增强型绿色荧光蛋白RNAi表达载体的构建和鉴定

目的构建靶向增强型绿色荧光蛋白基因（EGFP）的RNA干扰质粒载体。方法设计并合成针对EGFP编码区的模板寡核苷酸单链,克隆入pSilencerTM 3.1-H1 neo载体中,对重组质粒进行酶切鉴定和DNA序列测定。结果重组质粒经限制性内切酶酶切得到66 bp和4.3 kb条带;重组阳性克隆测序结果与实验设计的模板寡核苷酸序列相符。结论成功构建靶向EGFP的RNA干扰表达载体,为RNA干扰技术在实验室的进一步开展奠定基础。     

融合型B细胞淋巴瘤独特型DNA疫苗表达质粒的构建

已有研究证实,B细胞淋巴瘤病人自身免疫球蛋白独特型可视为肿瘤特异性抗原用于独特型疫苗。为探究带有细胞因子的融合型独特型肿瘤疫苗是否会提高免疫效果,制备小鼠B细胞型淋巴瘤细胞的独特型单链可变区片段,与免疫佐荆单核细胞趋化因子MCP3融合,同时融合报告基因EGFP,构建融合型独特型淋巴瘤DNA疫苗。用RT—PCR法扩增BALB／c小鼠源B细胞淋巴瘤细胞株A20的IgVH和IgVL基因,重组PCR法将编码（Gly4Ser）3的核苷酸片段连接两基因,制备scFv片段。用相同PCR方法,选用1段编码NDAQAPKS连接肽连接趋化因子MCP3基因与scFv片段,获得MCP3-scFv融合基因片段。将scFv和MCF3-scFv融合基因片段分别插入真核表达质粒pTARGET,并在融合基因的下游插入报告基因EGFP,构建真核表达质粒pTARGET／scFv—EGFP和pTARGET／MCP3-scFv—EGFP。结果表明,成功扩增A20细胞的IgVH和IgVL基因片段及scFv—EGFP、MCP3-scFv—EGFP融合基因片段;酶切鉴定表明,成功制备重组真核表达质粒pTARGET／scFv—EGFP和pTARGET／MCP3-scFv—EGFP。结论：成功构建带有鼠源scFv片段、趋化因子MCP3和EGFP融合的独特型抗B细胞淋巴瘤疫苗表达质粒pTARGET／MCP3-scFv—EGFP和pTARGET／scFv—EGFP。所构建的重组表达质粒为下一步的抗B细胞淋巴瘤基因疫苗的体内动物实验奠定了基础。     

人PTEN绿色荧光蛋白真核表达载体的构建及鉴定

目的 构建人PTEN绿色荧光蛋白真核表达载体并进行鉴定.方法 参照PTEN基因全序列,在cDNA两端各设计一条对应引物,并引入各自的酶切位点.从正常人胎盘组织中提取mRNA作为模板合成第一链,并扩增目的基因序列片段,经双酶切后定向克隆至绿色荧光蛋白真核表达载体pEGFP-C1中,筛选阳性重组质粒,分别经双酶切、测序法对重组质粒进行鉴定.结果 双酶切和特异PCR结果表明克隆的基因片段约1.2 kb;测序法进一步证实该基因为PTEN编码基因,经NCBIBLAST分析与Gene Bank中基因序列完全相同.结论 成功构建了人PTEN绿色荧光蛋白真核表达载体pEGFP-C1-PTEN,为研究在肿瘤发生发展中的作用奠定了基础.     

pEGFP-N1/VEGF真核表达质粒的构建与鉴定

目的构建pEGFP-N1/VEGF真核表达质粒,并进行鉴定。方法利用RT-PCR方法从组织中提取VEGF的基因片段,与pMD18-T载体连接,构建pMD18-T/VEGF重组质粒,酶切后与pEGFP-N1真核表达载体连接,构建pEGFP-N1/VEGF真核表达质粒,进行测序、酶切鉴定,表明质粒构建成功。结果本实验成功构建了pEGFP-N1/VEGF真核表达质粒。结论为进一步研究利用VEGF基因修饰骨组织工程骨,促进血管再生提供实验基础。     

