大鼠δ阿片受体基因小发卡RNA及人前脑啡肽原基因双表达慢病毒载体的构建与鉴定
SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响
SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响刘东升;崔岩;申仑;杜延平;李桂林;王任直;王岩;张波【摘要】Objective To construct and identify SHH-N gene adeno-associated virus vector and to detect its effect on genes controlling to proliferation in neural stem cells(NSCs). Methods The neural stem cells in the subventricular zone of postnatal rat brain were used to collect SHH-N. pSNAV2. 0-CMV-SHH-N-IRES-EGFP was established by enzyme cutting and ligation , and then transfected packaging cell line 293T. Real time PCR was performed after SHH-N transfection of neural stem cells by rAAV-SHH-N-EGFP vector for 48 hours, SHH-N gene expression of infected cells after 2 week examined by fluorescence microscopy. Results ( 1 ) SHH-N gene was coincident with NCBI report. (2) pSNAV2. 0-CMV-SHH-N-IRES-EGFP expression vector and rAAV-SHH-N-EGFP vector was successful established and packaged. (3)Real time PCR was performed after SHH-N infection for 48 hour, induction of Nmyc and Glil in rAAV-SHH-N-EGFP-treated group was enhanced compared to control group. Conclusion rAAV-SHH-N-EGFP vector has been successfully established and it can stably express in neural stem cells. Forced expression of SHH-N in NSCs resulted in enhanced induction of Nmyc and Glil.%目的构建rAAV-SHH-N-EGFP载体并检测其对神经干细胞增殖相关基因影响.方法分离并培养大鼠脑室下区神经干细胞,提取RNA、反转录、PCR得到SHH-N的cDNA,克隆入pSNAV2.0-CMV-IRES-EGFP,包装得到腺相关病毒rAAV-SHH-N-EGFP,感染神经干细胞48 h后,采用实时定量PCR检测SHH信号通路下游相关基因的mRNA水平变化,并观察rAAV-SHH-N-EGFP载体在神经干细胞内的表达情况.结果 (1)克隆得到SHH-N基因,测序结果与NCBI报道序列一致.(2)成功构建pSNAV2.0-CMV-SHH-N-EGFP表达载体并鉴定,包装得到rAAV-SHH-N-EGFP.(3)实时定量PCR 分析rAAV-SHH-N-EGFP感染神经干细胞48 h后,rAAV-SHH-N-EGFP处理组较感染rAAV-EGFP的对照组,Glil和Nmyc的mRNA水平上调.(4)rAAV-SHH-N-EGFP可有效感染神经干细胞,在感染14 d后稳定表达SHH-N.结论成功构建rAAV-SHH-N-EGFP过表达载体并使其在神经干细胞内稳定表达.过表达SHH-N 可使神经干细胞内SHH信号通路相关基因Glil转录水平上调,并使细胞增殖相关基因Nmyc转录水平上调.【期刊名称】《基础医学与临床》【年(卷),期】2011(031)003【总页数】6页(P291-296)【关键词】神经干细胞;SHH信号通路;Nmyc基因【作者】刘东升;崔岩;申仑;杜延平;李桂林;王任直;王岩;张波【作者单位】大连医科大学附属第一医院,神经外科,辽宁,大连,116011;大连医科大学附属第一医院,神经外科,辽宁,大连,116011;大连医科大学附属第一医院,神经外科,辽宁,大连,116011;大连医科大学附属第一医院,神经外科,辽宁,大连,116011;中国医学科学院北京协和医院,神经外科,北京,100730;中国医学科学院北京协和医院,神经外科,北京,100730;清华大学玉泉医院,神经外科,疼痛病区,北京,100730;大连医科大学附属第一医院,神经外科,辽宁,大连,116011【正文语种】中文【中图分类】R394.3音猬蛋白(sonic hedgehog,SHH)分泌蛋白在神经系统的发育时期中调节神经管腹侧的发育和脑背侧部神经管前体的增殖发育[1]。
某大学生物工程学院《生物化学》考试试卷(225)
某大学生物工程学院《生物化学》课程试卷（含答案）__________学年第___学期考试类型：（闭卷）考试考试时间：90 分钟年级专业_____________学号_____________ 姓名_____________1、判断题（95分，每题5分）1. DNA复制时，前导链按5′→3′合成，滞后链则按3′→5′合成。
（）[厦门大学2015研]答案：错误解析：DNA复制时，以5′→3′走向为模板的一条链合成方向为5′→3′，与复制叉方向一致，称为前导链；另一条以5′→3′走向为模板链的合成链走向与复制叉移动的方向相反，称为滞后链，其合成是不连续的，先形成许多不连续的片段（冈崎片段），最后连成一条完整的DNA链，前导链与滞后链的合成方向都是5′→3′。
2. 细胞水平的调控是一种最原始、最基础的调控机制。
（）答案：错误解析：3. 酮体包括丙酮，β羟丁酸和乙酰乙酸，前两种都衍生于乙酰乙酸，分别由乙酰乙酸脱羧酶和β羟丁酸脱氢酶催化。
（）答案：正确解析：乙酰乙酸、β羟丁酸和丙酮，统称为酮体。
乙酰乙酸在肝线粒体β羟丁酸脱氢酶催化下可还原生成β羟丁酸；由乙酰乙酸脱羧生成丙酮。
4. 用烟草花叶病毒构建植物表达载体时，外源基因可直接插入其基因组中，然后感染植物细胞，并在植物细胞中高水平表达。
（）答案：错误解析：因烟草花叶病毒为单链RNA病毒，应首先将其RNA逆转录成cDNA。
5. 载脂蛋白不仅具有结合和转运脂质的作用，同时还具有调节脂蛋白代谢关键酶活性和参与脂蛋白受体的识别的主要作用。
（）答案：正确解析：6. 琥珀酸脱氢酶是三羧酸循环中唯一掺入线粒体内膜的酶。
（）答案：正确解析：7. DNA重组修复可将DNA损伤部位彻底修复。
（）答案：错误解析：8. 原核细胞中，构成RNA聚合酶的σ因子的浓度低于核心酶的浓度。
（）答案：正确解析：原核细胞RNA聚合酶全酶中的σ因子只参与转录的起始，当起始完成以后即与核心酶解离，并可以重新利用参与新一轮的转录起始，因此它的浓度不需要与核心酶一样。
CASK基因过表达慢病毒载体的构建及鉴定
CASK基因过表达慢病毒载体的构建及鉴定覃觅;孙宇;刘俊;王延宁;赵文;周华富【摘要】Objective:To construct a lentiviral vector over-expressing calcium/calmodulin-dependent serine protein kinase (CASK).Methods:The whole length CASK gene fragment was amplified by polymerase chain reaction (PCR) and inserted into linearized GV358 lentiviral vectors using ligase.Positive clones of CASK-GV358 vectors were identified by PCR.The lentiviral vectors containing CASK gene were co-transfected into 293T cells with packaging plasmids.The virus titer was determined by ELISA.The recombinant lentivirus over-expressing CASK (LV-CASK) and negative control recombinant lentivirus CON238 were used to infect human NSCLC H1299 cells,respectively.Fluorescence quantitative PCR (FQ-PCR) and western blotting were used to detect the expression of CASK.Results:CASK gene was amplified and successfully inserted into the GV358 lentivirus vector.The sequences of the recombinant plasmid were confirmed by PCR and DNA sequencing.The expression of GFP could be detected in 293T cells infected with recombinant lentiviruses.The lentiviruses over-expressing CASK were harvested and concentrated to 2 × 108TU/mL.According to the results of FQ-PCR and western blotting,the mRNA and protein expression level of CASK significantly upregulated in H1299 cell infected with LV-CASK,compared to cells infected with CON238 (P ＜0.05).Conclusion:The lentivriral vector over-expressing CASK gene was constructed successfully and stably expressed CASK in H1299 cells.