重组逆转录病毒载体系统的建立及其感染滴度的研究

选择新霉素磷酸转移酶II（Neo^R)做为标记基因,将重组逆转录病毒载体经包装细胞馐后得到的重组病毒浓缩后,应用该重组逆转录病毒载体系统体外感染靶细胞NIH3T3以测定其滴度,其滴度可达到10^6,具有理想的感染靶细胞的能力。     

克隆内蒙古白绒山羊胸腺素β4基因并稳定转染胎儿成纤维细胞 #br#

 【目的】克隆内蒙古白绒山羊胸腺素β4(thymosin beta 4,Tβ4) 基因,构建皮肤特异性表达载体,转染内蒙古白绒山羊胎儿成纤维细胞,筛选出稳定表达红色荧光蛋白并可用于核移植的转基因细胞克隆。【方法】通过RT-PCR克隆Tβ4基因cDNA序列,然后与KAP6-1基因启动子片段以及红色荧光蛋白表达元件连接构成Tβ皮肤特异性表达载体pCDsRed-KT。外源表达载体以lipofectamineTM 2 000介导转染胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。【结果】 克隆了内蒙古白绒山羊Tβ4基因,cDNA全长142 bp,其中包含135 bp的完整ORF,编码44个氨基酸残基,氨基酸序列与已报道的牛胸腺素β4(XM002706880.1)同源性为100%。测序显示构建的表达载体pCDsRed-KT中,Tβ4基因正确连接在皮肤特异性启动子KAP6-1下游,顺序连接CMV启动子和红色荧光蛋白基因,载体构建正确。PCR检测显示外源KAP6-1启动子和Tβ4基因整合到细胞基因组中,筛选出的转基因细胞高效表达红色荧光蛋白。【结论】克隆得到内蒙古白绒山羊Tβ4基因并构建成功其真核表达载体,可稳定转染绒山羊胎儿成纤维细胞,为下一步通过核移植方法获得转胸腺素β4基因绒山羊提供了条件。     

Contribution of promoter to tissue-specific expression of the mouse immunoglobulin kappa gene

The immunoglobulin kappa (kappa) gene promoter was activated by a "neutral" enhancer derived from Harvey murine sarcoma virus (HaMuSV) in immunoglobulin-producing myeloma cells, regardless of the enhancer's orientation or position in the vector. In one fibroblast line (3T3) the immunoglobulin kappa gene promoter was completely inactive when linked to the HaMuSV enhancer, whereas in mouse L cells, promoter activity was observed only with the HaMuSV enhancer in tandem with the immunoglobulin kappa gene promoter. The differential behavior of the gene promoter, when activated by a neutral enhancer in these three murine cell lines, suggests that promoter sequences contribute to the tissue-specific expression of this gene.     

小鼠MCK基因启动子的克隆及其活性初步分析

本研究克隆了小鼠MCK基因5'侧翼区以及核心启动子区,并分析和研究了其调控活性。首先以小鼠尾部肌肉基因组DNA为模版克隆了小鼠MCK基因5'侧翼区序列,回收纯化,连接pEASY-T3 Cloning载体,测序,分析了其增强子与核心启动子序列。然后将核心启动子序列亚克隆到无启动子的荧光蛋白报告载体pGL3-Venus中,转染小鼠成肌细胞C2C12,观察其在荧光显微镜下的表达情况。结果表明,小鼠MCK基因-1354～+1 bp核心启动子序列能调控Venus荧光蛋白在C2C12细胞内表达,证实MCK启动子核心序列具有组织特异性调控能力。     

Requirement for positive selection of gamma delta receptor-bearing T cells.

The alpha beta and gamma delta T cell receptors for antigen (TCR) delineate distinct T cell populations. TCR alpha beta-bearing thymocytes must be positively selected by binding of the TCR to major histocompatibility complex (MHC) molecules on thymic epithelium. To examine the requirement for positive selection of TCR gamma delta T cells, mice bearing a class I MHC-specific gamma delta transgene (Tg) were crossed to mice with disrupted beta 2 microglobulin (beta 2M) genes. The Tg+beta 2M- (class I MHC-) offspring had Tg+ thymocytes that did not proliferate to antigen or Tg-specific monoclonal antibody and few peripheral Tg+ cells. This is evidence for positive selection within the gamma delta T cell subset.     

