重组人白细胞介素3同源双体分子基因在大肠杆菌的表达

白细胞介素３（ＩＬ－３）为造血生长因子，可以保进祖造血干细胞的增殖与分化。利用基因工程技术及蛋白质工程技术构建并高效表达人重组ＩＬ－３同源双体分子，此双体分子表现出高于单体ＩＬ－３分子的刺激骨髓细胞集落形成的生物学效应，为进一步的理论研究及今后的临床应用提供了广阔的前景。     

重组人干细胞因子-血小板生成素融合蛋白的表达及活性初步研究

目的研究重组人干细胞因子-血小板生成素(SCF-TPO)融合蛋白表达的最佳条件及其生物学活性。方法设计引物，利用RT—PCR从胎肝细胞中扩增到SCF和TPO氨基端功能区片段，采用基因融合新策略将其融合成SCF-TPO融合基因，克隆到pGEM—T载体中，构建pET32a／SCF-TPO融合基因原核表达载体，在宿主菌E.eoliBL21(DE3)plysS中经异丙基-β-D-硫代半乳糖(IPTG)诱导其高表达SCF-TPO，SDS-聚丙烯酰胺凝胶电泳和Western blot检测。目的蛋白经包涵体变性，金属螯合层析纯化，透析复性，用细胞因子依赖细胞系MO7e进行细胞增殖实验。结果获得SCF-TPO融合基因的高表达，表达量占菌体总蛋白的30％以上。Western blot法鉴定表达正确，MTT法证明该融合蛋白具有细胞增殖活性，浓度为100ng／ml时刺激活性更强。结论表达并复性后的重组人SCF-TPO融合蛋白具有刺激MO7e细胞生长活性。     

黄芪注射液影响贫血小鼠粒单系、红系造血及作用机制的探讨

目的：研究黄芪注射液对贫血小鼠粒单系和红系血发生的影响,并探讨其作用机制。方法：制备贫血小鼠模型,随机分为给药组和对照组,采用造血祖细胞体外培养等实验血液学技术。结果：一定浓度的黄芪注射液体内作用可使贫血小鼠血清集落刺激因子（CSF）水平明显升高,刺激贫血小鼠粒单系、红系造血祖细胞增殖和分化以及骨髓基质细胞增殖,使降低的骨髓有核细胞（BMC）数以及外周血象明显回升,黄芪注射液和造血细胞直接一起孵育对粒单系祖细胞（CFU－GM）和红系祖细胞（CFU－E、BFU－E）的产率无明显影响。结论：一定浓度的黄芪注射液体内作用可促进贫血小鼠粒单系和红系造血功能恢复。     

Flt3配体胞外域的原核表达纯化及对脐血CD34~＋细胞的扩增作用

背景：Fms样酪氨酸激酶3配体（Flt3配体）是一种重要的生长因子,通过激活特定的酪氨酸激酶受体,调控造血细胞的生长、生存和/或分化,具有促进造血干细胞体外扩增的应用潜力。目的：构建pET32a（＋）-hFLext原核表达载体,表达、纯化hFLext蛋白,观察其对脐血CD34＋细胞的扩增作用。方法：克隆hFLext,构建pET32a（＋）-hFLext重组表达载体。转化大肠杆菌BL21,IPTG诱导蛋白表达,镍珠亲合层析纯化蛋白。磁珠分选脐血CD34＋细胞,单独加入hFLext或联合干细胞因子、血小板生成素孵育1周,观察体外扩增作用。结果与结论：成功克隆hFLext,并构建了pET32a（＋）-hFLext重组表达载体。在大肠杆菌BL21成功表达Trx-hFLext融合蛋白,经8mol/L尿素变性包涵体蛋白,逐步透析复性,镍珠亲合层析纯化蛋白,成功获得高纯度的Trx-hFLext融合蛋白。Trx-hFLext融合蛋白不仅具有维持及轻度刺激CD34＋细胞体外扩增的作用,并且与干细胞因子及血小板生成素具有协同作用,为造血干/祖细胞体外扩增研究奠定了基础。     

