人白血病抑制因子和白细胞介素6融合基因的构建与表达

为了获得对白血病细胞有更强的抑制和诱导分化作用,本实验应用计算机优化设计,采用PCR扩增和分子克隆技术,分别扩增编码IL-6成熟蛋白的部分cDNA片段和编码LIF成熟蛋白的cDNA片段与人工设计的四肽接头连接到克隆载体,并将IL-6基因插入到LIF基因的N端,亚克隆到原核表达载体pBV220酶切位点之间,融合成编码单一的基因。采用30℃扩增菌体,42℃诱导,实现了在E.coli中表达预期的36kD蛋白谱带。表达量约占菌体总蛋白量的21％。经初步活性测定,重组融合因子对HL-60白血病细胞克隆生长的抑制明显地强于LIF和IL-6单独使用时的作用。     

可溶性HLA-A2-IgGFc融合蛋白在昆虫细胞的表达及鉴定

目的利用杆状病毒-昆虫细胞表达体系制备可溶性HLA-A2与免疫球蛋白IgGFc段的融合蛋白。方法首先将可溶性HLA-A*0201和IgG3分子Fc段的cDNA基因插入杆状病毒表达载体pFastHTa,形成重组载体pFHT-sHLA-A*0201-IgGFc。将该重组载体转化DH10Bac大肠杆菌,sHLA-A*0201-IgGFc基因同源重组入菌体内杆粒(Bacmid)中。抽提阳性杆粒,转染昆虫细胞Sf9,72 h收集病毒上清。再将病毒上清感染Sf9细胞,96 h收集细胞并裂解,SDS-PAGE观察其表达。细胞裂解液经Ni+-NTA树脂纯化Western-blot法鉴定其蛋白质序列,间接ELISA法鉴定是否形成正确构象。结果融合蛋白不仅与HLAⅠ类分子的构象特异性抗体(W6/32)结合,还与羊抗人IgG抗体结合。结论所制备可溶性HLA-A2-IgGFc融合蛋白序列正确,并在体外形成正确空间构象。     

稳定表达HLA-A*1101蛋白的K562细胞株的建立

本研究目的是建立稳定表达HLA-A*1101分子的K562细胞株,为研究慢性髓系白血病(CML)HLA-I限制性抗原特异T细胞的细胞毒作用提供靶细胞.利用RT-PCR从CML患者外周血单个细胞中扩增HLA-A*1101基因全长序列;利用重组PCR将2A肽连接子(D-V-E-X-N-P-G-P)基因连接到HLA-A+ 1101的3’端,并将其定向克隆入带有增强型绿色荧光蛋白基因的真核表达载体pEGFP-N3,构成HLA-A+ 1101-T2A-EGFP转录盒的重组表达载体;利用电穿孔技术将pEGFP-N3或重组质粒转染入K562细胞,利用荧光显微镜监测绿色荧光蛋白的表达,通过G418筛选出有效转染pEGFP-N3或HLA-A* 1101 -T2A-EGFP载体的K 562细胞株;利用RT-PCR和流式细胞术检测HLA-A* 1101基因和蛋白的表述情况.结果表明,成功构建了以2A肽基因为连接子的HLA-A* 1101-T2A-EGFP真核表达载体;重组质粒转染的K562细胞经G418筛选2个月后,获得2株表达绿色荧光蛋白的K562稳定细胞株HLA-A* 1101 +K562和pEGFP-N3+ K562.RT-PCR检测证明,HLA-A* 1101+ K562细胞表达HLA-A* 1101基因,而pEGFP-N3+ K562细胞则不表达HLA-A*1101;流式细胞术分析显示,HLA-A* 1101和GFP蛋白双阳性HLA-A*1101+K562细胞占88.5％,明显高于pEGFP-N3+ K562细胞(0.698％).结论:建立了一个简单有效地筛选HLA-A+ 1101+ K562细胞株的方法,并成功地建立了膜稳定表达HLA-A*1101蛋白的K562细胞株,为进一步研究CML的特异性细胞免疫提供工具细胞.     

