人HCN2真核表达载体的构建及在HEK293细胞中的表达

目的：构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞（HEK293）中的表达情况。方法：对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切（XhoI和BamHI）装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果：测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论：成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

人HCN2 真核表达载体的构建及在HEK293 细胞中的表达

目的:构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞(HEK293)中的表达情况。方法:对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切(XhoI和BamHI)装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果:测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论:成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

人星状病毒非结构蛋白基因nsP1a/1真核表达载体构建及其

目的 将人星状病毒非结构蛋白nsP1 a./1基因连接到真核表达载体上,转染人胚肾上皮细胞48 h后检测其表达.方法 设计特异性引物PCR扩增人星状病毒非结构蛋白nsP1 a/1片段,分别插入真核表达载体pcDNA3.1(+)和pEGFP-N2载体,构建重组表达质粒pcDNA3.1(+)-nsP1a/1-His和pEGFP-N2-nsP1a/1.在转染试剂PEI的介导下将重组表达质粒分别转染293T细胞,转染48 h后分别在荧光显微镜下观察EGFP的表达以及通过Western blot检测nsP1a/1基因的表达.结果 重组表达质粒pcDNA3.1(+)-nsP1a/1-His和pEGFP-N2-nsP1a/1构建成功;转染pEGFP-N2-nsP1a/1后48 h能够在荧光显微镜蓝色激发光下观察到较强的黄绿色荧光;转染pcDNA3.1(+)-nsP1a/1-His后48 h收集细胞进行Western blot检测,能够检测到nsP1a/1-His融合报告基因的表达.结论 成功构建了人星状病毒非结构蛋白nsP1a/1基因真核表达质粒,并在人胚肾上皮细胞293T细胞获得表达,为进一步深入研究nsP1a/1在人星状病毒抵御宿主细胞抗病毒天然免疫中是否发挥作用奠定了基础.     

Construction of a recombinant eukaryotic expression plasmid containing human PDLIM2 gene and its biological activity

In order to obtain a full-length expression plasmid for human PDLIM2 gene, fragment amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD 18-T vector and subcloned into the pIRES2-EGFP plasmid to form the pIRES2-EGFP-PDLIM2 eukaryotic expression vector. After it was transfected to the bladder cancer cell line, BIU-87, the biological activities of high expression were verified by RT-PCR and Western blotting. Meanwhile the mRNA and protein expressions of p65 were detected. Finally we analyzed the effect of overexpressed PDLIM2 on BIU-87 cell proliferation. In conclusion, a recombinant eukaryotic expression vector pIRES2-EGFP-PDLIM2 containing the complete CDS region of PDLIM2 was successfully constructed. PDLIM2 negatively regulated p65 expression and inhibited BIU-87 cell proliferation. We laid the foundations for further research into the function of the PDLIM2 gene in bladder cancer.     

大鼠瘦素基因重组腺相关病毒的构建及在原代神经元细胞中的过表达

目的构建携带大鼠瘦素(leptin)基因的重组腺相关病毒(adeno-associated virus,AAV),并鉴定其在原代鼠神经元细胞中介导的瘦素过表达,为肥胖症基因治疗研究奠定实验基础。方法提取大鼠脂肪组织总RNA,利用RT-PCR技术,获取目的基因瘦素cDNA,通过重组DNA技术,得到瘦素cDNA与pGEM-T载体的重组质粒,阳性重组子用PCR及测序分析鉴定。用Spe I和EcoR V双酶切将pGEM-Leptin中的瘦素基因片段切出,再克隆到AAV2表达质粒pTR-UF22中,构建瘦素重组AAV2载体pAAV2-CBA-leptin。以pDG作为辅助质粒用HEK293细胞包装AAV2-CBA-Leptin,并用一步重力流柱法纯化病毒,由荧光定量PCR测定病毒基因组DNA的拷贝数即为病毒滴度。然后将AAV2-CBA-Leptin及对照病毒AAV2-CBA-EGFP感染大鼠原代神经元细胞,分别用免疫染色和Western blotting鉴定外源基因在神经元的表达。结果测序证实瘦素基因与GenBank提供的原始序列完全一致。重组载体经酶切鉴定与预期结果完全一致,HEK293细胞包装病毒效果良好,得到滴度为1.5×1012vg/mL纯化的重组瘦素病毒AAV2-CBA-Leptin。Western blotting检测显示AAV2-CBA-Leptin能介导瘦素在大鼠神经元细胞中过表达,并随着病毒量的增加而增强。AAV2-CBA-EGFP感染鼠神经元细胞5d后95%左右的细胞有明显的绿色荧光,免疫染色和DAPI核酸染色显示荧光细胞均为神经元而神经胶质细胞无荧光。结论成功构建并包装了瘦素重组AAV2病毒并可介导瘦素在神经元细胞中高效、特异表达,从而为研究瘦素在中枢神经系统控制体重和糖尿病等方面的功能及基因治疗研究打下基础。     