rhTpo／GM-CSF融合蛋白的构建、表达及活性测定

本研究的目的是找出一个治疗由于放化疗等原因造成的造血组织损伤导致的贫血、感染和出血的有效方法。通过RT-PCR的方法从人胎肝中克隆了重组人血小板生成素(rhTpo)的基因,利用基因工程的手段将其与重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)的基因相融合,并在原核细胞中表达。研究结果表明,大肠杆菌JM101表达的融合蛋白rhTpo/GM-CSF在体外小鼠骨髓造血祖细胞的培养中保留了Tpo对巨核细胞系和红细胞系的刺激作用,并增加了GM-CSF对粒细胞系的刺激活性。结论提示,原核细胞表达的融合蛋白rhTpo/GM-CSF具有刺激骨髓红细胞系、粒细胞系及巨核细胞系造血的活性。     

成纤维细胞生长因子与造血调节研究进展

成纤维细胞生长因子（FGFs）属于肝素一结合多肽家族，具有影响细胞增殖、分化、凋亡和迁移等多种功能。FGFs受体表达于各类造血干／祖细胞和基质细胞。FGFs既可维持造血微环境稳定，也能促进造血干／祖细胞增殖，维持它们的原始表型。此外，FGFs还能协同其他造血生长因子增强造血干／祖细胞体外克隆的形成，有助于造血干／祖细胞的体外扩增，具有潜在的临床应用价值。     

造血干细胞移植建立血小板整合素β3胞内段截短体转基因小鼠模型

本研究通过逆转录病毒感染整合素β3缺陷小鼠（β3-/-）骨髓造血干细胞再进行骨髓移植建立β3及其胞内段不同截短体的转基因小鼠模型,为进一步研究整合素β3胞内段在血小板双向信号转导途径中的作用提供基础。将整合素β3野生型、β3-Δ759（缺失R760GT762氨基酸片段）和β3-Δ754（缺失T755NITYRGT762氨基酸片段）的cDNA分别克隆至带有GFP编码基因的MSCV MigR1逆转录病毒载体质粒中,用质粒转染BOSC23包装细胞株包装出带有β3全长及不同突变体的逆转录病毒,再感染β3-/-供体小鼠骨髓细胞,尾静脉注射移植入经致死剂量辐照的野生型C57BL/6小鼠体内,移植后6-8周流式细胞术检测GFP和β3的表达率以评估转基因效率。结果表明,成功建立了空载（Vector）、β3野生型和截短突变β3-Δ759、β3-Δ754等4种小鼠模型,血小板转基因成功的比率（即GFP阳性率）为18%-66%,转基因血小板β3表达可达到β3＋/-水平。结论：利用逆转录病毒感染的造血干细胞移植建立血小板转基因小鼠模型是一种高通量快速有效的建立转基因小鼠模型的方法,这为进一步研究整合素β3胞内不同区段对血小板整合素信号转导途径的影响及体内对血小板进行基因操作和基因治疗奠定了基础。     

TMSG-1蛋白不同功能域重组质粒构建及亚细胞定位

目的构建肿瘤转移抑制基因-1（TMSG-1）蛋白不同功能域重组质粒,并观察其亚细胞定位,探讨TMSG-1蛋白的转录活性。方法以pcDNA3-TMSG-1真核表达载体为模板,聚合酶链式反应（PCR）扩增TMSG-1基因全长及不同截短体片段,与带有Flag标签的真核表达载体pcDNA3连接,转化感受态细胞,氨苄青霉素筛选阳性克隆,提取质粒,酶切鉴定。用构建的TMSG-1基因各个截短片段的重组质粒转染Hela细胞,Western blot法鉴定蛋白表达。免疫荧光检测各个截短体亚细胞定位。结果 PCR成功扩增得到目的基因片段,并构建得到TMSG-1全长及不同截短片段T1（aa129-aa380）、T2（aa71-aa380）、T3（aa1-aa128）、T4（aa1-aa70）、T5（aa71-aa128）的真核表达载体,经酶切及基因测序鉴定序列完全正确。Western blot检测结果表明各重组质粒有预期长度的蛋白表达。免疫荧光检测结果表明,含有Homeodomain结构域的截短体T2、T3及T5能够入核,其中T2在核内弥漫分布,T3及T5主要定位于核仁。结论成功构建并表达了TMSG-1全长及不同截短片段的真核表达质粒,其中含有Homeodomain结构域的截短体定位于细胞核。     

