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亚克隆

亚克隆的相关文献在1990年到2021年内共计145篇,主要集中在分子生物学、基础医学、畜牧、动物医学、狩猎、蚕、蜂 等领域,其中期刊论文122篇、会议论文3篇、专利文献33382篇;相关期刊86种,包括四川大学学报(自然科学版)、生物工程学报、生物技术通报等; 相关会议3种,包括首届国际中西医结合变态反应学术会议暨全国中西医结合变态反应第三次学术会议、第20届欧洲血液病年会、中国科协首届学术年会等;亚克隆的相关文献由472位作者贡献,包括吴锦艳、尚佑军、田宏等。

亚克隆—发文量

期刊论文>

论文:122 占比:0.36%

会议论文>

论文:3 占比:0.01%

专利文献>

论文:33382 占比:99.63%

总计:33507篇

亚克隆—发文趋势图

亚克隆

-研究学者

  • 吴锦艳
  • 尚佑军
  • 田宏
  • F·A·斯图尔特
  • J·P·P·穆伊雷尔斯
  • Y·张
  • 刘湘涛
  • 张吉利
  • 张志东
  • 王光祥
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  • 会议论文
  • 专利文献

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    • LI Xiao-Jie; MIAO Hui; WANG Di; ZHAN Hao-Miao; WANG Lin-Lin; WU Chuan-Fang
    • 摘要: 为探究RNA与SAFB1蛋白相互作用过程中RNA二级结构的作用.采用足迹法确定SAFB1与LINC-PINT-1特定的结合区域,在此基础上根据RNA结构分析研究SAFB1结合区域所具有特定的二级结构.最后通过凝胶迁移率实验论证该二级结构在RNA与SAFB1蛋白结合中的作用.结果显示:在针对LINC-PIN T-1的亚克隆发现只有包含30~55 nt区域的亚克隆才能维持RNA结合区域的二级结构,并且证实了LINC-PINT-1与SAFB1蛋白相互作用依赖于特定的RNA二级结构.结果表明:长非编码RNA与SAFB1蛋白相互作用或依赖于特定的RN A二级结构,本研究为RN A与蛋白质相互作用的分子机制研究提供了有力的理论实验支撑.
    • 黄海峰; 曹瑞勇; 杨小健; 聂鑫; 杜丽; 王凤阳; 王成强; 张振兴; 李宝宝; 郑义盈; 安琪; 张萌萌; 章泸尹; 朱姝
    • 摘要: 试验旨在对羊源多杀性巴氏杆菌toxA-N基因进行克隆、原核表达及纯化,并对表达蛋白toxA-N进行生物信息学分析,为探索羊源多杀性巴氏杆菌毒素基因的相关特性提供参考依据.以羊源多杀性巴氏杆菌基因组为模板,参考GenBank中公布的多杀性巴氏杆菌HN06中toxA基因序列(登录号:CP003313.1)设计引物,通过PCR技术扩增出目的片段,构建重组质粒pET28a(+)-toxA-N,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达后,经考马斯亮蓝染色及Western blotting鉴定,纯化蛋白并运用生物信息学软件对表达蛋白进行特性分析.结果 显示,试验成功扩增出大小为1 515 bp的toxA-N基因片段,经BamH Ⅰ和NotⅠ双酶切得到大小约为5 369和1 515 bp两条片段,表明成功构建了pET28a(+)-toxA-N重组质粒,IPTG诱导表达的菌株经考马斯亮蓝染色及Western blotting鉴定后,成功表达出大小约为60 ku的toxA-N蛋白;生物信息学分析表明,toxA-N蛋白为包涵体,其分子式为C2635H4002N664O797S17,原子总个数为8 115,消光系数为84 480,不稳定指数为43.50,亲水性平均值为-0.381.在toxA-N蛋白二级结构中,α-螺旋、β-折叠、延伸链和无规则卷曲分别占53.47%、2.77%、11.28%和32.48%,与三级结构预测结果一致.本试验通过对toxA-N基因的初步研究,揭示了多杀性巴氏杆菌毒素的相关特性,对家畜的疾病预防、诊断、治疗具有重要意义.
    • 贺超; 邓璐; 汤承; 岳华
    • 摘要: 根据GenBank中猪表皮生长因子(pEGF)基因信息设计引物,通过PCR、亚克隆、基因重组等技术,构建得到pEGF拷贝数分别为1、2、3的原核重组表达质粒.用重组质粒转化E.coli BL21,IPTG诱导表达,SDS-PAGE和Westernblotting分析结果表明,在37°C诱导条件下,1、2、3拷贝pEGF重组质粒均能表达pEGF蛋白,灰度值分析表明:表达的融合蛋白占菌体蛋白总量分别为9.54%、20.37%、26.79%,表明在原核表达中,对pEGF进行同向串联,增加其拷贝数,可以提高pEGF在表达后融合蛋白中所占比例,相应提高pEGF蛋白产量.本研究为pEGF重组蛋白的高效表达和批量生产提供可靠的依据.
    • 刘爱玲; 陆学东; 吴润香; 贾雪
    • 摘要: Objective To construct subclones of hMPV frgm clinical samples and make preparation for full length cDNA clone and full understanding of it. Methods Primers were designed according to entire gene sequences of GenBank. 697 nasopha-ryngeal swabs of RRTI were collected, extracted virus nucleic acid. Target gene segments were amplified by long reverse transcriptase polymerase chain reaction. Purified the agarose gel, then cloned into PJET1. 2/blunt vector and transformed into TOP10. Positive cLones were selected with ampicillin. The recombinant plasmids containing the hMPV genome fragments were identified by PCR and sequence analysis. Results Genome segments were subclones of hMPV identified by PCR and sequencing. Conclution The recombinant plasmids carrying with hMPV gene segments was obtained successfully. And can be used to later research.%目的 从临床标本中扩增人偏肺病毒(hMPV)基因片段进行亚克隆,为进一步全面深入地研究人偏肺病毒奠定基础.方法 根据GenBank中已知的hMPV全基因序列设计引物.收集呼吸道感染住院患儿鼻咽抽吸物697例,提取病毒核酸,运用长片段RT-PCR技术扩增目的片段,切割含目的条带的凝胶进行纯化,并将目的片段连接到PJET1.2/blunt克隆载体,转化感受态细胞TOP10,经含氨苄青霉素的抗生素平板筛选,得到阳性克隆,提取质粒,最后对重组质粒进行PCR鉴定和测序鉴定.结果 随机选取5例所获的阳性条带经PCR和测序鉴定,证实确为hMPV基因片段.结论 成功构建了hMPV基因组亚克隆,可用于后续的实验研究.