可溶性HLA-A2-IgGFc融合蛋白在昆虫细胞的表达及鉴定

目的利用杆状病毒-昆虫细胞表达体系制备可溶性HLA-A2与免疫球蛋白IgGFc段的融合蛋白。方法首先将可溶性HLA-A^＊0201和IgG3分子Fc段的cDNA基因插入杆状病毒表达载体pFastHTa,形成重组载体pFHT-sHLA-A^＊0201-IgGFc。将该重组载体转化DH10Bac大肠杆菌,sHLA-A^＊0201-IgGFc基因同源重组入菌体内杆粒（Bacmid）中。抽提阳性杆粒,转染昆虫细胞Sf9,72 h收集病毒上清。再将病毒上清感染Sf9细胞,96 h收集细胞并裂解,SDS-PAGE观察其表达。细胞裂解液经Ni^＋-NTA树脂纯化Western-blot法鉴定其蛋白质序列,间接ELISA法鉴定是否形成正确构象。结果融合蛋白不仅与HLAⅠ类分子的构象特异性抗体（W6/32）结合,还与羊抗人IgG抗体结合。结论所制备可溶性HLA-A2-IgGFc融合蛋白序列正确,并在体外形成正确空间构象。     

链激酶基因的分离与表达及其单克隆抗体的制备(英文)

用PCR方法从链球菌染色体基因组中分离了链激酶基因,并把它克隆到表达载体pBV220中。诱导表达后重组蛋白以包涵体的形式存在并占菌体总蛋白的60％以上。纯化后的重组蛋白的纯度达90％以上。血纤维蛋白原平板测活方法表明,重组蛋白比活性为1.3×10~5U/mg。重组链激酶蛋白免疫小鼠后获得6株分泌抗链激酶单克隆抗体的杂交瘤。把重组链激酶基因克隆至M13mp19中进行DNA序列分析,结果表明与文献报道的有区别。     

恙虫病东方体Kato株56kD蛋白基因的克隆与表达

目的　表达恙虫病东方体 (Orientiatsutsugamushi) 5 6kD保护性抗原蛋白 ,探索其作为诊断抗原的可能性。 方法　采用PCR方法 ,从O .tsutsugamushiKato株菌种基因组DNA中扩增出 5 6kD基因片段 ,将该片段克隆于原核表达载体pQE30 ,构建成重组质粒pQE 5 6 ,IPTG诱导表达 ,SDS PAGE检测有无蛋白的表达。结果　 (1)获得长约 15 0 0bp的PCR片段 ,序列分析结果与已知Kato株 5 6kD基因序列相同 ;(2 )SDS PAGE检测表达产物 ,在相对分子量 5 6× 10 4处有表达带 ;(3)表达后菌体超声破碎后 ,目的蛋白主要以包涵体形式存在。结论　获得了 5 6kD基因片段 ,并在大肠杆菌中实现了表达。     

人血红蛋白α,β基因在大肠埃希菌中的表达

从人外周血红细胞中提取出总RNA,通过逆转录聚合酶链反应(RT-PCR)得到人血红蛋白的α和β基因片段,与pT7BLue质粒连接后,采用Sanger双脱氧终止法测序的结果与文献报道一致。将α,β基因克隆进pBV220原核表达载体中,宿主菌经热诱导,在1.6×105处有特异的蛋白带表达。表达量达总菌体蛋白的20-30%,并经Western-BLot验证。     

