神经营养素-3的克隆与真核表达

目的　构建人神经营养素 - 3(NT 3)的真核表达质粒 .方法　由于NT 3基因中无内含子 ,因此直接运用PCR技术从人基因组DNA中分离出人NT 3完整基因 ,与 pcDNA3.1(- )载体重组并测序 ,获得pcDNA NT3.用脂质体介导法将重组质粒转染人胚肾 2 93A细胞株 ,经G4 18筛选后 ,用RT PCR和免疫荧光法在转录和翻译两个水平上检测表达产物 .结果　测序证实所获得的是正确的重组质粒 .它的转录和翻译产物均可在细胞中检测到 .结论　成功地进行了NT 3的克隆与真核表达 ,NT 3基因修饰后的胚胎细胞有可能对神经轴突的生长起指导作用 .该实验结果为深入研究NT 3的生物学作用创造了条件 ,为基因修饰细胞移植治疗神经疾病的实验研究奠定了基础 .     

构建逆转录病毒PBabepuro-Bcl-2表达质粒及稳定转染细胞株

目的:构建pBabepuro-Bcl-2逆转录病毒表达质粒,鉴定表达,在小鼠胚胎成纤维细胞NIH3T3中建立过表达Bcl-2稳定转染细胞。方法:以人上皮细胞的cDNA为模板,利用X-tremeGENE HP(Roche)转染293T细胞,蛋白质免疫印迹法在蛋白水平检测表达,利用逆转录病毒感染NIH3T3细胞。结果:成功构建了逆转录病毒表达质粒pBabepuro-BCl-2,蛋白水平检测证实其在293T细胞可以有效地表达,成功建立了过表达Bcl-2的NIH3T3稳定转染细胞。结论:通过构建pBabepuro表达质粒及在NIH3T3建立过表达Bcl-2稳定转染细胞,为进一步研究Bcl-2的功能调控奠定了基础。     

PARP抑制剂诱导外源整合基因删除的作用特点

构建能表达LacZ基因和c-Ha-T24ras基因的两种真核表达重组质粒A13.4和B12.7（两基因相对位置不同），并将其转染NIH3T3细胞和HeLa细胞，经G418筛选，分别建立了四种稳定转化细胞系A13.4-NIH3T3、B12.7-NIH3T3、A13.4-HeLe及B12.7-HeLa。用聚ADP核糖聚合酶（PARP）的NAD位点抑制剂苯甲酰胺（BA）分别处理四种转化细胞后，检测细胞中整合的外源LacZ基因与c-Ha-T14ras基因的删除情况，结果如下：BA诱导的基因删除可发生于NIH3T3转化细胞系，但不能发生于HeLa转化细胞系；位于同一外源表达载体上的LacZ基因与c-Ha-T24ras基因的删除过程是同步的；外源整合DNA片段的转录强度可能直接影响其被BA诱导删除的敏感性。     

bFGF真核表达载体构建及表达

旨在构建可在哺乳细胞中表达碱性成纤维细胞生长因子（basic Fibroblast Growth Factor,简称bFGF）的真核表达载体，以便用于研究bFGF基因治疗在骨组织工程中的应用，应用基因重组技术，将已经克隆的bFGF基因从PBR322－bFGF载体上亚克隆到真核表达载体pcDNA3．1上，构建的重组质粒经脂质体介导转染3T3细胞，36h后观察瞬时表达情况，酶切，PCR和DNA测序结果均证实了插入片段的正确性，免疫组织化学检测bFGF的表达分泌情况，结果显示部分经转染的细胞呈阳性（棕色颗粒）。结果表明已成功构建了bFGF真核表达载体pcDNA3.1-bFGF，并且bFGF能够在3T3细胞表达。     