    • 李德鹏; 桑威; 李振宇; 黄一虹; 张奎; 徐开林
    • 摘要: 目的 克隆人肝细胞生长因子(human hepatocyte growth factor,hHGF)的编码基因,构建真核表达载体,获得hHGF蛋白.方法 从含hHGF的人肝脏组织总RNA中,利用RT-PCR方法扩增出hHGF cDNA;利用TA克隆技术,将该基因片段克隆至真核表达载体pcDNA3.1+质粒,转化E.coli DH5α细胞,鉴定pcDNA3.1+-hHGF重组质粒.将重组质粒pcDNA3.1+-hHGF转染293T细胞,应用ELISA方法检测hHGF在293T细胞中的表达.结果 ①测序结果证实本实验克隆的基因与人HGF基因序列一致.②重组质粒pcDNA3.1+-hHGF是具有表达功能的真核表达质粒.③ pcDNA3.1+-hHGF转染293T细胞,于转染后12、24、48、72 h均能检测到hHGF表达.结论 成功构建hHGF真核表达载体,获得分泌性hHGF蛋白.%Objective To clone the gene encoding of human hepatocyte growth factor (hHGF) , construct the eukaryotic expression vector , and harvest hHGF protein. Methods hHGF cDNA was amplified by RT -PCR from total RNA of human liver tissue containing hHGF, the gene fragment was cloned into the eukaryotic expression vector pcDNA 3. 1+ plas— mid by TA cloning technique and then transformed E. Coli DH5ααcells,identification of recombinant plasmid pcDNA3.1 — hHGF. Recombinant plasmid pcDNA3.1 + -hHGF transfected 293T cells. The expression of hHGF in 293T cells was detected by ELISA. Results ①Sequencing results confirmed that the cloned gene was identical to the hHGF gene sequence . ②The recombinant plasmid pcDNA3.1+ —hHGF was an expression of functional eukaryotic expression plasmid . ③The expression of hHGF could be detected , at 12, 24, 48 and 72 hours after pcDNA3.1 + -hHGF transfected 293T cells. Conclusion hHGF eukaryotic expression vector is successfully constructed and the secreted hHGF protein is obtained .
    • 宋志强; 李媛; 孙文静; 刘洋; 邹晓辉; 陈维; 周玉梅; 辛九庆
    • 摘要: 牛支原体(M.bovis)可以引起犊牛肺炎、关节炎、乳腺炎、角膜结膜炎等疾病,是一种牛的重要呼吸道病原体,目前M.bovis粘附宿主细胞的机制还不明确.研究表明,M.bovis表面存在的多种蛋白与致病菌在宿主体内的侵袭力与传播能力有关.我们通过分离表达多个M.bovis基因,最终获得了一个表达纤溶酶原结合蛋白的新基因,并命名为P18.该基因表达大小约为67ku的重组蛋白.通过western blot鉴定,重组表达的P18蛋白可以被M.bovis抗体识别.进一步的试验表明,P18蛋白还具有纤溶酶原结合活性,从而推测该基因表达的蛋白可能是M.bovis的一个粘附相关因子.%Mycoplasma bovis is the causative agent of M. Bovis-associated disease (MbAD). While the mechanisms by which M. Bovis adherent host cells is remain unclear, our recent study provided that M. Bovis express variable surface lipop proteins (Vsps) is facilitated it invasion and dissemination in the infected host and speculated that there are other adhesion-related factors in existence of M. Bovis. We cloned and expressed a novel gene of M. Bovis, designate P18, which express a profibrinolysin binding protein according to the domain sequence analysis binding test. The P18 gene express a 67 ku recombinant protein. The recombinant P18 protein were detected by SDS-PAGE and confirmed by western blot with positive serum. There fore, we conjectured that P18 protein is one of M. Bovis adhesion-related factors.
