯化

本研究将构建的抗人ＣＤ３单链抗体基因，克隆到融合蛋白表达载体ｐＧＥＸ－４Ｔ－１中，用ＩＰＴＧ诱导表达ＧＳＴ－ＳｃＦｖ融合蛋白，并以ＳＤＳ－ＰＡＧＥ分析，在Ｍｒ为５２０００左右出现一条新生蛋白带，表达量约占菌体总蛋白的４０％。经初步纯化和复性后，用ＧＳＴ亲和色谱纯化，再经凝血酶水解获得抗人ＣＤ３ＳｃＦｖ，竞争结合抑制实验证明，该表达产物具有ＣＤ３亲和活性     

A组轮状病毒外壳蛋白VP4的原核表达

选用原核表达载体ｐＧＥＸ２Ｔ，该载体表达谷胱苷肽－Ｓ转移酶融合蛋白，将完整的Ａ组轮状病毒外壳蛋白基因插入ｐＥＧＸ２Ｔ中，在ＳＤＳ－ＰＡＧＥ上没有明显的表达条带，而将部分ＶＰ４基因和６３１个碱基，插入ｐＧＥＸ２Ｔ，在ＳＤＳ－ＰＡＧＥ中有明显的表达条带。     

hIL—13融合表达载体的构建及其在大肠杆菌中的表达

通过ＲＴ－ＰＣＲ技术直接从激活的健康人外周血单个核细胞（ＨＰＢＭＣ）中扩增出的０．３ｋｂ的ｈＩＬ－１３基因片段，经ＤＮＡ重组技术，插入到融合蛋白表达载体ｐＧＥＸ－４Ｔ－２中，转化入感受态的大肠杆菌ＴＧ１中，成功地构建了ｈＩＬ－１３的基因工程表达菌株ｈＩＬ－１３「ｐＧＥＸ－４Ｔ－２／ＴＧ１。经异丙基硫代半乳糖苷（ＩＰＴＧ）诱导后表达的ＧＳＴ－ＩＬ－１３融合蛋白经ＳＤＳ－ＰＡＧＥ证明分子量约３６ｋＤ、     

抗人肿瘤坏死因子—α单链抗体基因在大肠杆菌中的融合表达

目的：将抗ｈＴＮＦ－α单链抗体基因克隆入融合表达载体ｐＧＥＸ４Ｔ－１中，以期得到ＧＳＴ－ＳｃＦｖ融合表达蛋白。方法：将限制性内切酶酶切拼接法获得的Ｅ６ＳｃＦｖ基因克隆入融合表达载体ｐＧＥＸ４Ｔ－１中，转化大肠杆菌ＤＨ５α，经异丙基－β－Ｄ－硫代半乳糖苷（ＩＰＴＧ）诱导，１２％ＳＤＳ－ＰＡＧＥ检测表达产物，光密度扫描和Ｗｅｓｔｅｒｎ－ｂｌｏｔ验证表达产物。结果：ＳＤＳ－ＰＡＧＥ显示，Ｅ６ＳｃＦｖ表达产物约为５２ｋｕ左右，与预期的结果相符；光密度扫描结果表明，ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白占菌体总蛋白的４０％；Ｗｅｓｔｅｒｎ－ｂｌｏｔ证实，在相应分子量处，有ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白的显色印迹；进一步对表达产物的形式分析，ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白的表达产物为包涵体形式。结论：在大肠杆菌中成功地表达了抗ｈＴＮＦ－α单链抗体基因与谷胱甘肽巯基转移酶（ＧＳＴ）基因的融合蛋白     

中国版纳猪MHCI类P1分子全长的原核表达与纯化

目的：获得原核表达的中国版纳猪ＳＬＡＩ类Ｐ１蛋白质分子。方法：ＰＣＲ扩增去信号肽的ＳＬＡＩ类Ｐ１ｃＤＮＡ序列，亚克隆至ｐＧＥＭＴ载体，测序。将亚克隆的Ｐ１　ｃＤＮＡ片段插入表达载体ｐＥＴ４２ｂ（＋），构建重组表达质粒ｐＥＴ－４２ｂ（＋）／ｓｌａ－ｐｌ，转化Ｅ·ｃｏｌｉ表达菌 ＢＬ２１－ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＬ，ＩＰＴＧ诱导 Ｐ１－８ ｘ ｈｉｓ融合蛋白表达，经包涵体洗涤，８ ｍｏｌ／Ｌ尿素变性溶解，Ｎｉ２＋亲和层析，梯度透析后，定量保存。ＳＤＳ－ＰＡＧＥ、ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ鉴定目的蛋白的表达与纯化。结果：目的蛋白（分子量３９．５ ｋＤ）表达量占细菌总蛋白 １５％，每升表达菌获得纯度９５％的目的蛋白 ４０ ｍｇ～６０ｍｇ。结论：成功建立猪 ＳＬＡ分子全长原核表达、纯化体系，为建立间接识别猪移植抗原ＳＬＡＩ类分子的人Ｔ细胞系及表位分析打下基础。     

