pQE-TGF-β1原核表达质粒的构建及在大肠杆菌中的表达

目的：为了解人的TGF-β1基因的功能及生物学活性，制备具有生物学活性的TGF-β1蛋白。方法：用基因重组技术构建pQE30-TGF-β1原核表达载体，并在M15大肠杆菌中进行表达，利用Ni-NTA琼脂柱纯化TGF-β1单体，通过复性得到双体蛋白，利用MTT方法对复性的蛋白进行活性测定.结果：经酶切鉴定及测序结果证明pQE30载体上成功地插入了TGF-β1成熟肽基因片段。pQE30-TGF-β1原核表达载体在大肠杆菌中得到了高效表达，表达量约占全菌蛋白的20％，表达得TGF-β1单体蛋白经纯化后进行SDS-PAGE电泳显示均得到了一条蛋白带，分子量为15KD左右。MTT测活证明TGF-β1单体蛋白经复性后具有良好的活性。结论：pQE30-TGF-β1原核表达载体的构建及重组TGF-β1蛋白的制备，为深入了解TGF-β1的生物学功能奠定了基础。     

在大肠杆菌中融合表达重组羧肽酶抑制剂

利用PCR技术扩增得到编码土豆羧肽酶抑制剂（carboxypeptidase inhibitor from potato，CPI）活性多肽基因，克隆于表达载体pGEX-4T-1的多克隆位点中，得到重组质粒pGCPI，测序后将重组质粒转化到受体菌Escherichia coli BL21中。重组菌经IPTG诱导表迭，超声波破菌后，通过谷胱甘肽sepheroseFF亲和层析柱一步纯化得到融合蛋白，纯度大于979，6。融合蛋白经凝血酶切割并分离除去谷胱甘肽-S-转移酶后得到纯度大于98％的重组CPI，ELISA鉴定产物具有免疫原性，体外检测表明其促进纤溶的生物功能与天然CPI无明显差异。     

木聚糖酶基因在大肠杆菌中的表达及表达蛋白的纯化

热海栖热袍菌Thermotogamaritima是一个嗜极端高温的厌氧细菌,其产生的耐高温和热稳定性的木聚糖酶B具有很好的工业应用前景.利用PCR技术将嗜热产乙醇菌T.maritima编码高度热稳定性木聚糖酶的基因克隆至原核表达载体pET-20b,使之与组氨酸标签融合,并在大肠杆菌JM109(DE3)中得到了高效表达.最后采用金属Ni2+螯和层析柱对基因表达产物进行了纯化.     

肺耐药相关蛋白基因及基因产物在膀胱移行细胞癌中的表达

目的探讨肺耐药相关蛋白(LRP)在膀胱癌组织中的表达及与临床病理参数的关系。方法用半定量逆转录聚合酶链反应(RTPCR)技术,检测66例(Ta期12例,T1期26例,T2期11例,T3期10例,T4期7例;G135例,G219例,G312例)术前未经任何治疗的原发膀胱癌患者癌组织及正常组织中LRP、多药耐药基因1(MDR1)和多药耐药相关蛋白1(MRP1)mRNA表达,并用免疫组化法检测LRP、P53和P63蛋白的表达情况。结果(1)膀胱癌标本中,,LRPmRNA的表达率最高(64%,42/66);(2)LRPmRNA水平,正常膀胱组织(142±036)高于膀胱癌组织(080±033,t=282,P<001);低分级膀胱癌组织(G1为139±042)高于高分级癌组织(G2、G3为072±031,t=414,P<001),浅表膀胱癌组织(136±039)高于浸润癌组织(068±030,t=358,P<005);(3)LRPmRNA表达与MDR1或MRP1表达无关,但与其自身的蛋白表达密切相关(r=089,P<001);(4)LRP蛋白表达与低分级、低分期相关(r=081,P<001;r=078,P<005),但与P53、P63表达无关(P>005)。结论膀胱癌组织中,LRPmRNA与蛋白表达一致,且与肿瘤进展有关,可能是膀胱癌内源性多药耐药的预测指标。     