    • 丁维进; 唐乙; 宋艳玲; 苏志达; 李翠; 刘安堂; 胡霞; 江华
    • 摘要: BACKGROUND: Muscle-derived stem cells (MDSCs), a newly discovered adult stem cell possessing utility potential in tissue engineering and regenerative medicine, have been isolated from skeletal muscle tissue. The MDSCs isolating method varies a lot. OBJECTIVE: To isolate and culture MDSCs and its clone as well as sub-clone through the use of modified preplate technique combined with limited dilution technique. METHODS: The muscle tissue was obtained from new born mice under microscopy and then digested and filtered, from which MDSCs were isolated by modified preplate technique with first round plating period of 1 hour. The muscle derived cells were counted and MDSCs were marked with immunohistochemistry method. The MDSC clone and its subclone were obtained with limited dilution technique. RESULTS AND CONCLUSION: During the isolation procedure with preplate technique, muscle derived cells made up a progressively higher ratio in the cell culture and the procedure with first round plating period of 1 hour provided plenty cells for MDSCs isolation. MDSCs presented with adherent growth 72 hours after the sixth suspension, grew into cell population of 300-500 cells in 10 days about, and proliferated with its small round and spindle morphology persisted. MDSCs clone and sub-clone were obtained through limited dilution technique and found desmin positively expressed and Sca-1 positively expressed at ratio of (92.3±4.1)%. The MDSCs and its clone from mice may provide proper cell resource for tissue engineering and regenerative medicine research.%背景:研究者们通过多种方法从肌组织中分离得到肌源干细胞,并应用于各类组织工程和再生医学研究.目的:结合改良的差速贴壁法和有限稀释技术分离小鼠来源肌源干细胞,并培养其单细胞克隆和亚克隆集落.方法:以新生C57BL/6小鼠四肢作为肌组织取材对象,经三重酶消化和细胞筛过滤,运用改良的差速贴壁法分离出肌源干细胞,予细胞特异标记物以免疫组织化学染色;以有限稀释技术克隆培养的方法,获得稳定的肌源干细胞单克隆和亚克隆集落.结果与结论:差速贴壁培养过程中,肌性细胞占比逐渐增高,首次贴壁1 h可以获得足够数量的细胞进行第6次贴壁培养;肌源干细胞需72 h左右贴壁生长,经10 d左右可以增殖为300~500细胞数量的集落,细胞形态以小圆形细胞为主,并有少量梭形细胞,肌源干细胞能够维持形态并持续增殖;应用有限稀释技术可获得肌源干细胞单克隆和亚克隆集落,肌源干细胞克隆细胞均呈现Desmin染色阳性,Sca-1染色阳性,阳性率为(92.3±4.1)%.提示应用preplate法和有限稀释技术可以分离得到小鼠来源肌源干细胞及其克隆集落.
    • 潘秀华; 王立梅; 李希; 冯玉梅
    • 摘要: 目的 筛选高转移潜能的人乳腺癌细胞亚克隆,为人类乳腺癌转移相关的体内外研究提供实验对象.方法 通过有限稀释法分离和培养人乳腺癌细胞MDAMB-435S的单克隆;采用细胞生物学方法体外评价亚克隆的克隆形成、增殖、运动和侵袭能力;将MDA-MB-435S亚克隆细胞通过乳腺脂肪垫接种于免疫双缺陷SCID鼠,验证体外筛选克隆的体内转移能力.定量资料采用独立样本t检验进行统计学分析.结果 筛选得到MDA-MB-435S的高转移亚克隆14-E5;14-E5与亲代细胞呈梭形、伪足较长的细胞形态明显不同,体积比亲代细胞小,呈多边形,触角增多;细胞的体外克隆形成能力、运动能力、侵袭能力及体内自发转移能力均较亲代显著增强(P0.050).结论 筛选并建立了高转移潜能人乳腺癌细胞亚克隆细胞株14-E5,可以用于乳腺癌转移基因筛选、转移机制研究、抗转移药物的研发和评价抗转移实验性治疗疗效.
    • 景志杰; 郭民; 刘田福
    • 摘要: 目的 构建带绿色荧光蛋白(GFP)的pcDNA6/myc-hisB的真核表达载体. 方法 采用亚克隆的技术扩增得到GFP的开放阅读框序列,Hind Ⅲ和Kpn Ⅰ双酶切后插入pcDNA6/myc-hisB,重组体经质粒PCR和酶切鉴定,并转染HEK 293T细胞后观察绿色荧光的表达,同时经Western blot检测GFP和标签蛋白myc的表达. 结果 PCR扩增得到特异的GFP开放阅读框序列,质粒PCR和酶切鉴定显示pcDNA6/myc-hisB-GFP构建成功,细胞转染结果 表明重组体可以表达GFP和myc蛋白.结论 获得带有绿色荧光蛋白的pcDNA6/myc-hisB-GFP的真核表达载体,为此载体的应用拓展了更大的空间.
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