hIL-15成熟肽段基因的克隆及其原核表达载体的构建

本文采用ＲＴ ＰＣＲ的方法自成人脾脏中扩增出ｈＩＬ １５成熟肽段基因，定向克隆入ｐＵＣ１９中，筛选带有插段的阳性克隆。经序列测定证实后，插入大肠杆菌融合表达载体ｐＧＥＸ ４Ｔ １中，构建重组表达质粒ｐＧＩＬ １５。ＳＤＳ ＰＡＧＥ证实ｐＧＩＬ １５大肠杆菌中可表达分子量为３８６ｋＤ的融合蛋白带，表达量约占菌体总蛋白的１８５％。目的蛋白经纯化后具有促进靶细胞ＤＮＡ合成的作用。     

氧化修饰的低密度脂蛋白和极低密度脂蛋白对单核细胞MCP-1表达的影响

为了研究氧化修饰的低密度脂蛋白（ＯＸ┐ＬＤＬ）和氧化修饰的极低密度脂蛋白（ＯＸ┐ＶＬＤＬ）对单核细胞的单核细胞趋化蛋白１（ＭＣＰ┐１）ｍＲＮＡ和蛋白表达的影响。在单核细胞的培养基中分别加入２５μｇ／ｍｌ的ＬＤＬ、ＯＸ┐ＬＤＬ、ＶＬＤＬ和ＯＸ┐ＶＬＤＬ，培养２４小时后分别收集单核细胞及其条件培养基。参照异硫氰酸胍法提取单核细胞总ＲＮＡ，并用γ３２Ｐ末端标记ＭＣＰ┐１寡核苷酸探针，进行ｓｌｏｔｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析。同时用夹心ＥＬＩＳＡ检测其条件培养基中ＭＣＰ┐１蛋白含量。结果显示单核细胞能表达ＭＣＰ┐１，ＯＸ┐ＬＤＬ和ＯＸ┐ＶＬＤＬ能明显增强单核细胞表达ＭＣＰ┐１。而ＬＤＬ和ＶＬＤＬ的作用却很轻微。说明单核细胞能通过表达ＭＣＰ┐１，从而进一步招引其它的单核细胞迁入内皮下间隙，而ＯＸ┐ＬＤＬ和ＯＸ┐ＶＬＤＬ能加强这种作用。     

人单核细胞趋化蛋白－1（MCP－1）基因的PCR扩增、克隆及序列分析

本文报道用植物血凝素（ＰＨＡ）诱导健康人外周血单个核细胞（ＰＢＭＣ，包括单核细胞和淋巴细胞），提取细胞总ＲＮＡ，反转录合成ｃＤＮＡ第一链，再以其为模板进行ＰＣＲ，得到了编码成熟单核细胞趋化蛋白－１（ＭＣＰ－１）的ｃＤＮＡ。该ｃＤＮＡ用ＥｃｏＲＩ、ＢａｍＨＩ双酶切后，回收含ＭＣＰ－１基因的长约２８０ｂｐ的ＤＮＡ片段，插入到经ＥｃｏＲＩ、ＢａｍＨＩ双酶切的ｐＵＣ１９载体中，进行序列分析。结果表明，ＭＣＰ－１中第１２个氨基酸的编码序列与国外报道的不同。由ＴＧＴ变成了ＴＧＣ，但编码相同的氨基酸即半胱氨酸，其余的编码序列则完全相同，说明ＭＣＰ－１的基因型可能存在着多态性。     

Expression of monocyte chemoattractant protein 1 (MCP-1) in adult periodontal disease: increased monocyte chemotactic activity in crevicular fluids and induction of MCP-1 expression in gingival tissues.

The present study shows that monocyte chemotactic activity in crevicular fluids increases with severity of the disease and that a monocyte chemoattractant, monocyte chemoattractant protein 1 (MCP-1), is expressed as the predominant cytokine of gingival tissues and their fibroblasts treated with Porphyromonas (Bacteroides) gingivalis lipopolysaccharide (P-LPS). High monocyte chemotactic activity in the crevicular fluids was neutralized significantly by antiserum specific for the JE/MCP-1 protein. Marked expression of the MCP-1 gene was observed in the gingival tissues of all adult periodontal patients tested, but not in those of healthy subjects. Monocyte chemotactic activity was observed in culture supernatants of human normal gingival tissues treated with P-LPS, and the chemotactic activity increased in a dose-related manner. Expression of MCP-1 in P-LPS-treated human gingival fibroblasts was further examined. P-LPS induced the MCP-1 gene expression in a dose- and treatment time-dependent manner. The MCP-1 gene product in the culture supernatant was detected as two forms with molecular masses of 11,000 and 15,000 Da by immunoprecipitation with the specific antiserum. The MCP-1 gene expression was induced in the fibroblasts treated with interleukin-1 beta and tumor necrosis factor alpha, but not with interleukin-6. These results suggest that gingival fibroblasts can participate in monocyte recruitment in gingival tissues of adult periodontal patients via the MCP-1 gene product and that MCP-1 plays an important role in the inflammatory reaction in the disease.     