%目的:构建人钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)基因过表达慢病毒载体并进行鉴定.方法:采用聚合酶链反应(PCR)扩增CASK基因片段,通过连接酶将CASK基因片段连接至线性化的GV358载体上,运用PCR方法鉴定阳性克隆的CASK GV358载体;将重组质粒和包装质粒共转染至293T细胞,并用酶联免疫吸附试验(ELISA)法进行滴度检测.将成功包装的CASK过表达慢病毒LV-CASK(OE组)和阴性对照病毒CON238(NC组)分别感染人非小细胞肺癌(NSCLC)H1299细胞,同时设空白对照组(MOCK组),用荧光定量PCR(FQ-PCR)和western blotting法检测CASK表达水平.结果:通过PCR技术成功地扩增CASK基因片段并连接到GV358病毒载体上,PCR及DNA测序鉴定结果证明CASK-GV358质粒构建正确;重组慢病毒转染293T细胞后可观察到绿色荧光及蛋白表达,包装过表达慢病毒并测定其浓缩滴度为2×108TU/mL.FQ-PCR和western blotting检测结果显示,与NC组相比,OE组CASK mRNA和蛋白的表达均显著上调(P＜0.05).结论:成功构建CASK基因过表达慢病毒载体,可在H1299细胞中稳定表达,为CASK基因的后期研究提供了实验基础.【期刊名称】《广西医科大学学报》【年(卷),期】2017(034)005【总页数】4页(P684-687)【关键词】CASK;慢病毒;载体构建;过表达【作者】覃觅;孙宇;刘俊;王延宁;赵文;周华富【作者单位】广西医科大学第一附属医院,南宁530021;广西医科大学第二附属医院,南宁530007;广西医科大学第一附属医院,南宁530021;广西医科大学第一附属医院,南宁530021;广西梧州市人民医院,梧州 543000;广西医科大学第一附属医院,南宁530021【正文语种】中文【中图分类】R349.8人钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase，CASK)属于膜相关鸟苷酸激酶(membrane associated guanylate kinase，MAGUK)家族，广泛分布于多种组织和细胞，参与蛋白质的分子组织、细胞连接组装、转录调节、发育调节等重要的细胞生理功能。
某大学生物工程学院《生物化学》考试试卷(3639)
某大学生物工程学院《生物化学》课程试卷（含答案）__________学年第___学期考试类型：（闭卷）考试考试时间：90 分钟年级专业_____________学号_____________ 姓名_____________1、判断题（100分，每题5分）1. 非循环式光合磷酸化既可产生ATP，也可产生O2和NADPH。
（）答案：正确解析：非循环式光合磷酸化电子沿Z形传递，直至NADP＋产生NADPH，电子传递过程中产生的H＋浓度差推动ATP的生成，同时分解H2O分子产生O2。
2. 所有核酸的复制都是在碱基互补配对的原则下进行。
（）答案：正确解析：3. 人体内若缺乏维生素B6和维生素PP，均会引起氨基酸代谢障碍。
（）解析：4. 原核生物和真核生物的染色体均为DNA与组蛋白的复合体。
（）[四川大学2015研]答案：错误解析：真核生物的染色体为DNA与组蛋白的复合体，而原核生物的染色体为DNA与碱性精胺、亚精胺结合。
5. 真核细胞的基因转录也具有抗终止作用。
（）答案：正确解析：抗终止作用即是转录水平上的通读，某些真核细胞基因的转录可在特定的条件下可以发生这种现象。
6. 2,4二硝基苯酚（DNP）可解除寡霉素对电子传递的抑制。
（）答案：正确解析：2,4二硝基苯酚（DNP）是一种解偶联剂，它可使呼吸链上的电子流动与氧化磷酸化失去偶联关系。
本来寡霉素是通过抑制氧化磷酸化而间接抑制电子流动的。
当氧化磷酸化与呼吸链不再偶联的时候，寡霉素将丧失对电子流动的抑制作用。
7. 放线菌素D既能抑制DNA的复制，又能抑制转录。
（）解析：8. 氨基酸活化时，在氨酰tRNA合成酶的催化下，由ATP供能，消耗一个高能磷酸键。
（）答案：错误解析：9. 嘧啶环和嘌呤环在分解代谢中均被水解开环，且降解产物均易溶于水。
（）答案：错误解析：嘧啶环分解过程中开环，降解产物易溶于水。
但嘌呤环不同。
10. 原核生物中的三种终止与释放因子分别识别三种终止密码子。
1.Beclin1
J OURNAL OF V IROLOGY, 0022-538X/98/$04.00ϩ0Nov.1998,p.8586–8596Vol.72,No.11Copyright©1998,American Society for Microbiology.All Rights Reserved.Protection against Fatal Sindbis Virus Encephalitis by Beclin,a Novel Bcl-2-Interacting ProteinXIAO HUAN LIANG,1LINDA K.KLEEMAN,1HUI HUI JIANG,GERALD GORDON,2JAMES E.GOLDMAN,3GAIL BERRY,2BRIAN HERMAN,2†AND BETH LEVINE1*Departments of Medicine1and Pathology,3Columbia University College of Physicians andSurgeons,New York,New York10032,and Department of Cell Biology andAnatomy,University of North Carolina at Chapel Hill,Chapel Hill,North Carolina275992Received5May1998/Accepted6July1998bcl-2,the prototypic cellular antiapoptotic gene,decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains,resulting in protection against lethal encephalitis.To investigate potential mechanisms by which Bcl-2protects against central nervous system Sindbis virus infection,we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library.We iden-tified a novel60-kDa coiled-coil protein,Beclin,which we confirmed interacts with Bcl-2in mammalian cells, usingfluorescence resonance energy transfer microscopy.To examine the role of Beclin in Sindbis virus pathogenesis,we constructed recombinant Sindbis virus chimeras that express full-length human Beclin(SIN/ beclin),Beclin lacking the putative Bcl-2-binding domain(SIN/beclin⌬Bcl-2BD),or Beclin containing a pre-mature stop codon near the5terminus(SIN/beclin stop).The survival of mice infected with SIN/beclin was significantly higher(71%)than the survival of mice infected with SIN/beclin⌬Bcl-2BD(9%)or SIN/beclin stop (7%)(P<0.001).The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells,fewer apoptotic cells,and lower viral titers than the brains of mice infected with SIN/beclin⌬Bcl-2BD or SIN/ beclin stop.Thesefindings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may playa role in antiviral host defense.The cellular antiapoptotic gene bcl-2represents a novel class of antiviral host defense molecules which function both by restricting viral replication and by preventing virus-induced cell death.Bcl-2blocks apoptosis in vitro induced by several different RNA viruses,including Sindbis virus(18,41),influ-enza virus(13,26),reovirus(31),Semliki Forest virus(34), LaCrosse virus(29),and Japanese B encephalitis virus(21). Previously,we have shown that Bcl-2overexpression in virally infected neurons in vivo also protects mice against fatal en-cephalitis caused by the prototypic alphavirus,Sindbis virus (17).The protective effects