Construction of eukaryotic vector pcDNA3.1-flag-pygo2 and expression in C6 cell

目的构建pcDNA3.1-flag-pygo2真核表达载体并在C6细胞中进行表达。方法从小鼠脑胶质细胞中提取总RNA,RT-PCR法反转录合成c DNA,设计引物,调取目的片段,与pcDNA3.1-flag载体连接后转化大肠杆菌DH5α,LB平板筛选菌落,提取质粒。重组质粒pcDNA 3.1-flag-pygo2经过酶切鉴定及测序后,阳离子脂质体法转染C6细胞并经免疫细胞化学染色及蛋白印迹检测重组体的表达。结果重构质粒pcDNA 3.1-flag-pygo2经限制性核酸内切酶EcoRⅠ,HindⅢ酶切分析及测序检查,表明真核表达载体构建正确;瞬时转染C6细胞后,免疫细胞荧光染色及蛋白印迹检测表明转染细胞能够表达外源Pygo2基因。结论成功构建了pcDNA3.1-flag-pygo2真核表达载体并能够在真核细胞中进行表达,这为今后研究pygo2基因在胶质瘤中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector pcDNA3.1-flag-pygo2 and express the combined protein in C6 cell line.Methods To extract total RNA from primary glial cell of mouse and to synthesize c DNA by RT-PCR,then design primer and clone whole segment of pygo2 gene.After the targeted gene was inserted into vector pcDNA3.1-flag,the recombined plamid was transformed into E coli DH5α for LB agar plate screening and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing.,the constructed eukaryotic expression plamid was transfected to C6 cell line by lipofectamin method,finally the protein expression was detected by immunocytochemical staining as well as western blot.Results The new constructed vector was confirmed by restricted enzyme Eco R I,HindIII digestion assay and correct Pygo2 was verified by sequenceing.finally,pcDNA3.1-flag-hpygo2 can express exogenous pygo2 gene in glioma C6 cell line after transient transfection by the determination of immunocytofluorescent staining and western blot.Conclusion The new plamid pcDNA3.1-flag-pygo2 was constructed successfully,and can express fused protein in eukaryotic cell,which establish the foundation for future research on pygo2 gene function in human glioma

Over-expression of Pygo2 Promotes C6 Cells Proliferation of Glioma

目的通过构建过表达PygO2的重组体上调PygO2表达,探讨其在大鼠胶质瘤C6细胞增殖中的作用及机制。方法重组体经ECOr I和HIndⅢ双酶切鉴定和dnA测序后,用脂质体2000将其转染大鼠胶质瘤C6细胞,采用WESTErn blOT检测外源PygO2蛋白表达,应用克隆形成实验和MTT法检测细胞增殖,流式细胞术检测细胞周期,采用WESTErn blOT检测过表达PygO2对C6细胞中CyClInd1、β-CATEnIn水平的影响,并采用细胞免疫荧光法检测其对C6细胞中CyClInd1、β-CATEnIn亚细胞定位的影响。结果双酶切和测序鉴定结果证实插入序列正确,重组体能有效上调PygO2表达。将重组体转染C6细胞上调PygO2表达后,细胞的生长增殖被显著促进,克隆形成显著增多,细胞周期进程从g1期至S期转变显著增强;且CyClInd1水平随之增高,亚定位无改变,β-CATEnIn水平和亚细胞定位无明显改变。结论成功构建了过表达PygO2的重组体,过表达PygO2通过增高CyClInd1水平,促进细胞从g1期进入S期,从而促进大鼠胶质瘤C6细胞增殖。Objective To up-regulate expression of Pygopus2(Pygo2) by construction of the recombinant vectors of over-expression of Pygo2 protein,and to explore the role and mechanism of over-expression of Pygo2 in C6 cells proliferation of glioma.Methods The recombinant plasmids were digested with EcoRⅠ and Hind Ⅲ to execute the restriction endonuclease identification,then the sequence analysis was assayed by DNA sequencing.The recombinant plasmids were transfected into cultured glioblastoma C6 cells using lipofectamineTM 2000.The exogenous Pygo2 protein level of C6 cells was detected by Western blot analysis.Colony forming assay and MTT assay were used to detect the cell proliferation,and cell cycle analysis was performed by flow cytometry analysis.The effect of Pygo2 over-expression on the level of cyclinD1 and β-catenin of C6 cells was detected by Western blot analysis,and the expression and subcellular location of cyclinD1 and β-catenin of C6 cells were further quantified by immunofluorescent staining.Results The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis,which up-regulated Pygo2 expression of C6 cells efficiently.After Pygo2 expression were up-regulated by transfected C6 cells with the recombinant plasmids,cells proliferation was promoted and colony forming was increased significantly,cell cycle progression from G1 to S transition was enhanced notably.Furthermore,the expression level of cyclinD1 was significantly increased without change of subcellular location,and the expression level and subcellular location of β-catenin were not changed obviously.Conclusion The recombinant vectors of Pygo2 over-expression were constructed successfully.Over-expression of Pygo2 promotes the growth of glioma cells by an increased expression of cyclinD1 to improve G1/S transition.重庆市自然科学基金资助项目(cstc2011jjA10110);重庆市教委科技基金资助项目(KJ100504);福建省自然科学基金资助项目(2009D002