人高迁移率族蛋白B1细胞内定位及移位研究

目的 观察人高迁移率族蛋白B1（HMGB1）在真核细胞内的定位和移位情况。方法 构建人HMGB1及增强型绿色荧光蛋白（EGFP）融合蛋白的哺乳动物细胞表达载体。通过两步亚克隆的方法，将HMGB1和EGFP的编码序列以融合表达形式克隆到带有血凝紊（hytohemagglutinin，HA）标记的载体pcDNA3-HA上，随后转染293A细胞，在荧光显微镜下观察结果。结果 重组质粒经酶切、聚合酶链反应（PCR）和测序鉴定证明构建正确，并在293A细胞中得到大量表达。荧光显微镜观察发现，融合蛋白主要分布于细胞核中，经肿瘤坏死因子-α（TNF-α）刺激后18h，部分细胞中融合蛋白从胞核移位到胞质，与预期结果一致。结论 HMGB1的绿色荧光蛋白融合表达载体构建成功。该载体能在哺乳动物细胞中有效表达并正确定位、移位，为下一步深入研究HMGB1作用细胞的信号通路提供了一个重要的工具。     

Mfn2基因克隆及pEGFPmfn2真核表达质粒的构建

目的利用基因工程技术克隆线粒体融合蛋白2(mitofusin-2,mfn2)基因并构建一种无细胞毒性、不激活原癌基因的真核表达的pEGFPmfn2质粒载体。方法采用高效Trizol试剂快速提取大鼠血管平滑肌细胞株A7r5总RNA,经RT-PCR获得mfn2cDNA,扩增、纯化、回收mfn2基因片段并将质粒pEGFP-N1和mfn2基因片段分别双酶切,前者纯化回收大片段,后者纯化回收2.2 kb片段,再将回收的pEGFP-N1大片段与mfn2基因片段(2.2 kb)重组。对重组pEGFPmfn2质粒进行PCR和双酶切鉴定并测序。结果成功地从A7r5中克隆了mfn2基因,并构建了以pEGFP-N1为载体的真核表达质粒。结论含mfn2基因新型表达质粒构建的成功将为研究mfn2功能、进一步研究该基因异常表达与心血管疾病发生的关系奠定基础。     

血管内皮生长因子165真核基因转染载体转染的骨骼肌细胞

背景：血管内皮生长因子基因转染治疗组织损伤的研究倍受关注,构建稳定可靠的人血管内皮生长因子真核表达载体有重要意义。目的：克隆人血管内皮生长因子基因血管内皮生长因子165片段,构建pcDNA4-HisMax-C/VEGF165真核表达质粒,并验证其转染大鼠骨骼肌细胞的可靠性。方法：采用反转录-聚合酶链反应技术,从人卵巢癌患者外周血中提取并扩增出血管内皮生长因子165基因片段,通过DNA重组技术将该基因片段重组于pcDNA4-HisMax-C真核表达载体上,构建成pcDNA4-HisMax-C/VEGF165重组质粒,聚合酶链反应扩增,分别用酶切电泳分析和DNA测序的方法对提取和重组DNA进行鉴定。pcDNA4-HisMax-C/VEGF165重组质粒转染骨骼肌细胞1周后反转录-聚合酶链反应提取血管内皮生长因子基因并酶切电泳鉴定。结果与结论：构建的重组质粒目的基因片段为人血管内皮生长因子165cDNA,对大鼠骨骼肌细胞转染后检测到血管内皮生长因子165基因片段。提示成功地克隆了血管内皮生长因子165基因并构建了其真核表达质粒,能以此为载体转染至骨骼肌细胞,并已整合到骨骼肌的基因组参与转录,证明了其转染的有效性。     