携带Fas基因重组腺病毒治疗瘢痕疙瘩的体外研究

目的 应用成功制备的携带人Fas基因的两种重组腺病毒，感染瘢痕疙瘩(keroid，K)成纤维细胞(fibroblasts，FB)，使其稳定高效地表达以替换原有无功能的Fas蛋白，并恢复正常的重建后Fas信号传导通道。方法 腺病毒感染KFB后，检测及比较两种腺病毒转染前后，KFB内Fas蛋白的表达变化、蛋白功能以及细胞增殖-凋亡状况。结果 腺病毒感染后的KFB均能稳定提高Fas蛋白的表达，并且在Fas单克隆抗体的诱导下可以发生明显的细胞凋亡。结论 ①构建的两种重组腺病毒Ad-Fas在体外实验中能有效地提高KFB蛋白的表达，并可以重建原来阻断的死亡信号传导通道；②细菌内重组腺病毒体外治疗效果更佳；⑧再次估证了Fas基因突变与K之间的联系，为K基因治疗展示了一条新途径。     

TIP30基因在肝癌组织中的表达及意义

1998年肖华等亦发现一种分子量为30kD的转录共分子，可特异性地提高HIV-1增殖调节蛋白Tat的转录，并命名为TIP30(Tat interacting protein30)。序列分析发现与CC3为同一蛋白。近年来的研究表明，TIP30基因具有抑制肿瘤转移、促进肿瘤细胞凋亡、抑制肿瘤血管形成等作用。目前TIP30蛋白在肝癌组织中的表达、可能存在的作用未见有文献报道。本实验利用免疫组织化学技术检测了     

人骨保护素基因在大肠杆菌中的表达及表达产物的初步活性分析

To express human osteoprotegerin (OPG) inE. Coli and analyze its bioactivity in vitro.     

基因重组BDNF感染脊髓神经干细胞及其表达

[目的]探讨脊髓神经干细胞（NSC）被重组逆转录病毒感染后目的基因的表达。[方法]用人脑源性神经生长因子（hBDNF）基冈重组逆转录病毒上清液感染脊髓NSCs，取感染后的NSCs培养液（BDNF条件培养液）培养背根神经结（DRG）和小鼠嗜铬瘤细胞（PC12细胞）检测培养液中BDNF活性；酶联免疫吸附反应（ELISA）检测培养液中BDNF浓度；逆转录聚合酶链反应（RT—PCR）检测BDNF mRNA。[结果]感染hBDNF基因重组逆转录病毒的NSCs上清中有BDNF、的表达，经hBDNF条件培养液培养的PC12细胞．数量增多，突起明显变长。经hBDNF条件培养液培养背根神经结，神经元突起伸出，逐渐增多，长度大于神经节直径。[结论]脊髓NSC可直接作为基因靶细胞，被BDNF基因重组逆转录病毒有效感染而表达和分泌有生物学活性的BDNF、     

国人瘦素基因原核重组表达载体的构建及鉴定

目的 构建并鉴定瘦素基因原核重组表达载体.方法 以RT-PER法自人脂肪组织制备目的 基因,应用DNA重组技术,克隆至pMD18T载体与pGEX-4T-1原核表达载体,转化大肠杆菌DH5α,EcoR I+Xho I双酶切及测序鉴定.结果 扩增得到520 bp目的 基因并构建原核重组表达载体pGEX-4T-1-leptin.双酶切电泳见520bp目的 条带,测序结果与GenBank收录序列一致.结论 成功构建原核表达载体pGEX-4T-1-leptin,为leptin功能研究及进一步的临床应用提供了条件.     

人瘦素蛋白在大肠杆菌中的融合表达和纯化

目的构建人瘦素因子（Leptin）的原核表达质粒pGEX-4T-3-LEP，并诱导该基因在原核细胞中大量表达。方法采用逆转录-聚合酶链反应（RT—PCR）和DNA重组技术，从人皮肤成纤维细胞内将编码人Leptin的cDNA序列克隆至原核表达载体pGEX-4T-3上，选取阳性克隆扩增后，提取DNA进行酶切鉴定和测序。同时应用SDS-PAGE和蛋白质印迹方法对其表达产物进行特异性鉴定。结果克隆出的Leptin基因的cDNA由469bp组成，包括翻译起始密码子、编码序列和终止密码子等部分，含有Letinp基因的cDNA的表达质粒pGEX-4T-3在DH5a细菌体内能够表达，并且随着诱导时间的延长，Leptin基因的表达量增加，重组Leptin蛋白主要存在于包涵体中。结论Leptin基因可以在原核细胞中获得表达，为深入研究该蛋白在创伤愈合中的具体作用奠定了基础。     