of Bcl-2against fatal Sindbis virus encephalitis were associated with a reduction both in neuronal apoptotic death and in central nervous system(CNS)viral replication.A similar antiviral effect of Bcl-2overexpression has been observed during Sindbis virus infection in cultured AT3cells(41)as well as during influenza virus infection of MDCK cells(26),Japanese B encephalitis virus infection of N18cells(21),and Semliki Forest virus infection of AT3cells (34).Although the role of endogenous Bcl-2in antiviral de-fense has yet to be evaluated,these studies support the hy-pothesis that Bcl-2may be important in protecting cells against viral infections.Most previous studies examining the effects of Bcl-2on viral infections have been with neurotropic RNA viruses(e.g.,Sind-bis virus,reovirus,Semliki Forest virus,LaCrosse virus,and Japanese B virus)(18,21,29,31,34,41).Although Bcl-2may affect viral replication and virus-induced apoptosis with non-neurotropic viruses(e.g.,influenza virus)(13,26),host mech-anisms to inhibit apoptosis may be of particular importance during viral infections of vital,nonrenewable cell populations such as neurons.In such instances,virus-induced apoptotic death of neurons may result in irreversible CNS pathology and death of the host organism(1,17,19,25).Therefore,while apoptosis in other cell types may be an adaptive host defense strategy that reduces total viral burden for the organism(re-viewed in references11,16,36,and39),unique strategies may have evolved to permit control of CNS viral replication without inducing apoptotic death of infected neurons.It is thus possi-ble that cellular genes that play a role in preventing apoptosis during normal neuronal development(e.g.,bcl-2and bcl-x L) (reviewed in reference23)may also be important in regulating CNS viral replication and in defending against apoptosis in-duced by neurotropic viruses.To further understand how the cellular gene bcl-2exerts antiapoptotic and antiviral effects during CNS viral infection, we performed a yeast two-hybrid screen to identify Bcl-2-in-teracting gene products in adult mouse brain.In this study,we describe the identification of a novel Bcl-2-interacting gene product,which we named Beclin because of its predicted coiled-coil structure(hence,the“-in”suffix)and its interaction with Bcl-2(Becl).Like Bcl-2overexpression,Beclin overex-pression in neurons in vivo can inhibit Sindbis virus replication, reduce CNS apoptosis,and provide protection against fatal Sindbis virus infection.A Beclin construct lacking the putative Bcl-2-binding domain provides no protection and has no anti-viral activity.Thus,ourfindings identify a novel protein,Bec-lin,which may play a role in host defense against Sindbis virus infection.In addition,they suggest that interactions with Bcl-*Corresponding author.Mailing address:Department of Medicine, Columbia University College of Physicians and Surgeons,630W.168th St.P&S8-444,New York,NY10032.Phone:(212)305-7312.Fax: (212)305-7290.E-mail:Levine@.†Present address:Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio,San An-tonio,TX78284.85862-like proteins may be important for the protective activity of Beclin.MATERIALS AND METHODSPlasmids.To construct vectors for transient expression in mammalian cells, the open reading frame of human bcl-2,flag epitope-tagged human beclin,and flag epitope-tagged beclin deleted of nucleotides238to453were cloned into pSG5(Stratagene).To construct viral cDNA clones,the previously described neurovirulent double subgenomic Sindbis virus vector dsTE12was used(17). Full-lengthflag epitope-tagged human beclin,human beclin containing a stop codon inserted at nucleotide position270,human beclin containing an in-frame deletion of nucleotides238to453,human bcl-2,and human bcl-2containing a stop codon at nucleotide position118were cloned into the Bst EII site of dsTE12 to generate plasmids SIN/beclin,SIN/beclin stop,SIN/beclin⌬bcl-2BD,SIN/bcl-2, and SIN/bcl-2stop,respectively.To construct vectors for yeast two-hybrid studies, the sequences encoding amino acids(aa)1to218of human bcl-2,1to212of bcl-x L,1to149of bcl-x S,and1to171of bax were cloned into pGBT9in frame with the GAL4DNA-binding domain.To avoid difficulties with targeting of proteins to the nucleus,the sequences encoding C-terminal transmembrane domains of bcl-2family members were omitted.Control pGBT9plasmids con-taining lamin(pLAM5Ј)and p53(pVA3)inserts were obtained from Clontech. Yeast two-hybrid screen.Saccharomyces cerevisiae SFY526cells were cotrans-formed with pGBT9/bcl-2and106cDNA