PDX-1腺病毒载体构建及其在脂肪间充质干细胞中的表达

研究通过重组腺病毒表达系统包装重组腺病毒Ad—PDX-1,通过重组腺病毒将Pancreaticduodenalhomeoboxl（PDX-1）导入大鼠脂肪间充质干细胞（ratadiposemesenchymalstemcells,rASCs）中,评价PDX．1在rASCs中的表达情况,为进一步探讨ASCs．PDX-1在体内修复糖尿病的潜能提供了实验基础。研究通过构建pAdEasy-CMV—PDX-1腺病毒质粒,在重组腺病毒表达系统PadEasy-1中成功包装出重组腺病毒Ad-PDX-1。培养及鉴定大鼠脂肪间充质干细胞,将携带PDX-1的重组腺病毒感染rASCs,通过RT-PCR、免疫荧光及免疫印迹（Westernblotting）检测PDX-1在rASCs中的表达及产物定位。结果显示,构建的重组腺病毒包装成功,具有较高滴度,并能成功感染大鼠脂肪间充质干细胞。PDX．1蛋白定位于细胞核,并可稳定表达,为后续将大鼠脂肪间充质干细胞向胰岛B细胞诱导分化奠定了基础,为糖尿病的干细胞治疗提供依据。     

HCMV截短UL83基因真核表达重组体的构建、转染及其免疫效力研究

为了构建人巨细胞病毒(HCMV)截短UL83基因真核表达重组体,实现其在Hep-2细胞中的稳定表达,研究该截短UL83基因真核表达重组体免疫效力,采用基因重组的方法,将HCMV AD169株截短UL83基因定向克隆到带有绿色荧光蛋白(GFP)作为报告基因的真核表达载体pEGFP-C1上,构建真核重组表达质粒pEGFP-C1-UL83;脂质体转染至Hep-2细胞中,G418筛选获得稳定表达pp65细胞表达系。经基因测序显示,重组体中截短UL83基因完全正确,RT-PCR和Western blot检测证实其可在Hep-2细胞中稳定表达。用该重组体和其表达产物在HCMV先天性感染小鼠模型上进行免疫保护试验显示,母鼠血清可检测到特异性抗HCMV pp65抗体,效价为:1∶2.51~1∶50.79;子鼠脑组织内未分离出病毒,亦未检测出病毒pp65蛋白抗原表达。初步结果表明,pEGFP-C1-UL83具有较好的免疫原性,可作为DNA疫苗刺激机体产生有效抗体,并具有阻止病毒垂直传播的保护性作用。     

重组高原鼠兔瘦素原核表达载体的构建、蛋白表达及纯化

瘦素(leptin)由ob 基因编码,对调节能量代谢起着重要作用。本研究建立了高原鼠兔瘦素的原核表达系统,并对其进行了原核表达。研究中作者从高原鼠兔瘦素基因的cDNA 文库中,扩增编码高原鼠兔瘦素的核酸序列,并利用DNA 基因重组技术将其克隆到原核表达载体pET30a(+ )中,构建了高原鼠兔瘦素原核表达载体pET30a(+ )/ ppleptin。对目的片段进行测序确认后,将其转化到大肠杆菌BL21 中,并利用IPTG 诱导外源性目的蛋白表达。表达的包涵体蛋白经溶解及变性后上柱纯化。重组质粒经测序检测后,表明原核载体构建正确。同时,SDS - PAGE 凝胶电泳结果显示,重组菌在16KD 处有明显新增条带,纯化后的目的蛋白条带纯度较高。该结果为高原鼠兔瘦素的后续基础研究提供了基础资料。     

