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。     

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.     

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.     

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.     

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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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.     

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?     

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。     

Destabilizing Domains Mediate Reversible Transgene Expression in the Brain

Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD) of the Escherichia coli dihydrofolate reductase (DHFR) that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinson''s disease) and should be further investigated.     

Expression of Tropomyosin-Encoding Gene in the Kidney Depends on Functioning of Vasopressin Gene

Brattleboro diabetes insipidus mutant rats and normal WAG rats were subjected to water loading or thirst during 3 days. It was found that tropomyosin-encoding gene expression has a tissue-specific pattern in the kidney. Northern blot and western blot analysis had shown that the main expression of Tpm3(3) takes place in the renal medulla, and its intensity differs in normal and mutant rats. The differences between mutants with an ineffective vasopressin synthesizing system and the rats having an intact vasopressin gene were more distinct under long-lasting dehydration. The ratio between renal medullary tropomyosin of Brattleboro and WAG lines of rats was 39.76 +/- 0.90 versus 18.29 +/- 0.86 under water loading, and 46.12 +/- 2.14 versus 13.83 +/- 0.66 in thirst.     

NOD1真核表达质粒的构建及表达

目的:构建天然免疫胞内识别受体核苷酸寡聚域1(NOD1)真核表达质粒。方法:NOD1基因片段经PCR扩增获得,经酶切后连接到真核表达载体pcDNA3/flag中,对挑选出的阳性克隆测序,将序列正确的重组质粒pflag-NOD1转染293T细胞,用Western印迹检测目的蛋白的表达,同时用NF-κB的萤光素酶报告基因检测NOD1蛋白的活性。结果:pflag-NOD1可以在真核细胞293T中表达,并可以增强NF-κB报告基因的转录活性。结论:构建了重组质粒pflag-NOD1,在细胞中表达NOD1后能够提高NF-κB转录的生物活性,为进一步研究NOD1的功能奠定了基础。     

含有绿色荧光蛋白基因的质粒载体的构建及其在水稻白叶枯细菌中的表达

本文用绿色荧光蛋白基因（green fluorescent protein）标记水稻白叶枯细菌,观察其在白叶枯细菌中的表达情况。光激发后,白叶枯细菌发出绿色荧光,表明gfp在白叶枯细菌中得到了高效表达。后续工作意在利用gfp所发出的绿色荧光,来追踪白叶枯细菌侵染水稻的路径,以及检测水稻在遭受白叶枯病害时的一些生理生态变化,进一步探讨水稻对白叶枯细菌的抗生机理,希望能够为水稻抗性品种的检测提供新的理论依据。文中重点介绍了对质粒pM2464的改造过程,经gfp标记后的水稻白叶枯细菌,在紫外或蓝光的激发下,发出绿色荧光,证明了用标记有gfp基因的白叶枯细菌来观察其侵染水稻过程的想法是可行的。     

CpG基序重组菌发酵培养基的优化

目的:为提高CpG基序重组菌的发酵水平,降低CpG重组质粒的生产成本,运用响应面法优化CpG重组菌发酵培养基。方法:利用Plackett-Burman试验筛选出影响发酵水平的3个最显著因素:糖蜜、玉米浆和工业蛋白胨。然后用响应面设计试验并优化得到最显著因素的最佳浓度,并进一步通过发酵试验验证优化后的培养基。结果:得到一组具有较高菌浓及质粒产量且价格低廉的发酵用培养基:糖蜜4.5g/L,玉米浆8.5g/L,工业蛋白胨8.5g/L,甘油10mL/L,KH2PO41.5g/L,K2HPO42.3g/L,MgSO4.7H2O 0.25g/L,在此培养条件下,OD600实测值为0.6771,理论值为0.6643,两者接近,与标准LB培养基相比,质粒产量提高了15%左右。结论:最终筛选到的培养基具有较高的性价比。     

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration,and Acquisition of the Plasmid Results in a Variable Cost of Fitness

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.