维生素K2对SD去卵巢大鼠基质Gla蛋白(MGP)及腰椎基因表达的影响

目的研究MGP在绝经后骨质疏松发病机制中的作用及维生素K2对MGP及基因表达的影响。方法 36只10个月龄雌性SD大鼠,随机分成3组,假手术组、去卵巢(OVX)组、去卵巢加维生素K2干预(OVX+Vitamin K2)组,每组12只。OVX+Vitamin K2组在手术2周后给予维生素K2灌胃(30mg/kg,每周5次,持续12周)。各组大鼠在术后每3周留取血清及尿液。18周后处死大鼠,酶联免疫吸附(ELISA)法检测血清及尿液中MGP的浓度;病理切片观察大鼠腰椎组织结构变化,免疫组化法观察腰椎未羧化MGP的表达;荧光实时定量PCR观察腰椎MGP基因的表达水平。结果 (1)去卵巢18周后,各组血清MGP含量逐渐上升,维生素K2组上升幅度较假手术组及OVX组大(P<0.05),尿液中MGP变化不明显;(2)免疫组化切片中OVX组未羧化MGP(ucMGP)呈阳性染色,维生素K2组较OVX组表达明显减少;(3)腰椎MGP mRNA的表达:OVX+Vitamin K2组及假手术组明显低于OVX组(P<0.05),且OVX+Vitamin K2组高于假手术组(P<0.05)。结论腰椎组织中MGP基因表达增高可能是绝经后骨质疏松发生的作用机制之一,调节MGP mRNA表达可能是维生素K2治疗骨质疏松的一种作用途径。     

Characterization of Osteocalcin (BGP) and Matrix Gla Protein (MGP) Fish Specific Antibodies: Validation for Immunodetection Studies in Lower Vertebrates

In fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma-carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.     

Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata

Matrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage. Although the precise molecular mechanism of action of this protein remains unknown, all available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata, by in situ hybridization, Northern and RT-PCR Southern hybridization, and immunohistochemistry. The results obtained were compared with the patterns of developmental appearance of cartilaginous and mineralized structures in this species, identified by histological techniques and by detection of mRNA presence and protein accumulation of osteocalcin (Bone Gla protein), a marker for osteoblasts known to accumulate in bone mineralized extracellular matrix. The expression of MGP mRNA was first detected at 2 days posthatching (dph) by Northern analysis, RT-PCR amplification, and in situ hybridization, and thereafter continuously detected at various levels of intensity, until 130 dph. In situ hybridization analysis performed in parallel with immunohistochemistry indicated that until ca. 45 dph, the MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and hemal arches, and heart and the protein accumulated in cartilaginous tissues. At 85 dph, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilaginous structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney, although in situ hybridization only detected MGP mRNA in heart, located in the arterial bulbus and not in the cardiac muscle. Our results are in agreement with those recently described for MGP localization in adult tissues of another teleost fish, as well as available data from higher vertebrates, strengthening the hypothesis of a conserved function for MGP from teleost fish to human, a period of more than 200 million years of evolution. In addition, Sparus aurata, a marine teleost fish routinely grown in captivity, appears to be a good model to further analyze MGP gene expression and regulation.     

The effect of E. Coli and E. Coli endotoxin on peristalsis in the canine ureter

Summary The association of ureteric dilatation and urinary infection has been attributed to a direct toxic effect of E. Coli endotoxin on ureteric muscle. A specific causal relationship could not be established in this study in dogs.     

人类睾丸精原细胞瘤组织中P_（53）基因突变和Rb基因蛋白产物表达的缺失

采用Ｗｅｓｔｅｒｎ印迹和ＤＮＡ聚合酶链反应－单股构造多态性分析法对１７例人睾丸精原细胞瘤组织标本的抗癌基因Ｒｂ表达的蛋白产物和Ｐ_（５３）基因外显子５～８的突变进行检测，发现有３例标本的Ｒｂ基因表达的蛋白产物缺失，有４例标本的Ｐ_（５３）基因有点突变，对照的正常人睾丸组织和视网膜组织均呈Ｒｂ基因蛋白产物表达阳性，对照的正常人睾丸组织中未发现Ｐ_（５３）基因突变。本结果提示人睾丸精原细胞瘤的发生与抗癌基因Ｒｂ蛋白产物表达的缺失和Ｐ_（５３）基因的突变有关。