共查询到19条相似文献，搜索用时 31 毫秒
1.
以含翻译起始密码子ATG的质粒pSⅪⅤⅥ Ⅹ3/4为转移载体,将多角体蛋白及切除部分5′和3′端的乙型肝炎核心抗原(HBcAg)基因同时插入粉纹夜蛾核型多角体病毒(TnNPV)基因组中,构建了形成多角体的重组毒株。该毒株能利用合成—多角体ⅪⅤ串联启动子,表达由截短的HBcAg序列及其上下游多接头与杆状病毒DNA部分序列组成的融合蛋白基因。SDS-聚丙烯酰胺凝胶电泳、Wcstern印迹与免疫电镜观察的结果表明,融合蛋白基因表达产物的分子量约20.5KD,与理论计算值相符,并保留了HBcAg的抗原性,亦能形成典型的HBcAg颗粒。组建含多克隆点及截去两端序列的HBcAg基因转移载体质粒,使编码多肽抗原的寡聚核苷酸易于克隆,以HBcAg融合蛋白方式在杆状病毒载体系统中表达,并能形成HBcAg为蛋白载体的抗原颗粒结构。 相似文献
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0 前言 人的PAI—2是一种通过抑制纤溶酶原激活物来调节纤溶酶原激活过程的特异抑制网子。生化性质研究和临床应用需要大量的PAI—2蛋白,但通过纯化大规模培养的细胞上清和胎盘抽提液而获取PAI—2是很困难的。在大肠杆菌和哺乳运动细胞中也尚未获得高效表达。我们考虑到用昆虫杆状病毒表达载体系统(Bacul—ovir us expression vector system:BEVS)进行表达。 BEVS是一种比较新的载体宿主表达系统,它与细菌、酵母及哺乳动物细胞表达系统相比具有以下几个方面的优点:1)允许插入的外源基因容量较大;2)能高效表达。重组蛋白达到1～500mg/L;3)应用范围广泛。可以表达各种胞内、分泌和膜蛋白。如用的是多角体蛋白启动于为晚期启动子,可表达一些具有细胞毒作用的蛋白;4)通过双启动于载体或两种不同重组病毒共同感染细胞,可获得多种蛋白的同时表达;5)重组病毒可通过穿刺感染或与野生病毒共包装,形成具有多角体外壳的重组病毒口服感染幼虫,从而在虫体中表达。表达量常比胞内高10～100倍;6)安全。杆状病毒具有严格的宿主专一性,不感染脊椎动物或植物;7)表达蛋白与天然蛋白相似。可以进行高等真核细胞内的蛋白修饰,剪接及运输。 BEVS中作为基因表达载体的病毒是苜蓿尺蠖核型多角体病毒(ACNPV)和家蚕核型多角 相似文献
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目的:构建唾液抗菌肽P113基因多拷贝串联重组体,并将其克隆到表达载体pET-28a.方法:根据大肠杆菌偏爱的密码子设计并合成人唾液抗菌肽P113基因,采用PCR技术构建P113基因的自融合多拷贝串联重组体polyP113,BAMH I和HindⅢ双酶切表达载体pET-28a和polyP113基因,将两者连接后转化大肠杆菌DH5α,氨苄青霉素筛选阳性菌落,PCR、酶切、测序鉴定重组质粒.结果:所构建的含有十拷贝P113基因的重组体正确克隆到表达载体pET-28a上,无突变.结论:成功构建含十拷贝唾液抗菌肽P113基因表达框的大肠杆菌表达载体. 相似文献
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《集宁师专学报》2016,(5):1-4
该文就农杆菌介导的vbp基因遗传转化调控机理进行研究。方法一:分析vpb1基因启动子序列;方法二:对探测载体中vpb1基因启动子的转录活性进行验证;方法三:设计vbpl基因启动子序列突变位点。结果:(1)基于荧光蛋白表达情况来看,在pRSET-A质粒中的vbpl基因启动子能够将荧光基因的表达予以正常启动。(2)能够正确构建pRSET-m1突变质粒,vbp1基因启动子的转录活性会随着SigmaA结合位点的去除而降低。(3)能够正确构建p RSET-m2突变质粒,vbp1基因启动子的转录活性会随着转录因子Fnr结合位点中-65位碱基的突变而增强。(4)能够正确构建pRSET-m3突变质粒,vbp1基因启动子的转录活性会随着转录因子Fnr结合位点中-66位和-68位碱基的突变而消失。结论:深入研究农杆菌介导的vbp基因遗传转化调控机理,能够找出影响农杆菌的VBP蛋白表达的相关因素,有可能使所获得的农杆菌菌株具有较高的转化效率,以便能够有效提高植物转基因效率。 相似文献
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程岑 《淮南职业技术学院学报》2022,(1)
L-酪氨酸属氨基酸的一种,可采用RED同源重组技术敲除大肠杆菌上编码分支酸变位酶/预苯酸脱水酶的pheA基因,使中心代谢由合成苯丙氨酸流向酪氨酸的生产;也可从大肠杆菌DH5α基因组中扩增到aroG和tyrA基因,把它们串联在一个质粒上,在E.coliK12 tyrA-pheA-中转化入质粒pEVC(含基因aroG、tyrA)后,酪氨酸大量生成,为基因工程改造大肠杆菌生产酪氨酸垫定了坚实的基础。 相似文献
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陆军 《常熟理工学院学报》1996,5(4):40-49
ｐＧＡ４６－４是一个带有谷氨酸棒杆菌妄动功能片断的质粒。用ＰＣＲ技术从ｐｇａ４６－４中扩增该启动子片断，将该启动子Ｔ４噬菌体Ｐ３２基因的终止子克隆到大肠杆菌质粒ｐＫ１８上，再接入棒状杆菌粒ｐＸＺ１０１４２构建一穿梭表达载体。 相似文献