血管内皮生长因子165真核基因转染载体转染的骨骼肌细胞☆

背景:血管内皮生长因子基因转染治疗组织损伤的研究倍受关注,构建稳定可靠的人血管内皮生长因子真核表达载体有重要意义.目的:克隆人血管内皮生长因子基因血管内皮生长因子165片段,构建 pcDNA4-HisMax-C/VEGF165真核表达质粒,并验证其转染大鼠骨骼肌细胞的可靠性.方法:采用反转录-聚合酶链反应技术,从人卵巢癌患者外周血中提取并扩增出血管内皮生长因子165基因片段,通过DNA重组技术将该基因片段重组于pcDNA4-HisMax-C真核表达载体上,构建成pcDNA4-HisMax-C/VEGF165重组质粒,聚合酶链反应扩增,分别用酶切电泳分析和 DNA 测序的方法对提取和重组 DNA 进行鉴定.pcDNA4-HisMax-C/VEGF165重组质粒转染骨骼肌细胞1周后反转录-聚合酶链反应提取血管内皮生长因子基因并酶切电泳鉴定.结果与结论:构建的重组质粒目的基因片段为人血管内皮生长因子165 cDNA,对大鼠骨骼肌细胞转染后检测到血管内皮生长因子165基因片段.提示成功地克隆了血管内皮生长因子165基因并构建了其真核表达质粒,能以此为载体转染至骨骼肌细胞,并已整合到骨骼肌的基因组参与转录,证明了其转染的有效性.     

人Regucalcin cDNA真核表达载体的构建及表达

目的构建人regucalcin（RGN）全长cDNA的真核表达载体，观察其在人肾皮质近曲小管上皮细胞（HK-2细胞）中表达。方法用RT-PCR技术扩增获得人HK-2细胞RGN全长cNDA，将扩增的产物连接入pMD18-T克隆载体上。将重绢质粒转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，再用双酶切方法将RGN基因定向连接到真核表达载体DECFP-NI中，构成pEGFP-RGN，将pEGFP-RGN转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确。再用双酶切方法将RGN基因与EGFP定向连接到真核表达载体pcDNA3．1（＋）-zeocin中，构建真核表达pcDNA3．1-RGN-EGFP-zeo的重组体。将pcDNA3．1-RGN．EGFP-zeo转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，用脂质体转染入HK-2细胞。荧光显微镜、激光共聚焦显微镜观察pcDNA3．1-RGN-EGFP-zeo表达情况。结果RGNcDNA全长是897bp。以此构建的pcDNA3．1-RGN-EGFP-zeo真核表达载体，经DNA测序与GenBank中的人RGNcDNA序列一致。荧光显微镜、激光共聚焦显微镜观察到pcDNA3．1-RGN-EGFP-zeo转染的细胞中有绿色荧光蛋白的表达。结论本实验成功构建了pcDNA3．1-RGN．EGFP．zeO真核表达质粒，并在HK-2细胞中表达。     

P—选择素凝集样区和绿色荧光蛋白融合基因的构建

目的 构建P-选择素凝集样区与绿色荧光蛋白融合基因（pEGFP-N1/L）,为进一步明确P-选择素不同表位与功能间的关系,为阐明P-选择素生理病理意义奠定基础。方法 应用PCR技术扩增质粒pCDM1/cDNA凝集素校区,用T4连接酶将其插入载体pEGFP-N1;经酶切和测序对插入片段进行分析、鉴定。结果 通过酶切和序列分析证实插入片段序列正确。结论 应用基因工程技术构建pEGFP-N1/L融合基因成功,为绿色荧光蛋白作为主生物标记分子来研究P-选择素分子的构效关系打下良好的基础。     

炭疽芽孢杆菌基因芯片探针的制备

目的制备炭疽芽孢杆菌(BA)基因芯片探针,应用于BA基因芯片的检测。方法采用PCR方法,以BA疫苗株A16R基因组DNA为模板,扩增pXO1质粒上的8个特异性片段,连接至pMD19-T Simple载体,构建分别包含8个探针序列的重组质粒,以其为PCR扩增模板获取探针DNA。结果基因测序证明,成功获得BA的3个探针。结论 BA基因探针的成功获取,为炭疽芽孢杆菌基因芯片的制备奠定了基础。     