molecules from an adult mouse brain library fused to a GAL4activation domain vector(pGAD10;Clontech),plated onto SD medium lacking tryptophan and leucine,incubated at30°C for4days, and then screened for LacZ activity by a colony liftfilter assay.Putative inter-acting clones were isolated by manipulation in leuB Escherichia coli,and further tested against pGBT9and control plasmids.A positive-galactosidase reaction between pGBT9/bcl-2and clone F1was obtained within10to15min.For analysis of interactions between Beclin and Bcl-2family members,pGBT9plas-mids containing bcl-2family members were cotransformed with fragments of human beclin(1to450,262to450,and1to708)fused to the GAL4activation domain in pGAD424,and transformants were screened for LacZ activity. Sequencing and analysis of human beclin.The partial nucleotide sequence of mouse beclin obtained from sequencing clone F1was aligned with an overlapping clone GT197isolated from human breast(32).Primers immediately upstream and downstream of the predicted open reading frame were used to amplify the coding sequence of human beclin from a normalized human infant brain cDNA library(37).The resulting PCR products from several independent reactions were cloned into pCRII and sequenced in both directions,using Sequenase(U.S. Biochemical)as well as automated sequencing.The resulting nucleotide se-quence and deduced amino acid sequences were used to scan various data banks (GenBank,EMBL,SwissProt,and PIR)for homologous sequences,using the BLAST algorithms(2).The amino acid sequence was also analyzed by the PROSITE program to identify functional motifs and by the COILS program to identify coiled-coil regions(22).Northern blot analysis.Human and mouse multiple tissue blots(Clontech) were hybridized with32P randomly labeled human or mouse beclin probes(nu-cleotides1to485)as instructed by the manufacturer(Clontech).Equal loading [2g of poly(A)RNA]was confirmed by hybridization to a-actin probe. Production of recombinant viruses.The viruses SIN/beclin,SIN/beclin stop, SIN/beclin⌬Bcl-2BD,SIN/bcl-2,and SIN/bcl-2stop were generated from viral cDNA clones as previously described(17).Briefly,5Ј-capped transcripts were synthesized from cDNA clones linearized with Pvu I(for beclin-containing vi-ruses)or Xho I(for bcl-2-containing viruses),transcribed in vitro with SP6DNA-dependent RNA polymerase,and transfected into BHK cells by using Lipofectin according to the manufacturer’s instructions.Twenty-four hours after transfec-tion,virus particle-containing supernatants of transfected cell monolayers were collected,frozen in aliquots at70°C,and used for all subsequent experiments. Titers of stock viruses were determined by plaque assay titration on BHK-21 cells.Animal experiments.Ten-day-old litters of CD1mice were inoculated intra-cerebrally into the right cerebral hemisphere with1,000PFU of each recombi-nant virus in0.03ml of Hanks’balanced salt solution.For mortality experiments, three to six separate litters were inoculated with each virus,and mortality was determined by daily observation of the mice for3weeks after infection.For virus titration and histopathology experiments,three to six mice per experimental group were sacrificed at days1,2,and4after inoculation.The right cerebral hemisphere was dissected and stored atϪ70°C,and freeze-thawed tissues were used to prepare10%homogenates in Hanks’balanced salt solution for plaque assay titration.The left cerebral hemisphere wasfixed by immersion in3% paraformaldehyde.Histopathology.Paraformaldehyde-fixed mouse brains were embedded in par-affin,and a series of4-m parasagittal sections were cut at the level of the olfactory bulb,extending caudally from the bulb to the cerebellum and medulla. For each brain,sequential sections were stained by hematoxylin and eosin to detect histopathology,in situ end labeling(ISEL)to detect apoptotic nuclei,in situ hybridization to detect Sindbis virus RNA,and immunoperoxidase to detect flag-Beclin protein expression.ISEL and in situ hybridization were performed by methods identical to those described previously for SIN/bcl-2-infected mouse brains(17).Immunoperoxidase staining to detectflag-Beclin protein expression in SIN/beclin-,SIN/beclin⌬Bcl-2BD-,and SIN/beclin stop-infected mouse brains was performed by using the monoclonal anti-flag antibody M2(5g/ml;VWR) and the avidin-biotin peroxidase method(Vectastain ABC kit;Vector Labora-tories)according to the manufacturer’s instructions.The number of virus RNA-positive and ISEL-positive cells in each brain section was quantitated with