Stathmin SiRNA表达载体抑制stathmin的表达

目的:构建stathmin特异性SiRNA质粒表达载体,探讨其对鼻咽癌5-8F细胞stathmin的沉默作用.方法:合成用于stathmin基因特异性干扰表达的DNA片段,经退火形成双链DNA片段,片段克隆到质粒表达载体pGenesil 1.1上.载体导入JM109菌株进行筛选与扩增,采用酶切和测序对克隆表达载体进行鉴定.应用脂质体将鉴定后的重组表达质粒载体转入鼻咽癌5 -8F细胞,RT-PCR与Western Blot分析stathmin基因表达.结果:经酶切和测序鉴定,插入SiRNA质粒表达载体的stathmin特异性碱基序列和方向正确.重组质粒表达载体转染鼻咽癌细胞后,细胞转染效率达78.8 ±6.8％,stathmin基因在鼻咽癌中的表达明显下降.结论:构建的stathmin基因SiRNA质粒表达载体能抑制stathmin的表达.     

大鼠Otos基因RNA干扰质粒体的构建

目的:构建其基因Otos的RNA干扰质粒载体,为研究Otospiralin在内耳的生理功能奠定基础。方法:在GenBank中查到大鼠Otos基因的序列,输入到相应的设计软件中,以此设计引物序列,经过PCR扩增,酶切后,克隆于pAVU6 27载体并行酶切鉴定。结果:构建的鼠Otos shRNA载体经过测序鉴定,所得和预期相符。结论:重组质粒pAVU6 27-Otos的成功构建为下一步研究打下良好的基础。     

烟草质体多顺反子定点整合表达载体的构建和转化

构建了烟草质体多顺反子定点整合表达载体pLM4(-psaA-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3'-psbC-).用基因枪将该载体轰击烟草叶片5次,用添加了壮观霉素的选择分化培养基筛选,获得质体转基因烟草6株.用PCR、激光扫描、Western blot和RFLP等方法检测都证实多顺反子表达盒中的3个基因甘露聚糖酶基因(man)、绿荧光蛋白基因(gfp)、氨基糖苷3'-腺苷酰基转移酶基因(aadA)已整合到烟草质体基因组中,且均得到表达.     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

鸡输卵管特异表达载体的优化及体内表达

为了实现鸡输卵管特异表达载体在相应组织特异高产表达, 并简化质粒DNA的制备过程, 本研究在已经构建的鸡输卵管特异表达载体pOV1基础上进行了优化改造, 为进一步进行重组药物蛋白的暂态表达及转基因鸡研究奠定基础。首先用限制性内切酶将克隆在pOV1载体的鸡卵清蛋白基因5￠-和3￠-调控区切出, 同时克隆到切除neo基因及CMV启动子的pcDNA3.0载体, 构建成另一输卵管特异表达载体pOV2; 将鸡卵清蛋白基因5￠-调控区单独克隆入同样载体, 获得第三个鸡输卵管特异表达载体pOV3。为了检验三个输卵管表达载体驱动外源基因在鸡体内输卵管细胞中表达的有效性和特异性, 将LacZ报告基因分别克隆入pOV1、pOV2、pOV3中5'-调控区的下游, 获得的重组载体pOV1LacZ、pOV2LacZ和pOV3LacZ经聚乙烯亚胺包裹后, 经翅静脉注射产蛋鸡。用RT-PCR和酶活性检测法对LacZ基因在载体注射鸡体内的表达进行检测, 结果显示肝、脾、肾、心等组织中无LacZ基因的表达, 而输卵管膨大部不仅有LacZ基因的表达, 而且表达的重组酶能分泌到蛋清中, 雌激素注射对报告基因的表达具有促进作用, 其中pOV3LacZ的表达水平较高。这些试验结果表明, 鸡输卵管特异表达载体pOV3具有结构相对简单、表达水平较高、组织特异性较好等优点, 能用于鸡输卵管生物反应器的研制。     

Mutants of the Killer Plasmid of SACCHAROMYCES CEREVISIAE Dependent on Chromosomal Diploidy for Expression and Maintenance

Mutants of the killer plasmid of Saccharomyecs cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.—Haploids carrying these mutant plasmids (called d for diploid-dependent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K⁺R⁺) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.—Sporulation of the phenotypically K⁺R⁺ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.     

植物的MAR及其对转基因表达的效应

与核基质结合的DNA序列称为基质附着区（matrix attachment regions,MARs）,可提高转基因的表达水平并降低转基因在不同转基因系间的表达差异,因其在植物基因工程中的巨大应用潜力而引起了研究者的极大兴趣。对植物中MAR的研究尚处于早期阶段,本文综述了植物中MAR的分离鉴定、序列特征及MAR对植物中转基因表达的影响,并进一步讨论了MAR对转基因效应的可能机制。 Abstract：DNA sequences called matrix attachment regions (MARs) have recently attracted mu ch attention because of their perceived capacity to increase levels of transgene expression and to reduce transformant-to-transformant variation of transgene ex pression in plants. Work with MARs in plants is in its early stage .In the prese nt paper ,we reviewed the procedure to isolate and identify MAR sequences from higher plants, the sequence characteristics of the plant MARs and the effect of MARs on the transgene expression in plants. Funthermore, the possible mechanism to explain how MARs affect transgene expression in transformants was discussed.     

Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.     

Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1

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用质粒pUC18在大肠杆菌中表达人蛋白质二硫键异构酶

 用质粒ｐＵＣ１８在大肠杆菌中表达人蛋白质二硫键异构酶高音,王志珍（中国科学院生物物理研究所,生物大分子国家重点实验室,北京１００１０１）蛋白质二硫键异构酶（ｐｒｏｔｅｉｎｄｉｓｕｌｆｉｄｅｉｓｏｍｅｒａｓｅ,ＰＤＩ）催化蛋白质分子内天然二硫键的形成,...     

Transient Inhibition of CD28 and CD40 Ligand Interactions Prolongs Adenovirus-Mediated Transgene Expression in the Lung and Facilitates Expression after Secondary Vector Administration

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced β-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.     

Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rif^r, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rif^r, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.     

Persistence of Transgene Expression Influences CD8+ T-Cell Expansion and Maintenance following Immunization with Recombinant Adenovirus

Previous studies determined that the CD8⁺ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8⁺ T-cell maintenance following immunization with a recombinant adenovirus.Recombinant human adenovirus 5 (rHuAd5) vector vaccines have garnered considerable attention as platforms for eliciting CD8⁺ T-cell immunity due to their strong immunogenicity in numerous studies, including primate studies and preliminary human trials (30, 32, 53). While these vectors may not represent the optimal serotype for use in humans, due to the high prevalence of preexisting immunity, the robust immunogenicity of rHuAd5 in preclinical models merits further investigation, since the biological information derived from these studies will offer important insights that can be extended to other vaccine platforms.CD8⁺ T cells play an important role in host defense against tumors and viral infections. During the primary phase of the CD8⁺ T-cell response, the activated precursors undergo a rapid and dramatic expansion in cell number, followed by a period of contraction where 80 to 90% of the antigen-specific population dies off, leaving the remaining cells to constitute the memory population (44). CD8⁺ T cells mature over the course of the primary response and acquire the ability to produce gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and, to a lesser degree, interleukin 2 (IL-2). Memory T cells can be divided into central memory and effector memory T cells based on phenotype and anatomical location (44). These phenotypic differences have also been linked to functional differences; however, these relationships remain controversial (2, 16, 20, 46, 55).Various reports have revealed some unexpected qualities of the CD8⁺ T-cell response generated by intramuscular immunization with rHuAd5. The rHuAd5-induced CD8⁺ T-cell response exhibited a protracted contraction phase, and the memory population was composed primarily of effector and effector-memory cells (23, 38, 39, 41, 51). The phenotype of the rHuAd5-elicited CD8⁺ T-cell population was more consistent with the CD8⁺ T-cell population observed in persistent infections, such as polyomavirus (25), murine herpesvirus-68 (35), and murine cytomegalovirus (MCMV) (1) infections, than with that observed in acute infections, such as lymphocytic choriomeningitis virus (LCMV) (44), vaccinia virus (15), and influenza virus (24) infections. Further investigation demonstrated that, as in a persistent infection, antigen presentation persisted for a prolonged period following intramuscular immunization with rHuAd5, and transgene expression could persist at low levels for more than 1 year following infection (41, 51). These data suggest that the sustained effector phenotype may arise from prolonged, low-level transgene expression from the rHuAd5 vector, although this connection was not formally proven. It is difficult to fully appreciate the implications of these observations at this time, since chronic exposure to antigen is often associated with CD8⁺ T-cell dysfunction, yet rHuAd5 vectors have been used successfully to elicit protective immunity in many models of pathogen infection and tumor challenge (5, 54). Nevertheless, other reports have provided evidence that rHuAd5 vectors can, indeed, lead to dysfunctional CD8⁺ T-cell immunity (27, 36). Therefore, further investigation is necessary in order to properly assess the implications of the prolonged antigen expression following rHuAd5 immunization in terms of sustaining a functional memory CD8⁺ T-cell response.In the current report, we sought to determine the relationship between transgene expression and CD8⁺ T-cell maintenance and memory. To this end, we constructed an Ad vector with a doxycycline (DOX)-regulated expression cassette that would permit attenuation of gene expression at various times postinfection. Using this reagent, we addressed two key questions. (i) How does the duration of antigen expression affect the magnitude of primary CD8⁺ T-cell expansion? (ii) Is antigen expression required beyond the peak expansion to maintain the memory CD8⁺ T-cell population?     

Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression

A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.     

CpG基序重组质粒增强小鼠免疫和抗病力的效应

为研制安全高效免疫调节剂增强动物免疫抗病能力,本实验设计合成含11个CpG基序的寡聚核苷酸,重组构建含CpG的VR1012质粒(VR1C);制备壳聚糖纳米颗粒包裹重组质粒(CNP-VR1C),肌注接种3周龄昆明小鼠;接种后28天口服大肠杆菌攻毒观察小鼠天然免疫的变化和对强毒感染的抵抗力,Sandwich ELISA测定血清免疫球蛋白和白细胞介素含量。结果表明:CNP-VR1C能显著提高小鼠的天然免疫水平,增强对强毒感染的抵抗力,具有研制为高效安全分子免疫增强剂的应用潜力。