基因转染新方法--声化学基因转染

目的 基因转染效率低下一直是限制基因研究与基因治疗进一步发展和广泛应用的瓶颈问题。笔者将生物技术和超声技术有机结合，提出一种基因转染新方法即声化学基因转染或声化学内在化，为提高基因转染效率提供新的思路和新手段。     

Liposomal gene transfer of multiple genes is more effective than gene transfer of a single gene

Liposomal gene transfer is an effective therapeutic approach for the treatment of several pathophysiologic states. The purpose of the present study was to define whether gene transfer of multiple genes is a feasible approach and whether this approach would be more effective than the single transfer of cDNA. Rats were inflicted an acute wound and divided into four groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the insulin like-growth factor-I (IGF-I)cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the keratinocyte growth factor (KGF) cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the IGF-I/KGF cDNA (2.2 microg) and Lac Z gene (0.22 microg). Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, dermal and epidermal regeneration. IGF-I/KGF cDNA transfer increased IGF-I and KGF protein concentration and caused concomitant cellular responses, for example,by increasing IGFBP-3, P<0.05. IGF-I/KGF cDNA gene transfer improved epidermal regeneration by exhibiting the most rapid area and linear wound re-epithelization by almost 250% compared to control and each growth factor given individually, P<0.001, which was probably due to promitogenic and antiapoptotic effects on basal keratinocytes when compared to controls, P<0.001. Dermal regeneration was improved in IGF-I/KGF cDNA-treated animals by an increased collagen deposition and morphology when compared with vehicle, IGF-I and KGF, P<0.001. IGF-I/KGF cDNA increased VEGF concentrations and thus neovascularization when compared with vehicle, IGF-I and KGF, P<0.001. In the present study, we showed that exogenous gene transfer of multiple cDNA sequences have an additive effect on intracellular and biological responses when compared to the same gene administered as a single cDNA sequence. Our findings demonstrate that gene therapy with multiple genes is feasible, and that the gene transfer of multiple genes can enhance and accelerate physiologic and biological effects.     

Contributions of gene marking to cell and gene therapies

The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34(+) hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystrophy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches.     

Virus-mediated gene transfer for cutaneous gene therapy

Cutaneous gene therapy offers unique opportunities and limitations in the use of viral vectors for corrective gene transfer. Skin presents a formidable barrier to microbial invasion and is nourished by small blood vessels, thus ruling out the possibility of directed virus delivery through cannulated blood vessels. However, skin is physically accessible and its resident keratinocyte stem cell population is susceptible to direct in vivo transduction with retroviral vectors. Furthermore, keratinocyte stem cells transduced in culture have been shown to persist and to express the encoded transgene when grafted to immunocompromised mice. Cutaneous gene therapy trials are likely to involve virus-mediated transduction as a principal means of gene transfer.     

金黄色葡萄球菌耐消毒剂基因及5类抗生素耐药相关基因检测

目的了解本地区临床分离的金黄色葡萄球菌（SA）的耐药特点,探讨SA中耐消毒剂基因（qacA）、β-内酰胺类、氨基糖苷类、大环内酯-林可霉素-链阳菌素B类（MLSB）、四环素类、糖肽类药物耐药基因的存在情况。方法采用聚合酶链反应（PCR）法对20株SA进行β-内酰胺酶基因、氨基糖苷类修饰酶基因、MLSB类基因、四环素类基因、糖肽类基因、耐消毒剂基因检测。结果 PCR结果显示20株SA中13种耐药相关基因检出率分别为mecA75%、TEM90%、aac（6＇）/aph（2″）70%、ant（4＇,4″）15%、ant（6）-Ⅰ25%、ermA100%、ermC55%、msrA20%、msrB55%、tetM65%、qacA60%,vanA、vanB基因均为阴性。结论多数SA菌株具有耐多药特征,存在耐β-内酰胺类、氨基糖苷类、MLSB类、四环素类等多种抗生素耐药基因,且对胺类、胍类消毒剂耐受。