骨髓基质细胞对人造血CD_34~+细胞体外扩增及基因转导的作用

目的：探讨造血基质细胞对人外周血干细胞体外培养扩增及基因转导的促进作用。方法：应用基质细胞支持培养扩增体系进行人造血干细胞体外培养扩增及基因转导。结果：骨髓基质细胞和造血生长因子共同支持组的造血ＣＤ＋３４细胞在体外培养３周后,其造血祖克隆形成能力较单纯造血生长因子支持组高３０．７％（Ｐ＜０．０５）。在有基质细胞支持时,逆转录病毒载体上清转导人造血ＣＤ＋３４细胞后,其造血细胞克隆中Ｎｅｏ基因阳性克隆是无基质支持对照组的２倍。结论：基质细胞的支持有维持造血干细胞原始造血活性及促进基因转导的双重好处。     

转化生长因子β1基因转染鼠脂肪间充质干细胞的可行性

背景:脂肪间充质干细胞在转化生长因子β_1等生长因子的调控下可被诱导分化为软骨细胞.然而,外源性转化生长因子β_1存在引起趋化性、激发炎症反应、造成局部损伤、有效成分容易被稀释或丢失、半衰期短、缺少理想的转运蛋白等缺陷.因此,利用基因重组技术,使种子细胞表达转化生长因子β_1,对软骨组织工程的进一步发展具有重要的指导意义.目的:探讨真核表达载体pcDNA3.1-转化生长因子β_1质粒的构建方法,并观察其转染脂肪间充质干细胞的可行性.方法:利用基因重组技术,将大鼠转化生长因子β_1全长基因的PCR产物与克隆载体pT7Blue连接,并用大肠杆菌筛选阳性重组体构建真核表达质粒,采用XboⅠ、Hind Ⅲ双酶切及测序方法对质粒进行鉴定;将已经纯化的pcDNA3.1-转化生长因子β_1质粒和空载体pcDNA3.1质粒分别采用阳离子脂质体试剂LipofectamineTM2000转染脂肪间充质干细胞,经G418抗性筛选后扩大培养,同时用pcDNA3.1-绿色荧光蛋白观察转染效率.结果与结论:重组质粒经XboⅠ、Hind Ⅲ双酶切后出现1.35 bp和5.4 kb 2条带,测序结果显示重组质粒所包含的转化生长因子β_1全长碱基序列完全正确,绿色荧光蛋白显示其转染脂肪干细胞的转染效率> 80%,且转染细胞后有转化生长因子β_1 mRNA和蛋白的表达上调.提示利用基因重组技术可成功构建能转染脂肪间充质干细胞的真核表达载体pcDNA3.1-转化生长因子β_1质粒.     

Full-length cloning and 3'-terminal portion expression of human perforin cDNA.

BACKGROUND: Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. We report the full length of human perforin cDNA, which was cloned from liver tissue. RESULTS: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP. The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid. The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein. Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E. coli BL21 (DE3). The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose. The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes. CONCLUSION: These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perforin molecule. The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies.     