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为了研究鱼生长激素基因在大肠杆菌中的表达,采用聚合酶链反应（ＰＣＲ）技术对鲑鱼生长激素ｃＤＮＡ的５端和３端进行定向修饰．将修饰后的三种基因分别克隆到大肠杆菌表达质粒ｐＢＶ２２０,构建成重组鲑鱼生长激素表达质粒ｐＢＶＧＨ１１,ｐＢＶＧＨ１８和ｐＢＶＧＨ２４,转化大肠杆菌ＤＨ５α进行诱导表达．结果表明：核糖核蛋白体结合位点（ＳＤ）与起始密码子ＡＴＧ之间的距离对生长激素的表达水平影响极大．鲑鱼生长激素基因的终止密码子ＴＡＧ不能有效终止该基因在大肠杆菌中翻译的进行,造成部分通读．加入大肠杆菌强终止子ＴＡＡ可避免通读和产生超长蛋白,提高表达产量 相似文献
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灰斑古毒蛾核型多角体病毒几丁质酶基因及其分子进化 总被引：5，自引：0，他引：5
通过对构建的灰斑古毒蛾Orgyia ericae单粒包埋型核型多角体病毒(OrerSNPV)基因组DNA的EcoRI酶切片段质粒文库的测序分析,在EcoRI—J片段(6．4kb)中鉴定出了编码几丁质酶(ChiA)基因开放阅读框(ORF),chiA基因编码区由1695bp组成,编码564个氨基酸,预计蛋白质分子量为62kD。这也是首次在基因序列水平上研究OrerSNPV。将OrerSNPV ChiA氨基酸序列与其它已知的18种杆状病毒ChiA氨基酸序列联配比较,结果表明OrerSNPV与其它杆状病毒ChiA氨基酸有60．3—69．5％同源性,其中与BmNPV、CfDEFNPV和LdMNPV的同源性最高。根据氨基酸序列绘制的分子进化树表明杆状病毒chiA基因至少可以分为SlMNPV、GV和其它NPV三个分支,OrerSNPV与LdMNPV在进化树上关系最为接近。 相似文献
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目的 克隆、表达土豆环氧化物水解酶基因,分析其活性包涵体.方法 提取土豆总RNA,以反转录获得的cDNA为模板进行聚合酶链式反应(PCR),扩增得到土豆环氧化物水解酶基因,构建大肠杆菌表达质粒pET-REH,将重组表达质粒pET -REH转化E.coli BL21(DE3).结果 SDS-PAGE分析表明,IPTG诱导表达的重组土豆环氧化物水解酶在各种温度下均主要以包涵体形式表达,只在15℃有微量可溶表达.酶活性分析表明,28℃表达的重组酶包涵体酶活性性为0.7 U/mg.结论 初步证实了存在活性包涵体的观点. 相似文献
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研究小单孢菌产抗生素的生物合成机理,利用基因克隆方法从棘孢小单孢菌(Micromonospora echinospora)中克隆出产庆大霉素生物合成的关键酶基因-2-脱氧青蟹肌糖合成酶基因(gntB),并将其通过大肠杆菌――链霉菌穿梭质粒pIJ699转化原菌株,采用硫链丝菌素抗性基因启动子带动2-脱氧青蟹肌糖合成酶基因表达,在培养条件不变的情况下重组菌产抗率较原菌株提高3.5%. 相似文献
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Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasmid pGEMEX-1-Humanin was digested with restriction endonucleases BamH I and Hind III and the Humanin gene fragments, about 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3, l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA successfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed. 相似文献
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为分析LPS刺激前后对RIPK2基因甲基化的影响,试验采用凝胶电泳、亚硫酸氢盐、双荧光素酶报告系统和qRT-PCR方法分析LPS刺激前后RIPK2基因的甲基化模式及甲基化相关试剂对鸡RIPK2启动子活性及表达水平影响。结果表明：LPS刺激后鸡RIPK2基因甲基化水平从53.8%下降至15.4%;5-氮杂胞苷显著性提高鸡RIPK2启动子活性,促进基因表达;而CpG甲基转移酶M. SssI显著性抑制鸡RIPK2启动子活性,抑制基因表达。研究结果对提高家禽免疫和控制过度性炎症反应具有十分重要的现实意义。 相似文献
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Zhang Q Chen QH Fu ML Wang JL Zhang HB He GQ 《Journal of Zhejiang University. Science. B》2008,9(7):527-535
The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis ofgenome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h-ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer. 相似文献