Effect of acute inflammatory lung injury on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar macrophages.

Using a well-characterized rat model of immune complex-mediated acute inflammatory lung injury, we determined that there is a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Monocyte chemotactic activity is also significantly enhanced in culture supernatants from pulmonary alveolar macrophages (PAMs) from injured rat lungs. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein 1 (MCP-1) mRNA in PAMs obtained from rats with immune complex-induced lung injury. The increased expression of MCP-1 mRNA and associated increase in monocyte chemotactic activity present in culture supernatants of PAMs from injured rat lungs suggest that PAMs may participate in the pathogenesis of acute inflammatory lung injury by the secretion of monocyte chemoattractants including MCP-1.     

Monocyte chemoattractant protein 1 in a rat model of pulmonary granulomatosis.

Monocyte chemoattractant protein 1 (MCP1), also known as monocyte chemotactic and activating factor, possesses potent chemotactic activity for monocytes and can augment monocyte tumoristatic activity against some tumor cell lines. While these activities suggest a role in inflammatory and immunologic processes, the biologic role of MCP1 has not been studied in vivo. Glucan-induced pulmonary granulomatosis in the rat is an ideal model in which to study the role of MCP1 because the granulomas are monocyte/macrophage rich. Intravenous infusion of particulate yeast cell wall glucan resulted in the synchronous development of angiocentric pulmonary granulomas. Early lesions (6 hours) were characterized by intravascular glucan aggregates surrounded by neutrophils and foci of alveolar hemorrhage while later appearing granulomatous lesions (48 to 96 hours) were dominated by monocytes and macrophages. Granuloma formation was paralleled by a peripheral blood monocytosis. Analysis of bronchoalveolar lavage (BAL) fluid revealed an early, transient rise in tumor necrosis factor activity followed by a marked rise in monocyte-specific chemotactic activity. The rise in BAL fluid monocyte chemotactic activity, which coincided with the development of the monocyte/macrophage-rich granulomas, was preceded by a marked increase in whole lung MCP1 mRNA expression. BAL fluid monocyte chemotactic activity could be nearly completely neutralized with antibody directed against rat MCP1. These studies demonstrate that MCP1 mRNA expression is upregulated in glucan-induced pulmonary granulomatosis and that MCP1 is present in BAL fluid. Intrapulmonary granulomatosis may be important in the pathogenesis of granuloma formation.     

Expression of monocyte chemoattractant protein-1 in Kawasaki disease: the anti-inflammatory effect of gamma globulin therapy

We investigated the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA, protein, and its bioactivity in plasma, mononuclear cells (MNC) and polymorphonuclear leukocytes (PML) in patients with Kawasaki disease, including those who were treated with intravenous gamma globulin. MCP-1 mRNA expression was increased in the MNC and the plasma level of MCP-1 and monocyte chemotactic activity in plasma in the acute phase as compared with healthy control levels and decreased after the gamma globulin therapy. The infused gamma globulin contained MCP-1 protein with monocyte chemotactic activity and did not show a neutralising effect against MCP-1 protein in vitro . Our results suggest that the infusion of gamma globulin may reduce the production of MCP-1 in MNC in patients with Kawasaki disease, subsequently reducing its level in plasma. The changes in MCP-1 level after gamma globulin therapy may serve to alleviate the symptoms in the acute phase in patients with Kawasaki disease.     

Chemotactic Cytokine (IL-8 and MCP-1) Gene Expression by Human Whole Blood

The salient features of systemic or local inflammation are the myriad of cellular and humoral interactions that result in elicitation of inflammatory leukocytes. In this study using specialized connective tissue, intact whole blood, we demonstrate the gene expression of two novel chemotactic factors. The buffy-coat cellular expression of neutrophil chemotactic/activating factor/interleukin 8 (IL-8) and monocyte chemotactic/activating protein (MCP-1) mRNA were time and dose-dependent in response to either lipopolysaccharide or zymosan stimulation. This system with the complexity of tissue provides a unique model for the determination of chemotactic cytokine gene expression.     