GST-IRS-4原核表达载体的构建、表达及抗GST-IRS4多克隆抗体的制备

目的:在大肠杆菌中表达胰岛素抵抗受体底物-4(insulinreceptorsubstrate4,IRS4)与谷胱甘肽-S转移酶的融合蛋白,并制备抗IRS4的多克隆抗体(pAb)。方法:从肝癌细胞系HepG2提取总RNA,用RT-PCR扩增出IRS4PTB结构域的cDNA,克隆入表达载体pGEX-Teasy中,重组载体酶切鉴定后,在大肠杆菌中经IPTG诱导表达获得GST-IRS4蛋白,SDS-PAGE分析表达产物,包涵体经变性复性后,通过亲和层吸法纯化表达的GST-IRS4融合蛋白,并以此为抗原制备pAb。Westernblot检测重组抗原的免疫活性。结果:酶切及测序鉴定证明,IRS4基因已正确插入到pGEX-Teasy中,经IPTG诱导后,表达出相对分子量为44ku的融合蛋白。用Westernblot鉴定所制备的多克隆抗体可以与IRS4特异性结合。结论:IRS4片段在大肠杆菌中的成功表达及制备得到多克隆抗体,为检测IRS4及其在其他组织中的表达,进一步研究磷酸化酪氨酸结合结构域(PTB)的结构和生物学功能奠定基础。     

人von Willebrand因子A1区基因的克隆和表达

为了深入研究血栓形成的机制以及开发新的抗血栓药物 ,将人vonWillebrand(vWF)因子A1区cDNA在大肠杆菌中表达 ,以获得高效表达的具有生物学活性的重组A1区蛋白 ,应用PCR方法从含有人全长vWFcDNA质粒中扩增A1区基因片段 ,并将其克隆至pUCm T载体中 ,经DNA序列分析后 ,将其重组于有 6×HisTag的融合蛋白表达载体pQE 31中 ,并在大肠杆菌M1 5中诱导表达。采用Ni NTA柱进行纯化 ,用Western印迹鉴定。结果表明 ,PCR扩增得到 854bp的A1区基因片段 ,序列测定结果与文献报道一致。IPTG诱导 5小时后表达的vWF因子A1蛋白占菌体总蛋白的 30 %左右 ,经Ni NTA纯化后其纯度在 95 %以上。Western印迹检测显示A1蛋白具有很好的抗原性和特异性。结论 :成功地在大肠杆菌中高效表达了人vWFA1区蛋白 ,为进一步研究vWF在血栓形成与止血中的作用奠定了基础     

Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex

About 50% of patients with the polymyositis/scleroderma (PM-Scl) overlap syndrome are reported to have autoantibodies to a nuclear/nucleolar particle termed PM-Scl. The particle is composed of several polypeptides of which two have been identified as autoantigens. In this report, human cDNA clone coding for the entire 75-kD autoantigen of the PM-Scl particle (PM-Scl 75) was isolated from a MOLT-4 lambda gt-11 library. The deduced amino acid sequence of the cDNA clone represented a protein of 355 amino acids and 39.2 kD; the in vitro translation product of this cDNA migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at approximately 70 kD. The aberrant migration of the polypeptide in SDS-PAGE was shown to be related to the COOH half that was rich in acidic residues. Authenticity of the cDNA coding for PM-Scl 75 was shown by immunoreactivity of PM-Scl sera with in vitro translation products and recombinant fusion proteins encoded by the cDNA. In addition, rabbit antibodies raised to recombinant fusion protein reacted in immunofluorescence, immunoblotting, and immunoprecipitation with the characteristic features displayed by human anti-PM-Scl sera.     

Fcγ-Der f2载体构建及融合蛋白表达

目的构建人IgG Fcγ1片段Fcγ与粉尘螨Ⅱ类抗原Der f2嵌合基因真核表达载体pDisplay-Fcγ-Der f2,并转染入HEK293T细胞系瞬时表达,获得Fcγ-Der f2融合蛋白。方法以pMD19-T-Der f2载体为模板,设计引物并加入linker序列,经PCR扩增得到linker-Der f2 DNA片段。经限制性内切酶酶切后,先后将人Fcγ及linker-Der f2基因片段接入pDisplay真核表达载体。用Attractene转染试剂将其转染至HEK293T细胞使之表达融合蛋白。免疫荧光检测转染后γ2 h的HEK293T细胞并裂解细胞进行Western Blot检测。结果 pDisplay-Fcγ-Der f2质粒经双酶切鉴定及DNA测序鉴定证实序列完全正确,真核表达载体构建成功。免疫荧光鉴定转染细胞可见明显红色荧光。Western Blot检测证明融合蛋白相对分子质量为40×10~3,与理论预期值相符合,并证明了Fcγ与Der f2双功能特性。结论构建的融合蛋白Fcγ-Der f2符合目的要求。     