HBD-3基因的乳腺特异性表达载体的构建及真核表达

为了制备高效表达人β-防御素-3基因的转基因奶牛提供可靠的核移植供体细胞,本试验采用RT-PCR从人胎盘组织获得人β-防御素3cDNA,通过双酶切法将目的基因片段hBD插入到质粒pBCP之间,最后再通过双酶切法将目的片段BCD插入到真核表达载体pEGFP-C1中,最终构建乳腺特异性表达载体pEBCD。脂质体介导法转染奶牛胎儿成纤维细胞G418,抗性筛选3~4周后,经PCR、RT-PCR扩增检测和报告基因EGFP表达检测,得到稳定整合外源基因的转基因供体细胞;同时检测稳定转染的奶牛乳腺上皮细胞系的上清液,检测到重组人β-防御素-3蛋白可以在奶牛乳腺上皮细胞组织特异性表达。     

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.     

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.     

米非司酮调控的单链鼠白介素12真核表达载体的构建及鉴定

构建含有单链鼠白细胞介素12(mIL-12)基因,并可经米非司酮调控表达的真核载体,在体外鉴定其调控表达效果.用PcR技术从GCp35Ep40PN质粒上获取白细胞介素12的p40和p35亚基的基因,通过2次PCR技术引入linker后得到单链白细胞介素12基因,将其与米非司酮诱导调控系统克隆入pDC312质粒,构建成真核表达载体pDC-RumIL-12,通过限制性内切酶和PCR鉴定其正确性.在体外用脂质体转染法将pDc-RumIL-12载体转染HEK293细胞株,以不同的剂量米非司酮诱导载体表达,然后用ELISA法检测培养液上清中mIL-12蛋白的含量.测序结果表明所得鼠白细胞介素-12融合单链基因序列与设计相一致.酶切分析和PCR证实载体pDC-RumIL-12构建正确.将pDc-RUmlL-12转染HEK293细胞并经米非司酮诱导表达后,ELISA检测结果表明该栽体有良好的调控能力.没有诱导剂米非司酮时,只有极少量的mIL-12蛋白表达,而加入诱导剂米非司酮后,mIL-12表达量明显增加,并在一定范围内与米非司酮剂量呈正比.成功构建了含有单链鼠白细胞介素12(mIL-12)基因,并可经米非司酮调控表达的真核栽体,可用于肿瘤的基因治疗研究.     

Ischemia/reperfusion in rat heart induces leptin and leptin receptor gene expression

Concentrations of leptin, an adipocyte-derived hormone, are elevated in obesity. Recently, leptin has been shown to participate in multiple biological actions including inflammation, reproduction, and angiogenesis. Leptin has also been documented as a critical component in the process of wound healing; however, leptin involvement in cardiovascular disease is poorly understood. We examined the expression of leptin (ob) and leptin receptor (ob-R) genes in the rat heart following ischemia/reperfusion, which was induced by coronary artery ligation, and mRNA was obtained from hearts 0.5 to 36 h after initiating reperfusion. Expressions of ob and ob-R mRNA were examined by real-time quantitative RT-PCR and immunohistochemistry. The ob and ob-Ra mRNA and protein expressions were significantly increased (p<0.01) and ob-Rb mRNA was significantly decreased (p<0.01) in hearts after 8 h of reperfusion. Furthermore, ob and ob-R proteins were expressed in injured myocytes where inflammatory cells infiltrated. In contrast, those expressions were not influenced in hearts after 8 h of ischemia stress only. To determine the functional effects of leptin on the ischemic/reperfused heart, rats were treated with anti-leptin antibodies prior to ischemia/reperfusion; however, this treatment did not affect the elevation of mRNA expression levels of inflammatory markers such as TNF-alpha and IL-1beta in ischemic hearts. Our results demonstrated for the first time that ischemia/reperfusion induced leptin and leptin receptor gene expression in the rat heart. This study helps to elucidate the mechanisms behind the onset and development of ischemic heart disease concomitant with obesity.     