小鼠趋化因子CCL19Ig融合蛋白表达载体的构建、表达及鉴定

通过特异引物扩增出去掉终止密码的mCCL19编码序列,经酶切、亚克隆、拼接构建了真核表达载体pcDNA3.1-mCCL19Ig,酶切和测序鉴定插入序列;将重组质粒转染CHO细胞进行体外表达,通过RT-PCR、Western blot鉴定目的基因的表达,利用趋化小室法测趋化活性。结果测序证实重组表达载体含mCCL19编码序列和人IgG1 Fc段序列,其序列分别与GenBank中公布序列比对一致,IgG1 Fc段读码框未发生改变;Western blot结果证实转染了pcDNA3.1-mCCL19Ig的CHO细胞培养上清中有相对分子质量为38 000的融合蛋白mCCL19Ig表达;体外趋化实验表明融合蛋白mCCL19Ig对小鼠脾细胞有趋化活性,且呈剂量依耐关系。     

Increase of CCR1 and CCR5 expression and enhanced functional response to MIP-1 alpha during differentiation of human monocytes to macrophages

Chemokines and their receptors regulate migration of leukocytes under normal and inflammatory conditions. In this study, we analyzed the CC chemokine receptor (CCR) expression of monocytes differentiating in vitro to macrophages. We observed a time-dependent change of expression and functional responsiveness of CCR1, CCR2, and CCR5 within 48 h. Whereas freshly harvested monocytes were strongly attracted by monocyte chemotactic protein 1 (MCP-1), a specific ligand for CCR2, only a weak response was observed to macrophage inflammatory protein 1alpha (MIP-1alpha), which binds to CCR1 and CCR5. In striking contrast, differentiated macrophages displayed a strong chemotactic response to MIP-1alpha and only a weak response to MCP-1. These findings were paralleled by intracellular calcium shifts. During the time course of monocyte to macrophage differentiation, mRNA levels and surface expression of CCR2 decreased, whereas that of CCR1 and CCR5 increased. The time-dependent switch from CCR2 on monocytes to CCR1 and CCR5 on mature macrophages reflects a functional change belonging to the differentiation process of monocytes to macrophages and may form the basis for a differential responsiveness of monocytes and macrophages to distinct sets of chemokines.     

Induction of monocyte chemoattractant protein-1 (MCP-1) production by Pseudomonas nitrite reductase in human pulmonary type II epithelial-like cells

Monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes, is presumed to play a pivotal role in the recruitment and accumulation of monocytes in various diseases including pulmonary infections. We examined here whether or not Pseudomonas nitrite reductase (PNR), a recently identified IL-8 inducer in various respiratory cells, could stimulate human pulmonary type II epithelial-like cells (A549) to induce MCP-1 production. A time- and dose-dependent induction of MCP-1 protein synthesis associated with an increase of MCP-1 mRNA expression by A549 cells was observed in response to PNR. New protein translation was not required for PNR-mediated MCP-1 mRNA expression in the same cells. When anti-human MCP-1 monoclonal antibody was used for neutralizing of monocyte chemotactic factor (MCF) activities in the culture supernatants of these cells stimulated with PNR, significant reductions of MCF activities (the mean reduction rate; 49-59%, P<0. 05) were observed. These data suggest that PNR may contribute to monocyte migration, through inducing pulmonary epithelial cell-derived MCP-1 production in the airway of patients with pneumonia due to P. aeruginosa.     

高迁移率族蛋白B1对磷酸钙诱导巨噬细胞释放炎症因子的协同作用

背景：研究表明,巨噬细胞及其炎症反应参与了肾结石的发生发展。前期实验发现结石晶体可刺激巨噬细胞释放高迁移率族蛋白B1。 目的：观察高迁移率族蛋白B1对磷酸钙诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1的协同作用。 方法：实验分两部分：①将成功诱导为巨噬细胞的U937细胞分为空白组、100 mg/L磷酸钙组、100 μg/L高迁移率族蛋白B1组、100 mg/L磷酸钙+100 μg/L高迁移率族蛋白B1组,干预1,2,4 h后收集细胞上清液。②将已成功诱导为巨噬细胞的U937细胞分为100 mg/L磷酸钙组、磷酸钙+10 μg/L高迁移率族蛋白B1组、磷酸钙+50 μg/L高迁移率族蛋白B1组、磷酸钙+100 μg/L高迁移率族蛋白B1组,干预4 h后收集细胞上清液。Elisa法检测白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1水平。 结果与结论：ELISA结果显示,磷酸钙组,100 μg/L高迁移率族蛋白B1组上清液白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1质量浓度均高于空白组,磷酸钙+100 μg/L高迁移率族蛋白B1组上清液上述因子质量浓度均显著高于其他3组(P < 0.05),且呈时间依赖性。不同质量浓度高迁移率族蛋白B1+磷酸钙组细胞上清液白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1水平均显著高于磷酸钙组(P < 0.05),且呈浓度依赖性。结果表明,磷酸钙及高迁移率族蛋白B1均可诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1;高迁移率族蛋白B1可协同磷酸钙诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：