稳定表达T_7RNA聚合酶的猪睾丸细胞系的建立

根据GenBank中登录的T7RNA聚合酶基因参考序列,设计合成了1对特异性引物扩增对T7RNA聚合酶基因进行扩增,将测序正确的T7RNA聚合酶基因和真核表达载体pIRES2-EGFP双酶切后进行连接构建pIRES2-EGFP-T7RNA RNA质粒。再将构建正确的pIRES2-EGFP-T7RNA质粒经用脂质体法转染猪睾丸细胞,通过G418筛选和单细胞克隆化,同时构建pET-32a-RED原核表达质粒载体,用其检测T7启动子控制下的红色荧光蛋白的表达。结果表明,建立的ST/T7RNA细胞系经20次传代仍然能稳定表达T7RNA聚合酶。结果显示,成功建立能稳定表达T7RNA聚合酶的猪睾丸细胞系,为猪瘟病毒反向遗传操作平台奠定了基础。     

Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.     

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.     

内蒙古白绒山羊VEGF164基因毛囊特异性表达载体的构建及胎儿成纤维细胞的稳定转染

 【目的】构建绒山羊血管内皮生长因子164（vascular endothelial growth factor 164,VEGF164）基因的毛囊特异表达载体并稳定转染胎儿成纤维细胞,筛选获得稳定表达红色荧光蛋白和毛囊特异表达VEGF164并可用于核移植的转基因细胞克隆。【方法】以pCDsRed2载体为基本骨架将VEGF164基因亚克隆到KAP6-1启动子下游,接续连接红色荧光蛋白表达元件,构建VEGF164基因毛囊特异表达载体pCDsRed2-KV（6.3 kb）。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。【结果】在构建的表达载体pCDsRed2-KV中,VEGF164基因被正确连接在毛囊特异性启动子KAP6-1下游,基因下游按顺序连接CMV启动子和红色荧光蛋白基因;外源KAP6-1启动子和VEGF164基因整合到细胞基因组中。【结论】成功构建稳定表达红色荧光蛋白和毛囊特异表达VEGF164的真核表达载体,稳定转染绒山羊胎儿成纤维细胞,为下一步通过克隆技术获得转VEGF164基因绒山羊提供了条件。     

Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells

All human gamma delta T cells coexpressing the products of the variable (V) region T cell receptor (TCR) gene segments V gamma 9 and V delta 2 recognize antigens from mycobacterial extracts and Daudi cells. Exogenous and endogenous ligands on the cell surface, homologous to the groEL heat shock family, induced reactivities that resembled superantigen responses in this major subset of human peripheral blood gamma delta T cells. Stimulation of human V gamma 9/V delta 2 T cells is not restricted by human leukocyte antigens (HLA), including nonpolymorphic beta 2-microglobulin (beta 2M)-associated class Ib molecules. These data may be important for understanding the role of gamma delta T cells in autoimmunity and in responses to microorganisms and tumors.     

猪生长激素启动子的克隆及功能分析(英文)

[目的]克隆猪生长激素启动子,确定其启动子核心序列和主要的顺式作用元件。[方法]根据NCBI上公布的序列设计引物,PCR扩增了猪生长激素5’端-1821~+61bp的序列,并通过移步缺失的方法,获得9段长短不一的启动子序列,将其分别构建到双荧光素酶表达载体pGL3-basic上。通过重组质粒瞬时转染大鼠垂体瘤细胞(GH3)、猪髋动脉血管内皮细胞(PIEC)和猪肾细胞(PK15)和转染后细胞荧光素酶活性的测定,检测这些5’末端缺失质粒在垂体及非垂体细胞中的相对转录活性。[结果]成功扩增了猪GH基因5’上游启动区1882bp的片段并构建了9个pGL3-mGHpromoter报告基因载体;双荧光素酶报告基因检测系统证实插入报告基因载体中的启动子具有非常强的细胞特异性。[结论]猪生长激素特异性在垂体细胞中表达,其最小启动子位于-110bp以内,启动子区-218~-110bp和-429~-218bp间存在正向调控元件。     