新型重组IL6D24-PE40KDEL外毒素融合蛋白的理化分析

为了对构建成功的新型重组IL6D24-PE40KDEL外毒素融合蛋白的部分物化表征进行分析鉴定，采用Edman法、SDS-PAGE法、胰蛋白酶消化的MALDI—TOF—MS质谱法、蛋白印迹法和MTT法分别测定该融合蛋白的N-末端6个氨基酸、相对分子量、肽谱、抗原特异性以及靶向杀伤生物学活性。结果表明：所构建表达纯化的新型重组IL6D24-PE40KDEL外毒素融合蛋白N末端6个氨基酸序列为Met—lie—Asp—Lys—Gin—Ile-，而IL-6缺失24个氨基酸后的原序列为Ile—Asp—Lys—Gin—Ile-，由于采用了大肠杆菌表达系统，因此在N末端第1位多出了1个Met，经12％SDS-PAGE蛋白电泳和凝胶成像系统分析计算，其相对分子量为58．75kD，与理论值56．9kD相比误差在5％之内。MALTI—TOF—MS质谱测定共获得10个与理论预测值相符的肽段，经瑞士生物信息研究所的EXPASY分子生物服务网站的Peptident数据库搜索证明，在分子量为57kD左右的范围内未检索到与上述务件相符的已知蛋白，证明本融合蛋白为全新蛋白，与预测的目的蛋白相符。WB实验显示IL6D24-PE40KDEL外毒素融合蛋白能与IL-6及PEA抗体特异性结合。MTT生物活性测定表明，该融合蛋白对表达IL-6R的多发性骨髓瘤细胞系U266产生特异性杀伤，而对不表达IL6R的CEM淋巴细胞系无任何杀伤。结论：所研制成功的重组IL6D24-PE40KDL外毒素融合蛋白的确为一全新的具有靶向杀伤生物活性的蛋白质，与设计的完全一致，同时也证实了构建策略的可行性。     

CML28树突状细胞核酸疫苗的构建及其表达研究

为了构建负载CML28的核酸疫苗，并在树突状细胞中进行表达，用分子克隆的方法，从K562细胞中克隆出CML28的cDNA全长，将其亚克隆至pGEM—T载体，经测序后酶切，克隆至真核表达载体pcDNA3．1HisA，将重组质粒pcDNA3．1HisA—CML28进行酶切分析和测序鉴定；从正常人外周血中分离外周血单个核细胞（PBMC），在IL-4和GM—CSF条件下诱导分化为树突状细胞（DC），并进行鉴定；用电穿孔的方法将重组质粒pcDNA3.1 HisA—CML28导入到DC中，Western Blot检测His-CML28融合蛋白的表达。结果表明：经酶切分析和测序鉴定，重组质粒pcDNA3.1 HisA—CML28含有正确的CML28全长序列；经电穿孔导入Dc后，重组质粒能表达His—CML28蛋白。结论：成功构建了CML28树突状细胞核酸疫苗。     

大鼠凝血因子Ⅶ EGFP-EGF1融合蛋白的表达及其结合功能初步研究

本研究通过融合蛋白EGFP-EGF1的表达,探讨大鼠凝血因子Ⅶ上EGF1片段与组织因子（TF）的结合功能。用RT-PCR方法自大鼠肝脏组织获得EGF1编码区基因并将其插入EGFP原核表达载体中,构建pET28a-EGFP-EGF1融合蛋白表达载体;将该重组质粒转化大肠杆菌BL21中,用IPTG诱导表达EGFP-EGF1融合蛋白;采用亲和层析方法Ni柱纯化融合蛋白,将融合蛋白作用于经脂多糖刺激表达TF的大鼠血管内皮细胞;通过荧光显微镜及流式细胞术观察和检测融合蛋白与细胞结合情况。结果表明：EGFP-EGF1在转化的大肠杆菌中获得高效表达并成功纯化,SDS-PAGE证实分子量约为36kD。荧光显微镜及流式细胞术检测显示该融合蛋白能与内皮细胞膜上的TF结合。结论：大鼠凝血因子Ⅶ上EGF1区可介导FⅦ与TF特异性结合,从而可能为分子靶向抗栓治疗研究提供部分基础。     

The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL- 6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat- expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA- protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT- induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6- dependent mouse cell line, stably expressing the tat gene. These tat- positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.     

Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein

Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.     

Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-6-dependent growth of human myeloma cells

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)- transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL- 6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.     

The contribution of amino acid residues 1508-1515 of factor V to light chain generation.