Image-ProPlus software.To calculate the number of positive cells per brain section,nonoverlapping0.25-mm2microscopicfields spanning the entire brain section were scanned with a10ϫobjective and constant settings for brightness,contrast,and threshold values for positive events.The number of positive events(i.e.,RNA-positive cells or ISEL-positive cells)for each brain section was determined by adding the sum of all individualfields analyzed.The total number of positive events per brain section was divided by the total area of the brain section to yield the average number of RNA-positive or ISEL-positive cells per square millimeter of brain.Sections of hippocampus and anterior cortex from an adult human were stained with843,a polyclonal antibody against a human Beclin peptide corre-sponding to aa1to15(1:200dilution;Eurogenetics,Seraing,Belgium),and human Beclin was detected by the avidin-biotin peroxidase method.Plasmid transfections.Plasmids pSG5/bcl-2and pSG5/flag-beclin or pSG5/ bcl-2and pSG5/flag-beclin⌬Bcl-2BD(1g of each)were transiently transfected into COS7cells by using Superfect(Qiagen)according to the manufacturer’s instructions.Protein detection.For immunofluorescence studies,COS7cells werefixed24h after transfection with100%ethanol.Expression offlag-Beclin andflag-mutant Beclin⌬Bcl-2BD mutant constructs was detected with an anti-flag epitope anti-body(M2;1:20;VWR)andfluorescein isothiocyanate(FITC)-conjugated horse anti-mouse immunoglobulin G.Bcl-2expression was detected with a polyclonal rabbit anti-Bcl-2antibody(1:100;Pharmingen)and rhodamine-conjugated goat anti-rabbit antibody.SERCA(endoplasmic reticulum Ca2ϩATPase)was de-tected with an anti-SERCA antibody(1:500;Research Design,Inc.).Western blot analysis to detectflag-Beclin expression in BHK cells infected with SIN/ beclin,SIN/beclin stop,and SIN/beclin⌬Bcl-2BD was performed with either an-tibody M2(20g/ml)or anti-human Beclin peptide antibody843(1:200)and enhanced chemiluminescence detection as instructed by the manufacturer(Am-ersham).FRET.Fluorescence resonance energy transfer(FRET)microscopy was per-formed as previously described on COS7cells cotransfected with bcl-2and beclin expression vectors(9).The donor(FITC)filter set had the following parameters: excitation(ex)ϭ450to490nm;dichroic mirror(dm)ϭ510nm;emission(em)ϭ590long pass.The acceptor(rhodamine)filter set had the following param-eters:exϭ546nm;dmϭ580nm,emϭ590long pass.Images obtained with these twofilter sets were used to directly quantify the intensities of eachfluoro-phore.The signal recorded from the FRETfilter set(exϭ450to490nm;dmϭ580nm;emϭ590nm long pass)is from energy that has transferred from FITC to rhodamine molecules.A mapping program described previously(9)was used to mapfluorescent cells and to quantify the intensity within each cell.Quanti-tative analysis of these mapped images required solving three equations,one for eachfilter set,which accounted for the excitation and detection of both labels in all threefilter sets as well as the concentrations of the donor and acceptor molecules and the probability of transfer.The measured quantities are expressed as follows,in which thefirst letter(uppercase)indicates thefilter set(A,accep-tor;F,FRET;D,donor)and the second letter(lowercase)indicates the labels present(a,acceptor alone;f,acceptor and donor;d,donor alone).A solution of the equation is Eϭ1/[aconc(RKϩ1)],where aconcϭ(AdFfϪFdAf)/[(AdFa/ Aa)ϪFd];Rϭ(DaFf/FaϪDf)/[aconc((Fa/Aa)ϪFdDa/DdAa)ϪFϭFdDf/ Dd];and K is proportional to the product of the ratio of the quantum yield of the two labels and the ratio of the absolute detection efficiencies of the two labels. Nucleotide sequence accession numbers.The GenBank accession numbers for human and mouse beclin are AF077301and AF077302,respectively.RESULTSIdentification of beclin,a novel gene on chromosome17q21. Previously,we demonstrated that Bcl-2inhibits Sindbis virus replication and prevents Sindbis virus-induced apoptosis in mouse neurons(17).To further understand the mechanisms by which Bcl-2protects against Sindbis virus infection in neurons, we performed a yeast two-hybrid screen of an adult mouse brain library for complementary DNAs encoding proteins that bind to the cell death inhibitor Bcl-2.We constructed a bait plasmid(pGBT9/bcl-2)by fusing human bcl-2(lacking the C-terminal signal-anchor sequence to ensure translocation to the nucleus)to the GAL4DNA-binding domain,which was co-transformed with an oligo(dT)and random hexamer-primed adult mouse brain cDNA fusion library in a GAL4activation domain