重组质粒pPIC9K-pZP3α-hCGβ CTP109～145的构建

目的：为发展多特异性避孕疫苗,制备重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,用基因工程技术构建重组质粒pPIC9K－pZP3α－hCGβCTP^109-145,方法：根据pZP3α全长和hCG 0633CTP109-145编码的DNA序列设计两对引物,通过部分重聚合酶链式反应（overlaping PCR）,扩增出pPIC9K－pZP3α－hCGβCTP^109-145片断,克隆到载体质粒pPIC9K中,然后导入大肠杆菌DH5α,用PCR和双酶切反应鉴定重组质粒,并用DNA测序仪对重组质粒测序,结果：3步PCR扩增出pPIC9K－pZP3α－hCGβCTP^109-145DNA片段,插入到载体质粒pPIC9K的克隆位点,获得重组pPIC9K－pZP3α－hCGβCTP^109-145表达质粒,测序结果显示插入序列与设计预期完全一致。结论：重组质粒pPIC9K－pZP3α－hCGβCTP^109-145构建成功,为表达重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,探讨重组多特异性避孕疫苗抗生育效能的研究建立基础。     

人TPO基因转染肝癌细胞后的表达及其受体水平的变化

从hTPO工程菌株中提取的pcDNA3/TPO质粒用脂质体包裹后转染人肝癌细胞株HepG2,并以未转染的HepG2和转染pcDNA3野生型质粒的HepG2作为对照，研究hTPO和其受体c-mpl在三组细胞中的表达情况以及HepG2细胞的生长情况，发现（1）RT－PCR法检测结果表明pcDNA3/TPO基因在转染HepG2细胞后，mRNA能有效表达，但对c-mpl的表达基本无影响；（2）细胞的生长计数和MTT法检测结果说明hTPO的有效表达对人肝癌细胞株HepG2的生长、繁殖有一定的抑制作用，同时还发现脂质体导入及pcDNA3型质粒的转染能够促进HepG2的生长。     

pEGFP-N3-t-PA真核表达质粒的构建及其在成纤维细胞中的表达

目的利用基因工程技术克隆人组织纤溶酶原激活物（t—PA）基因并构建一种能高效、安全表达t—PA的pEGFP—N3-t—PA真核表达重组质粒,为进一步研制转基因动物奠定基础．方法采用高效Trizol试剂快速从黑色素瘤细胞中提取总RNA,RT—PCR获得t—PAcDNA,并将真核表达质粒pEGFP—N3和t—PA基因片段分别双酶切,将回收的pEGFP—N3大片段（4．7kb）与t—PA基因片段（1．1kb）重组．对pEGFP—N3-t—PA质粒进行酶切鉴定和基因测序鉴定．脂质体介导pEGFP—N3-t—PA转染成纤维细胞（NIn313）,并用RT—PCR法从mRNA水平检测t—PA的表达情况,用倒置荧光显微镜检测、分析其在NH 3T3细胞中的表达．结果成功地从黑色素瘤细胞中克隆了t—PA基因,并构建了以pEGFP—N3为载体的真核表达质粒载体,并能在真核细胞中表达、分泌t—PA．结论含t—PA基因真核表达质粒构建成功．     

人纤溶酶原K1—3区基因在大肠杆菌中的表达

将人纤溶酶原Krigle 1-3(K1-3)基因插入融合表达载体pET-17b，获得重组质粒pET-K13,转化E.coli BI21(DE3),在IPTG诱导下，人纤溶酶原K1－3基因在E.coli BI21(DE3,pET-K13)中获得高效融合表达，表达量占菌体总蛋白的24％，表达产物以包函体存在，Western blot证明重组 蛋白对人纤溶酶原抗血清有特异免疫原性。     