牛朊蛋白基因prnp敲除载体的构建及真核细胞转染

利用正负筛选策略(Positive-negative selection,PNS)对中靶细胞进行富集是提高体细胞基因打靶效率常用的策略之一。将动物的朊蛋白基因prnp敲除,使其不能表达朊蛋白(传染性海绵状脑病的致病蛋白),从而使其具有抵抗Prion病感染的能力。本研究采用正负筛选策略,构建了牛prnp基因的双等位基因敲除载体,经内切酶Sac Ⅱ线性化后,再通过电穿孔转染牛胎儿成纤维细胞,分别用600μg/mL G418、200nmol/mL Ganciclovir(GCV)进行正负药物筛选,最终获得了176个药物抗性细胞克隆,进一步采用PCR、测序、间接免疫荧光试验及Western blotting试验对细胞克隆进行鉴定,结果表明,其中的9个细胞克隆为中靶细胞,证明牛prnp基因被成功敲除。本研究为牛prnp的敲除提供了可行性依据,并为体细胞核移植生产敲除朊蛋白基因的转基因动物提供供体细胞。     

端粒酶shRNA慢病毒载体的构建及转染人类胚胎干细胞

目的：构建端粒酶shRNA慢病毒载体及建立端粒酶稳定干扰的人类胚胎干细胞系。方法：将端粒酶基因特异性shRNA靶序列与慢病毒载体PLKO.1-puro连接、转化、挑取阳性克隆进行PCR及测序鉴定;利用包装细胞293T获得重组的慢病毒,感染人类胚胎干细胞,分为干扰组ShTert、载体组vector和野生型组wt;Realtime-PCR检测端粒酶mRNA的表达。结果：经PCR和DNA测序鉴定,成功构建端粒酶特异性shRNA慢病毒载体,并感染人类胚胎干细胞;经检测shTert组端粒酶mRNA表达较vector和wt组明显降低,vector组和wt组之间无明显差异。结论：通过成功构建的端粒酶特异性shRNA慢病毒载体对人类胚胎干细胞的转染实现了对其端粒酶mRNA的调控。     

小鼠Oct4基因真核表达载体的构建及表达鉴定

目的:构建小鼠Oct4基因真核表达载体并对其表达进行鉴定.方法:以小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)cDNA为模板克隆Oct4基因编码区序列,将其通过定向克隆插入pcDNA3.1(+)栽体多克隆酶切位点(multiple cloning sites,MCS)中,形成pcDNA3.1(+)/Oct4重组载体.将pcDNA3.1(+)空载体和pcDNA3.1(+)/Oct4重组载体分别转染293T细胞,通过Western Blotting检测Oct4蛋白的表达.结果:成功检测到Oct4蛋白的表达.结论:成功构建了小鼠Oct4基因真核表达载体.     

携带白介素-18基因的人脐带间充质干细胞的构建及其意义

目的：构建携带白介素-18（IL-18）基因人脐带间充质干细胞（hUMSCs）,为肿瘤靶向性基因治疗研究提供一种工具。方法：体外分离培养hUMSCs,流式细胞仪（FACS）检测hUMSCs 的细胞免疫表型。应用基因重组技术将表达IL-18 基因的慢病毒转染至hUMSCs,利用RT-PCR 及Western blot 法检测IL-18 的蛋白mRNA表达水平。结果：成功在体外分离和培养了hUMSCs,流式细胞仪检测结果显示hUMSCs 表达CD29、CD44 和CD105,而不表达CD34 和CD45, 符合hUMSCs 的表型。成功构建携带IL-18 基因的hUMSCs,RT-PCR 及Western blot法检测结果提示IL-18基因转染至hUMSCs并能稳定表达。结论：构建携带IL-18基因的hUMSCs并稳定表达IL-18,为肿瘤靶向性基因治疗实验性研究提供了一种新实验工具。