vector,pGAD10.Of1million transformants,one positive colony was identified by the5-bromo-4-chloro-3-in-dolyl--D-galactopyranoside(X-Gal)filter assay.SequencingV OL.72,1998PROTECTION AGAINST SINDBIS VIRUS INFECTION BY BECLIN8587analysis of the cDNA plasmid rescued from this colony(F1) revealed a termination codon42bp downstream from the GAL4activation domain,several predicted short open reading frames between nucleotides124and1843and a longer pre-dicted open reading frame(with a good Kozak consensus se-quence and multiple stop codons upstream)spanning from nucleotide1855to the3Јend of the insert.Thus,either the 14-aa fusion protein was interacting with Bcl-2or one of the downstream open reading frames encoded a protein that con-tains its own activation domain and interacts with Bcl-2.To identify the Bcl-2-interacting region of F1,we fused nucleo-tides1to1854and1855to2500to the GAL4activation do-main in pGAD424and tested for interactions with Bcl-2.Nu-cleotides1855to2500,but not1to1854,encoded a protein that interacts with Bcl-2fused to the GAL4DNA-binding do-main(Table1)but not with control GAL4DNA-binding do-main plasmids containing p53,lamin(Table1),or Sindbis virus glycoproteins(data not shown).A database search revealed that the nucleotide sequence of F1(1855to2500)overlapped with sequences of several clones isolated from a normalized infant human brain cDNA library in the Merck EST(epitope tag sequence)database as well as clones from human breast(GT197)(32)and humanfibroblast (B32)cells(8).These clones contain only partial open reading frames of a novel gene that encodes a protein with coiled coils. As explained above,we assigned the name beclin to this gene because of the interaction of its encoded protein with Bcl-2 and the predicted coiled-coil structure of its encoded protein. Clones GT197and B32were both isolated in the generation of transcription maps of the breast cancer susceptibility locus on chromosome17q21and are mapped to a region located ap-proximately100kb centromeric to the gene BRCA1.They lie within a400-kb minimal deletion unit mapped by Tangir et al. in sporadic epithelial ovarian cancers(38).We aligned the overlapping partial clones in GenBank with our mouse beclin sequence to obtain a predicted sequence of the full-length open reading frame of human beclin and iso-lated human beclin from a normalized human infant brain cDNA library(37).Human beclin is predicted to encode a novel450-aa,60-kDa protein containing a coiled-coil region with25to28%homology with myosin-like proteins(Fig.1).It shares93%identity at the nucleotide level and98%identity at the amino acid level with the mouse beclin sequence identified in the yeast two-hybrid screen.Human Beclin is also homolo-gous with the Caenorhabditis elegans T19E7.3gene product (GenBank accession no.U42843)and the S.cerevisiae gene product Lph7p(GenBank accession no.U43503)(38and37% identical over145and137residues,respectively),indicating a high degree of evolutionary conservation.PROSITE analysis of human Beclin identified several potential glycosylation, phosphorylation,and myristoylation sites but no other func-tional sequence motifs.Ubiquitous expression of beclin mRNA in mouse and human tissues.To examine the tissue-specific pattern of beclin expres-sion,we hybridized mouse and human multiple-tissue North-ern blots with a beclin-specific probe.RNA blot analysis re-vealed that expression of beclin mRNA is widespread in both mouse and human adult tissue(Fig.2A).A beclin-specific probe hybridized to a2.2-kb transcript present at highest levels in human skeletal muscle but at detectable levels in all tissues examined.The size of this transcript is approximately the same as that observed previously for clones GT197and B32(8,32). In some tissues,additional1.7-and1.4-kb transcripts were observed,suggesting the presence of alternatively spliced tran-scripts.Beclin is expressed in human neurons.To examine whether Beclin protein is expressed in neurons(the primary CNS target cell type for Sindbis virus infection),we performed immuno-peroxidase staining of adult human brain sections.We used rabbit immune serum843,generated against a human Beclin peptide corresponding to aa1to15.843reacts with a61-kDa protein in lysates prepared from BHK cells after infection with a recombinant Sindbis virus containing aflag epitope-tagged human beclin insert(SIN/beclin)(Fig.3B).This protein mi-grates identically to the major band detected with an anti-flag epitope antibody in SIN/beclin-infected BHK cell lysates(Fig. 3A)and to in vitro translatedflag-Beclin(data