Spindlin 1编码的蛋白质定位于细胞核并使NIH3T3细胞发生恶性转化

研究了卵巢癌中高表达的新基因spindlin1的亚细胞定位及其对小鼠NIH3T3细胞生物学行为的影响, 并探讨了人spindlin1基因的生物学功能. 采用脂质体转染法将构建的融合蛋白表达载体pEGFP-N1/ pEGFP-N1-spindlin1瞬时转染COS-7细胞,观察spindlin1基因的亚细胞定位;同时稳定转染NIH3T3细胞,经G418抗性筛选后,用RT-PCR筛选并鉴定表达spindlin1的稳定细胞株;通过细胞增殖活性、流式细胞检测、软琼脂克隆形成、迁移实验及裸鼠成瘤等对转染细胞的生物学行为进行检测. 结果表明,spindlin1定位于胞核;并促进NIH3T3细胞的增殖,使其处于G2/M期的细胞比例显著增加;同时证实过表达spindlin1的NIH3T3细胞不仅具明显的克隆形成及迁移能力,而且能使裸鼠成瘤. 由此我们得出结论:spindlin1基因过表达可促使NIH3T3细胞发生恶性转化,提示spindlin1可能是一种与肿瘤形成相关的原癌基因.     

利用人内皮抑素基因构建植物表达载体及对烟草的遗传转化

将人内皮抑素(endostatin)基因重组于植物双元表达载体pGA 643,得到重组质粒pGE.用三亲融合法将其导入农杆菌LBA 4404中,采用叶盘法转化烟草,经诱导与筛选,获得了卡那霉素抗性植株.提取抗性植株总DNA,通过PCR扩增和Sou thern杂交检测,筛选出了整合有外源基因的转化植株.为进一步研究人内皮抑素在烟草中的表达及生物学功能奠定了基础.     

新基因hSSB1逆转录病毒载体的构建、鉴定和稳定株的筛选

 hSSB1 (Human Single strand DNA binding protein) 是参与细胞DNA损伤应答的一个重要信号分子。根据GenBank 提供的hSSB1基因序列扩增其cDNA序列,插入到pBABE逆转录病毒载体中, 连接后的质粒转化后经过双酶切,PCR扩增及测序来鉴定pBABE-hSSB1阳性克隆。将阳性表达的pBABE-hSSB1和包装质粒转染到HEK293T细胞中,产生病毒液。将包装好的病毒感染细胞并用嘌呤霉素(puromycin)筛选稳定表达hSSB1的细胞株。重组pBABE-hSSB1质粒经双酶切,PCR扩增鉴定及DNA测序分析等方法证实克隆成功。Western blotting检测发现转染重组质粒pBABE-hSSB1的细胞株中hSSB1蛋白的表达水平高于对照组。该研究成功构建了针对hSSB1基因的逆转录病毒载体(pBABE-hSSB1),并得到了稳定高表达hSSB1的细胞株, 为深入研究其功能奠定了基础。     

点突变ras癌基因全cDNA真核表达重组体的构建及诱导NIH3T3细胞转化的研究

将活化癌基因Ｔ２４－ｒａｓ的全ｃＤＮＡ序列正向插入真核载体ｐＭＡＭｎｅｏ，构建成重组质粒ｐＭＡＭｎｅｏ－Ｔ２４－ｒａｓ．将该质粒转染ＮＩＨ３Ｔ３细胞，通过药物筛选，建成细胞系３Ｔ３（Ｔ２４）．Ｓｏｕｔｈｅｒｎ杂交证明外源Ｔ２４－ｒａｓ基因已整合于受体细胞染色体中．３Ｔ３（Ｔ２４）细胞表现出形态学方面的明显变化：具失去接触抑制能力，且在裸鼠体内致瘤等恶性行为．本文构建的Ｔ２４－ｒａｓ基因真核表达重组体和建立的转化细胞系可用于肿瘤的诊断、预防、治疗及抗肿瘤药物的筛选、评估等研究．     