基于核磁共振氢谱技术研究电针“内关”穴对心肌缺血再灌注损伤大鼠血清及心肌组织代谢物的影响

目的:基于核磁共振氢谱(1 H NMR)研究电针\"内关\"穴对心肌缺血再灌注损伤(MIRI)大鼠血清和心肌组织代谢物的影响,探讨电针\"内关\"穴对MIRI大鼠代谢模式的影响。方法:30只SD雄性大鼠随机分为对照组、模型组、电针组。对照组:不予电针,仅用鼠板束缚大鼠,每次20min,每日1次,持续7d;模型组予心肌缺血再灌注造模:至第7天后结扎冠状动脉左前降支40 min后恢复血流60 min;电针组(频率10Hz/50Hz,脉冲宽度0.5ms,强度1mA):每日电针双侧\"内关\"穴1次,时间20min,持续7d,于第7天电针后造模。造模结束后,收集3组大鼠的血清和心肌组织。利用1 H NMR进行代谢物检测并利用多元统计方法主成分分析(PCA)、正交偏最小二乘分析(OPLS-DA)进行模式识别。结果:与对照组相比,模型组大鼠血清的亮氨酸、异亮氨酸、缬氨酸、3-羟基丁酸、乳酸、丙氨酸、赖氨酸、醋酸盐、N-乙酰糖蛋白、丙酮、乙酰乙酸、琥珀酸、谷氨酰胺、多不饱和脂肪酸、肌酸、甘油磷酸胆碱、甘氨酸均浓度下降,低密度脂蛋白/极低密度脂蛋白、葡萄糖浓度上升;其中除丙酮、乙酰乙酸和多不饱和脂肪酸外,低密度脂蛋白/极低密度脂蛋白在电针后下降到接近对照组水平,其它代谢物浓度均在电针后上升到接近对照组水平,电针组大鼠血清的代谢模式更接近对照组。与对照组相比,模型组大鼠心肌组织的乳酸、丙氨酸、赖氨酸、谷氨酸、谷氨酰胺、天冬氨酸、甘油磷酸胆碱、肌酸、牛磺酸、甘氨酸、苏氨酸、腺苷一磷酸、烟酰胺腺嘌呤二核苷酸浓度下降,葡萄糖浓度明显上升;其中肌酸、甘油磷酸胆碱、烟酰胺腺嘌呤二核苷酸浓度在电针后上升,葡萄糖浓度在电针后下降;此外,电针组大鼠心肌组织苏氨酸、腺苷一磷酸浓度较模型组进一步下降;电针组大鼠心肌的代谢模式虽然有向对照组偏移的趋势,但与对照组差异还是较大。结论:电针\"内关\"穴可调节MIRI大鼠糖代谢、脂肪酸代谢、氨基酸代谢及能量代谢,对心肌具有一定的保护作用,但其具体的代谢通路及机制需要进一步研究。国家自然科学基金面上项目(No.81574080);;湖南省中医药管理局课题(No.201521);;湖南省高校创新平台开放基金项目(No.15k093

一种产油微生物RNA的提取方法

本发明公开了一种从产油微生物中提取RNA的方法。该方法包括以下步骤：产油微生物菌体采用液氮研磨和玻璃珠振荡相结合的方式进行细胞破碎，在细胞破碎液中加入有机溶剂去除油脂，加入酸性苯酚/氯仿/异戊醇除去蛋白，离心取水相加入异丙醇沉淀，最后经乙醇洗涤沉淀，真空干燥，可成功提取产油微生物RNA。本发明操作简单，成本低，完整性好。采用本方法获得的RNA，可以满足产油微生物分子生物学如Northern杂交、mRNA分离、RACE、定量PCR、cDNA合成及体外翻译等方面的研究。待填