猪生长激素启动子的克隆及功能分析

[目的]克隆猪生长激素启动子,确定其启动子核心序列和主要的顺式作用元件。[方法]根据NCBI上公布的序列设计引物,PCR扩增了猪生长激素5’端-1 821～+61 bp的序列,并通过移步缺失的方法,获得9段长短不一的启动子序列,将其分别构建到双荧光素酶表达载体pGL3-basic上。通过重组质粒瞬时转染大鼠垂体瘤细胞(GH3)、猪髋动脉血管内皮细胞(PIEC)和猪肾细胞(PK15)和转染后细胞荧光素酶活性的测定,检测这些5’末端缺失质粒在垂体及非垂体细胞中的相对转录活性。[结果]成功扩增了猪GH基因5’上游启动区1 882 bp的片段,并构建了9个pGL3-mGH promoter报告基因载体;双荧光素酶报告基因检测系统证实插入报告基因载体中的启动子具有非常强的细胞特异性。[结论]猪生长激素特异性在垂体细胞中表达,其最小启动子位于-110 bp以内,启动子区-218～-110 bp和-429～-218 bp间存在正向调控元件。     

单核细胞增生性李斯特氏菌溶血素基因克隆及序列分析

构建单核细胞增生性李斯特氏菌 (L isteria Monocytogenes,L MO)溶血素基因重组质粒。文章采用 PCR方法扩增出 L MO 0 5 86株溶血素 (Hemolysin,hly)基因 ,将其克隆到 p MD18- T中 ,转化 E.coil TGI。经酶切及 PCR鉴定 ,而后进行测序。 hly基因体外扩增产物大小约为 16 4 6 bp。重组质粒经酶切及 PCR鉴定表明为正确重组子。核苷酸序列鉴定表明 ,其核苷酸序列与国外报道的 L MO F6 789株、L MO F2 36 5株同源性分别为 99.70 %和 99.39%。推导出的氨基酸序列与其相应菌株比较 ,同源性分别为 99.82 %和 98.90 %。在国内首次克隆到 L MO hly全基因 ,为研究hly的功能和探讨 hly蛋白作为特异性诊断靶抗原的研究奠定了基础。     

Characterization of T cell receptor gamma chain expression in a subset of murine thymocytes

While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.     

bcl-2/EGFP融合基因真核表达质粒的构建

为深入研究bcl 2对细胞凋亡的调控机制和肿瘤发生发展的影响,应用基因重组手段,根据表达载体pEGFP C1上的多克隆位点和bcl 2基因序列设计了1对引物,对含有bcl 2基因的质粒pWB bcl进行了PCR扩增,得到了708bp的目的片断。将其克隆到pMD18 Tvector,筛选阳性克隆并测序,序列分析表明,其与原初序列同源性达99%。将bcl 2插入到载体启动子下游,与报告基因绿色荧光蛋白(EnhancerGreenFluorescentProtein,EGFP)融合,经限制性内切酶酶切和PCR鉴定,证明Bcl 2/EGFP真核表达质粒构建成功。     

PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域 rtTA 通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。     

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.     

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.     

旋毛虫肌幼虫ES抗原基因TSPGⅡ在真核细胞中的表达

用限制性内切酶ＥｃｏＲⅠ和ＨｉｎｄⅢ从克隆载体ｐＵＴＳⅡ中工出一个０．９８８ｋｂＴＳＰＧⅡ基因,经ＥｃｏＲⅠ和ＢｓｔＸⅠ酶切鉴定;表达载体用相同的酶切,经琼脂糖凝胶电泳,分别回收ＴＳＰＧⅡ基因和ＰＳＶ．ＳＰＯＲＴⅠ表达载体,用Ｔ４ＤＮＡ连接酶定向接连,转化大肠杆菌感受态细胞,挑取重组,经ＥｃｏＲⅠ,ＢｓｔＸⅠ和ＨｉｎｄⅢ酶切鉴定,构建了重组表达质粒ＰＳＶ．ＴＳⅡ。利用脂质体作载体,将重且表达质粒转