BACKGROUND: Factor (F) V is activated by alpha-thrombin following cleavages at Arg(709), Arg(1,018) and Arg(1,545). Amino acid region 1,490-1,520 of FV is essential for procofactor activation. AIM: To ascertain which amino acid residues from this region are important for light chain formation and procofactor activation, site-directed mutagenesis was used to create recombinant FV molecules missing amino acid 1,508-1,510 (FV(Delta1,508-1,510)) and 1,508-1,515 (FV(Delta1508-1515)). We have also created recombinant FV molecules with mutations (1508)DDY(1510)-->AAF (FV(AAF)), (1514)DY(1515)-->AF (FV(AF)) and Y(1510)-->F (FV(Y1510F)). METHODS AND RESULTS: The recombinant mutant molecules were expressed and purified to homogeneity. The clotting activities of all clotting recombinant mutant FV molecules were tested in a two-stage assay following activation by alpha-thrombin and were found to be impaired compared with the clotting activity observed with wild-type recombinant FV or plasma-derived FV, with the exception of FV(Y1510F), which had normal clotting activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FV demonstrated that incubation of 100 nm recombinant wild-type or plasma-derived FV with 1 nmalpha-thrombin for 5 min was sufficient to generate heavy and light chains and completely activate the procofactor. In contrast, similar experimental conditions were ineffective in fully activating the two deletion mutant molecules as well as FVa(AAF) and FVa(AF), resulting in accumulation of a M(r) 220,000 fragment representing amino acids 1,019-2,195. CONCLUSION: Our data demonstrate that amino acid residues 1,508-1,515 of FV are required for efficient cleavage by alpha-thrombin and light chain formation.     

Sensitization of cancer cells to interleukin 13-pseudomonas exotoxin-induced cell death by gene transfer of interleukin 13 receptor alpha chain

We have demonstrated that primary interleukin 13 (IL-13) binding protein IL-13 receptor (IL-13R) alpha chain plays an important role in IL-13 binding and internalization in the IL-13R system. Although IL-13R alpha chain is expressed on many cancer cell lines, some cancer types do not express or express low levels of this receptor chain. Consequently, these cells show no or low sensitivity to the cytotoxic effect of a recombinant chimeric protein composed of IL-13 and a mutated form of a Pseudomonas exotoxin, IL13-PE38QQR. Here we demonstrate that pancreatic cancer, renal cell carcinoma, head and neck cancer, and glioblastoma cell lines that were genetically altered to express high levels of IL-13R alpha chain increase their binding affinity for IL-13, and increase their sensitivity to IL13-PE38QQR by at least 6-fold to 1000-fold compared with mock-transfected control cells. This observation was made by protein synthesis inhibition assay and confirmed by clonogenic assay. Our studies provide a proof of principle for a novel strategy for cancer therapy that combines gene transfer and targeted cytotoxin therapy.     

丙型肝炎病毒核心蛋白酵母双杂交诱饵载体的构建及鉴定研究

目的构建含丙型肝炎病毒(hepatitis C virus,HCV)核心(core,C)蛋白基因的酵母双杂交诱饵载体,为C蛋白与宿主蛋白相互作用机制研究奠定基础。方法聚合酶链反应(PCR)法扩增HCV 1b基因型C蛋白基因,经EcoR I和Pst I双酶切后插入到诱饵载体pGBKT7中,构建含HCV C蛋白基因的酵母双杂交诱饵载体pGBKT7-Core,经酶切分析及核酸测序分析加以鉴定。醋酸锂法将pGBKT7-Core转化入酵母菌株Y2HGold,蛋白印迹法检测C蛋白的表达,通过表型筛选检测诱饵蛋白对酵母细胞的毒性作用及对报告基因的激活作用。结果重组质粒pGBKT7-Core中的C蛋白基因为576 bp,经核酸测序分析与NCBI中注册的HCV 1b基因型C蛋白基因序列一致;转化酵母菌后检测到C蛋白的表达;HCV C蛋白对酵母菌Y2HGold无毒性,且对报告基因无激活作用。结论含HCV C蛋白基因的酵母双杂交诱饵载体构建成功,表达稳定。     

An Interleukin 5 Mutant Distinguishes between Two Functional Responses in Human Eosinophils

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5–dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5–dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.