Transformation of NIH 3T3 cells by a human c-sis cDNA clone

The mechanism of leukaemogenic transformation by human T-cell leukaemia/lymphoma virus (HTLV), a retrovirus implicated in the aetiology of certain adult T-cell leukaemias and lymphomas, is unknown but is conceivably associated with the expression of the cellular analogues of retroviral oncogenes. The HUT-102 cell line, derived from a cutaneous T-cell lymphoma and infected with HTLV, expresses several cellular oncogenes. It is unusual among haemopoietic cell lines in that one of these is c-sis, the gene from which the oncogene v-sis of the simian sarcoma virus was derived, and perhaps the gene for platelet-derived growth factor (PDGF). To explore the possible role of c-sis expression in HTLV-induced disease, we have obtained cDNA clones of c-sis from HUT-102 cells. Here we describe two such clones and report that one of them transforms NIH-3T3 cells. This is the first example of transformation of NIH-3T3 cells by a human onc gene other than c-ras or Blym, as well as the first demonstration of transformation by a human cDNA clone.     

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.     

A novel human gene spindlin1, encoding a protein localized in the cell nucleus and inducing NIH3T3 cell''s transformation

A novel human gene, spindlin1, recently cloned in our laboratory, is highly expressed in the tissue of ovary cancer. To study its biological function, a vector expressing green fluorescent-spindlin1 fusion protein was constructed and transfected into COS-7 and NIH3T3 cells by lipofectamine methods. The results showed that the fusion protein pEGFP-N1-spindlin1 was localized in the nucleus of COS-7 and NIH3T3 cells. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the parental NIH3T3 cells displayed a complete morphological change, improved the cell growth and increased the percentage of cells in G2/M phase (12.6% vs control cells at 3.4%). Furthermore, overexpressed spindlin1 cells formed colonies in soft agar, more motile in migration assay in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may be a prooncogene which is associated with tumorigenesis.     

逆转录病毒载体介导的Anti-ras核酶对T24ras转化3T3细胞的抑制作用

突变ｒａｓ基因存在于多种人类肿瘤细胞中,本文将可特异性切割１２位点突变ｒａｓ（Ｔ２４ｒａｓ）之核酶Ｒ８通过逆转录病毒载体导入Ｔ２４ｒａｓ转化的ＮＩＨ３Ｔ３细胞,以期检测核酶在细胞内对其靶基因的作用．结果表明Ｒ８可特异性地切割突变ｒａｓｍＲＮＡ,导致转化细胞生物学特性如细胞形态、生长速度等在一定程度上发生逆转     

Rapid senescence induced by overexpression of p53 in NIH3T3 cells

An NIH3T3 cell line which overexpresses temperature-sensitive p53Val135 was constructed by introduction of p53Val135 gene. It exhibited rapidly characteristic morphological and biochemical alterations related to repli-cative senescence when being cultured in 32℃. We suggested that the overexpression of p53 activated probably the onset of senescence in NIH3T3 cells, which induced a rapid cellular senescence.     

Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells

Patients with certain genetic disorders can be cured by bone marrow transplantation. However, as prospective donors do not exist for most patients with potentially curable genetic abnormalities, an alternative treatment for such patients involves the transfer of cloned genes into the patient's haematopoietic stem cells followed by re-infusion of the treated cells. Retroviral vectors provide an efficient means for transferring genes into mammalian cells and have been used to transfer genes into mouse haematopoietic cells. We have now produced amphotropic retroviral vectors containing either the bacterial gene for neomycin resistance or a mutant dihydrofolate reductase gene that confers resistance to methotrexate and have used these vectors to infect and confer drug resistance to human haematopoietic progenitor cells in vitro. Transfer could be demonstrated in the absence of helper virus by using an amphotropic retrovirus packaging cell line, PA12 (ref. 9). These studies are an important step towards the eventual application of retrovirus-mediated gene transfer to human gene therapy and for molecular approaches to the study of human haematopoiesis.     

定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA, 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列, 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo, 转化大肠杆菌BL21(DE3), 筛选表达菌株BL21-Mute, 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明, 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%, 失去抑制人脐静脉内皮细胞增殖的活性.