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文章以清洁级纯系雄性SD大鼠为实验模型,应用组织病理学、RT—PCR技术,研究高脂饮食诱发动脉粥样硬化过程中血管内膜细胞大电导钙激活的钾通道（large conductance Ca—activated K＋ channels,BKCa）、中电导钙激活的钾通道（Intermediate condutance Ca—activated K＋ channels,IKCa）基因表达的动态改变情况,从基因表达水平探讨高脂饮食诱发动脉粥样硬化的作用机制。同时指出,IKCa可能为动脉粥样硬化的早期启动因子,此观点有助于动脉粥样硬化的早期检测与防治。 相似文献
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1IntroductionRibosome-inactivating proteins(RIPs)occur natu-rally in a variety of higher plant species,and theyfunction by catalytic depurination of a specific aden-osine residue located near the3*terminus of eukary-otic large ribosomal subunit rRNA,preventing EF-2/GTP binding and thereby blocking peptidyl-tRNAtranslocation during protein synthesis[1].Many RIPsare potent antiviral and antifungal proteins in vitro[2,3],but it may beinsufficient for field application,as com-pared to the ac… 相似文献
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Qin Guo Wei Zhang Liu-Liu Ma Qi-He Chen Ji-Cheng Chen Hong-Bo Zhang Hui Ruan Guo-Qing He 《Journal of Zhejiang University. Science. B》2010,11(1):41-51
The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer. 相似文献
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This paper examines listening comprehension skills of visually impaired students (VIS) using computerised adaptive testing (CAT) and reader-assisted paper-pencil testing (raPPT) and student views about them. Explanatory mixed method design was used in this study. Sample is comprised of 51 VIS, in 7th and 8th grades. 9 of these students were interviewed for determining student views about tests. Results indicated that scores obtained from CAT are significantly lower than scores obtained from raPPT. Additionally, a positive and high correlation was found between scores of CAT and raPPT. This result suggests that similar ability estimations were made by CAT and raPPT. Another finding is CAT made more reliable predictions, and was completed in shorter duration using fewer items. In qualitative part, student views were gathered through interviews and content analysis revealed three themes as technical features, test features, and psychological effects. In general, students reported positive views about CAT. VIS prefer CAT due to its listening/control options, shorter test durations, clarity of reading, and fairness of test, elimination of dependency to reader. Study provides implications for test developers and test-users to consider CAT as a multi-accommodation for VIS through its advantages. 相似文献