Cloning and characterization of the isocitrate dehydrogenase gene from oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids over 65% of its cell dry weight [1]. Mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrare to α-ketoglutarate in eukaryotic cells, an essential and rate-limiting step in citric acid cycle. Yeast S. cerevisiae IDH is composed of an octamer of four IDH1 and four IDH2 subunits [2]. Early biochemical study showed that IDH activity correlates with the lipid storage capacity [3]. In this work we cloned the full length cDNA sequence of the mitochondrial IDH1 and IDH2 gene using methods of degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends from L. starkeyi AS 2.1560, a useful oil-producing yeast. We further determined the expression level of both lsidh1 and lsidh2 by real time PCR during the lipid storage process. Our results showed the encoded products, LsIDH1 and LsIDH2, are similar in size with a molecular weight of 40,277 and 40,708, respectively, and have high similarity with IDH sequences from other yeast species. More importantly, putative mitochondrial transit peptides have been identified at the N-terminus of each sequence, suggesting that both proteins are likely located within the mitochondria. Further gene expression analysis by real time PCR showed that both idh1 and idh2 expression decreased concurrently with the evolution of cellular lipids. Our data may be valuable for further engineering oleaginous yeasts

基于矿石纳米材料的DNA转化的机理初探及方法改进

一种价格便宜、资源丰富和对人体无害的矿石纳米材料——海泡石,用于微生物DNA转化。该法简便、快速、对人体健康无害,但对这种转化方法机制的理解还有很多问题。通过小片段RNA竞争试验,发现了与先前报道不同的转化机制。同时,对该法进行了优化,结果可以实现对冷藏1个月的大肠杆菌EscherichiacoliDH5α单菌落直接转化,无需感受态制备和处理后的温育过程,可得到比钙转高的转化率。由于可优化的参数很多,所以这种转化方法可以提升的空间很大。同时,该方法可用于探索其他用钙转和电转未成功的微生物

基于矿石纳米材料的DNA转化的机理初探及方法改进

一种价格便宜、资源丰富和对人体无害的矿石纳米材料——海泡石,用于微生物DNA转化。该法简便、快速、对人体健康无害,但对这种转化方法机制的理解还有很多问题。通过小片段RNA竞争试验,发现了与先前报道不同的转化机制。同时,对该法进行了优化,结果可以实现对冷藏1个月的大肠杆菌EscherichiacoliDH5α单菌落直接转化,无需感受态制备和处理后的温育过程,可得到比钙转高的转化率。由于可优化的参数很多,所以这种转化方法可以提升的空间很大。同时,该方法可用于探索其他用钙转和电转未成功的微生物

组氨酸标签位置对重组鹰嘴豆孢克鲁维酵母外切菊粉酶活性的影响

为了研究His_6-tag标签位置对鹰嘴豆孢克鲁维酵母CBS4857外切菊粉酶(Kluyveromyces cicerisporus CBS4857 exo-inulinase,KcINU1)酶活性的影响,实验首先根据Swiss-Model构建的KcINU1三维模型预测了组氨酸标签在KcINU1的位置,然后将编码His_6-tag的基因通过PCR方法分别引入到kcINU1的N端和C端,并将重组的kcINU1基因克隆到毕赤酵母表达载体pPICZ&alpha;A,重组质粒进一步经电转化法整合到毕赤酵母宿主菌X-33中,最后用甲醇诱导进行分泌表达,镍柱亲和层析法纯化目的蛋白,DNS去进行外切菊粉酶的活性测定。结...</p

甲烷氧化菌Methylomonas sp．GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶，常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp．GYJ3中溶解性甲烷单加氧酶基因和16S rDNA进行了测序与分析。利用已知相关基因数据库信息，设计了PCR引物和测序引物，获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp，部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较，结果表明MMOX组份中氨基酸序列的同一性为78％到99％，基因序列的同一性为71％到97％，6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明，MMOX序列具有高度保守性，特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp．GYJ3与γ蛋白细菌是相关联的，基于MMOX氨基酸序列的进化分析证明，与Methylomonas sp．GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp．KSWⅢ。综合分析表明，菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp．属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6．28，理论分子量为248874．41Da