融合蛋白hJagged1^ext-Fc毕赤酵母表达载体的构建及表达

本研究旨在构建人IgG1 Fc蛋白及融合蛋白hJagged1^ext-Fc，并在毕赤酵母GS115中表达。用RT—PCR法从人骨髓细胞中扩增hJagged1基因的胞外段。在测序正确后，将hJagged1基因的胞外段插入构建好的PIC—Fc载体，得到PIC—hJagged1^ext-Fc。用MD平板筛选重组子，G418法筛选高拷贝转化子，经甲醇诱导表达后，采用SDS—PAGE分析表达蛋白。结果表明：hJagged1基因的胞外段被有效地扩增。序列分析显示所构建的含phJagged1^ext-Fc融合基因的质粒与设计相同，人IgG1Fc蛋白及融合蛋白hJagged11^extFc得到正确表达。结论：成功地构建了hJagged1^ext-Fc融合基因的毕赤酵母表达载体，并在毕赤酵母中正确表达，为进一步研究Jagged1在造血干／祖细胞的体外扩增奠定了基础。     

哺乳动物极性蛋白mInscuteable-C末端肽段/GST融合蛋白的原核表达与纯化

目的:构建哺乳动物极性蛋白mInscuteable C末端257～532位氨基酸结构域与谷胱甘肽巯基转移酶(GST)的融合蛋白GST-mInsc 257～532的原核表达载体,在大肠杆菌中表达并纯化该融合蛋白。方法:将已经构建好的mInsc 257～532位氨基酸序列克隆至原核表达载体pGEX-4T中,构建重组的质粒pGEX-4T/mInsc 257～532;将重组质粒转化感受态细菌BL21,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达GST蛋白;经谷胱甘肽-琼脂糖球珠分离纯化;产物经SDS-PAGE电泳及Western Blot鉴定。结果:获得高表达及纯化的pGEX-4T/mInsc 257～532融合蛋白。结论:成功构建重组pGEX-4T/mInsc 257～532原核表达载体;诱导表达pGEX-4T/mInsc 257～532融合蛋白并纯化。     

Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

Members of the mammalian rab family of GTPases are associated with specific subcellular compartments, where these proteins are postulated to function in vesicular transport. By screening a human umbilical vein endothelial cell library with degenerate oligonucleotide probes, we have isolated a 1.6-kb cDNA clone encoding a 215-amino-acid protein belonging to the rab family of GTPases. This newly identified rab protein is 81% identical to human rab5, the canine counterpart of which has been localized to the plasma membrane and early endosomes. In light of this homology, we have named this new member of the GTPase superfamily "rab5b." Northern analysis using the rab5b cDNA as a probe revealed a 3.6-kb mRNA in a variety of cell types, including human umbilical vein endothelial cells, K562 erythroleukemia cells, U937 monoblastic cells, and HeLa cells. A fusion protein between glutathione-S-transferase (GST) and rab5b was expressed in bacteria and purified to homogeneity. The recombinant protein was shown to bind GTP and GDP. As is typical of other recombinant rab proteins, the rab5b-GST fusion protein displayed a low intrinsic rate of GTP hydrolysis (0.005/min). An antiserum to rab5b was prepared and used to determine the apparent molecular size and subcellular distribution of the protein. Western blotting with this antibody revealed a 25-kD protein in COS cells transfected with rab5b and in nontransfected HeLa cells. Indirect immunofluorescence and subcellular fractionation showed that rab5b localizes to the plasma membrane. We speculate that rab5b plays a role in vesicular trafficking at the plasma membrane in various cell types.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral- mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.