not shown). Immunoperoxidase staining of human brain sections from the hippocampus and frontal cortex revealed Beclin immunoreac-tivity in many neurons throughout these regions as well as in some glial cells.In neurons,Beclin demonstrated a granular, punctate pattern of staining that was found almost exclusively in the region of the perikaryon(Fig.2B).Beclinimmunoreac-FIG.1.Deduced amino acid sequence of human Beclin.The boxed area represents the Bcl-2-binding domain of human Beclin(Table1),and the under-lined area corresponds to the region that is predicted to have a coiled-coil conformation.TABLE1.Summary of yeast two-hybrid assay resultsGAL4activation domain plasmid b-Galactosidase reaction with GAL4binding domain construct aEmpty Bcl-2Bcl-x L Bcl-x S Bax Lamin p53EmptyϪϪϪϩϪϪϪF11–2563ϪϩϩNDϪϪϪ1–1855ϪϪϪNDϪϪϪ1856–2563(MusBeclin1–708)ϪϩϩNDϪϪϪHuman Beclin1–708ϪϩϩNDϪϪϪBeclin1–450ϪϩϩNDϪϪϪBeclin1–258ϪϪϪNDϪϪϪBeclin262–450ϪϩϩNDϪϪϪBeclin451–708ϪϪϪNDϪϪϪBeclin1–1353ϪϪϪNDϪϪϪaϩ,positive reaction within15min;Ϫ,lack of positive reaction at24h;ND,not determined.b Nucleotide positions of genes fused to the plasmid.8588LIANG ET AL.J.V IROL.tivity was also observed in the media of blood vessels,in the ependymal cells,and in the choroid plexus.No staining was observed in human brains stained with rabbit preimmune se-rum 843(data not shown).Interaction of Beclin and Bcl-2.We performed additional yeast two-hybrid studies to confirm that human beclin ,like mouse beclin ,encodes a protein that interacts with human Bcl-2and to further define the Bcl-2-interacting region of human Beclin (Table 1).We found that the region of human Beclin that corresponds to the mouse gene product isolated in the yeast two-hybrid screen (aa 1to 236)also interacts with Bcl-2.Further deletion mutation analysis revealed that aa 88to 150of Beclin were sufficient to mediate an interaction with Bcl-2.Interestingly,the coding sequence for this region of Beclin is deleted in some human infant brain cDNA clones in the Merck EST database,suggesting that Beclin exists in at least two forms,including one that contains a Bcl-2-binding domain and one that lacks this domain.Full length-Beclin does not interact with Bcl-2in the yeast two-hybrid system.As noted below,when expressed in transient transfection assays,flag-tagged full-length human Beclin displays a punctate immuno-reactivity pattern suggestive of association with intracellular organelles and is associated with the insoluble fraction after cell lysis.In contrast,a flag-tagged truncated Beclin (aa 1to 236)(corresponding to the region isolated in the yeast two-hybrid screen)displays a diffuse cytoplasmic staining pattern and is soluble after cell lysis.These differences between full-length and truncated Beclin are thought to account for differ-ences in ability to translocate to yeast nuclei and interact with Bcl-2in the yeast two-hybrid assay.To directly examine whether full-length human Beclin and Bcl-2interact in mammalian cells,we performed FRET studies of COS7cells cotransfected with Bcl-2and flag epitope-tagged Beclin.Beclin is predicted to be a coiled-coil protein that may be associated with the cytoskeleton,and it partitions with the insoluble fraction following cell lysis.For this technical reason,biochemical analyses of in vivo interactions between Bcl-2and Beclin cannot be performed.FRET is a fluorescence technique that can be used as a spectroscopic ruler to study and quantify the interactions of cellular components with each other (re-viewed in references 6,12,and 35).In FRET,a fluorophore (donor)in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor)nonradiatively through dipole-dipole interactions.The efficiency of this process varies as the inverse of the sixth power of the distance separating the donor and acceptor chromophores and,in practice,requires the distance between the donor and acceptor fluorophores to be short (usually less than 50Å).The dependence of the energy transfer efficiency on the donor-acceptor separation provides the basis for the utility of this phenomenon in the study of cell component interactions.FRET has been used by a number of investigators to examine interactions ofcellularconstituents (reviewed in 6,12,and 35)such as endosomal fusion events,ligand-dependent growth factor receptor aggre-gations,interactions of viral and cellular proteins with regula-tors of apoptosis (20),and interactions of cellular cytoskeletal components (33).Prior to measuring FRET,we first confirmed the colocaliza-tion of full-length Bcl-2and Beclin in transfected COS7cells,using confocal laser microscopy (Fig.4).Bcl-2is known to associate with the outer mitochondrial membrane,endoplas-mic reticulum,and perinuclear membranes,and it displays a punctate pattern of cytoplasmic immunoreactivity (reviewed in reference 28).We found that flag-Beclin invariably displayed a pattern of immunoreactivity identical to that of Bcl-2in all cotransfected COS7cells examined by confocal laser micro-scopic analysis (Fig.4A to C).Furthermore,we found that deletion of the putative Bcl-2-binding domain from Beclin did not alter its pattern of immunoreactivity;flag-Beclin ⌬Bcl-2BD appeared to have a pattern of staining similar to that of full-length Beclin,and like full-length Beclin,it colocalized with Bcl-2in cotransfected cells (Fig.4D to F).This granular pat-tern of Beclin immunoreactivity in the perinuclear region is similar to that observed for endogenous Beclin in human neu-rons (compare Fig.2B,4A,and 4D).After confirming the colocalization of Bcl-2and Beclin and of Bcl-2and Beclin ⌬Bcl-2BD,we used FRET analysis to de-termine whether Bcl-2and Beclin physically interact.We com-pared in transfected COS7cells the amounts of FRET between Bcl-2and full-length Beclin,Bcl-2and Beclin ⌬Bcl-2BD,and Bcl-2and a control protein,SERCA.Quantitative analysis of microscopic images (following corrections for cross-talk be-tween filter sets and donor and acceptor concentrations)showed significantly more FRET in cells with labeled full-length Beclin and Bcl-2(E Beclin-Bcl-2ϭ0.000578Ϯ0.000262;mean Ϯstan-dard error of the mean [SEM],n ϭ410)than in cells with labeled Beclin ⌬Bcl-2BD and Bcl-2(E Beclin ⌬Bcl-2BD-Bcl-2ϭ0.000189Ϯ0.000151;n ϭ2,946)(P ϭ0.0068,t test)or in cells with labeled SERCA and Bcl-2(E SERCA-Bcl-2ϭ0.0000639Ϯ0.0000390;n ϭ775)(P ϭ0.0021,t test).These quantitative analyses indicate that Beclin and Bcl-2exhibit FRET and pro-vide evidence of an interaction between these two proteins in mammalian cells.Furthermore,deletion of the Bcl-2-binding domain of Beclin mapped in yeast two-hybrid studies does not alter the spatial orientation of transfected Beclin,but it does significantly decrease FRET.This observation suggests that the FRET observed between full-length Beclin and Bcl-2re-flects a specific association of these proteins in vivo,rather than an artifact secondary to overexpression.Selective interaction of Beclin with death repressor mem-bers of the Bcl-2family.To investigate whether Beclin inter-acts with other Bcl-2family members that positively or nega-tively regulate apoptosis,we fused bax ,bcl-x S ,and bcl-x L into the GAL4binding domain vector and tested for interactions with Beclin in the yeast two-hybrid system (Table 1).The Bcl-x S GAL4binding domain construct activated transcription by itself and therefore could not be tested for interactions with Beclin.The same region of Beclin (aa 88to 150)that inter-acted with Bcl-2also interacted with Bcl-x L ,a related Bcl-2family member that inhibits apoptosis (4).In contrast,Beclin did not react with Bax,a family member that promotes apo-ptosis (27).This pattern of interaction,i.e.,with Bcl-2and Bcl-x L ,but not Bax,is identical to that observed for all previ-ously identified Bcl-2-interacting proteins outside the Bcl-2family (reviewed in reference 30).Mutations in Bcl-2and Bcl-x L that block death repressor activity also block binding to Beclin.Cheng et al.have shown that Bcl-2and Bcl-x L overexpression can delay Sindbis virus-induced death of BHK cells (5).To evaluate whether Bcl-2–Beclin and Bcl-x L –Beclin interactions may be related to this ability of Bcl-2and Bcl-x L to inhibit Sindbis virus-induced apoptosis,we constructed pGBT9vectors containing bcl-2and bcl-x L constructs with mutations in the conserved BH1domain that are known to block death repressor activity.A Gly-Ala mutation at amino acid position 145of Bcl-2completely abro-gates Bcl-2death repressor activity in interleukin-3depriva-tion-,␥-irradiation-and glucocorticoid-induced apoptosis (42)and also blocks Bcl-2binding to Beclin in the yeast two-hybrid system (Table 2).Similarly,substitution of aa 136to 138of Bcl-x L (VNX 3AIL)completely abolishes death repressor ac-tivity in Sindbis virus-induced apoptosis (5)and also blocks Bcl-x L binding to Beclin (Table 2).These mutations inBcl-2FIG.3.Expression of flag-Beclin protein constructs by the virus vectors SIN/beclin ,SIN/beclin stop,and SIN/beclin ⌬Bcl-2BD.(A and B)Western blot analyses of virus-infected BHK cell lysates with either anti-flag epitope antibody M2(A)or polyclonal rabbit anti-Beclin antiserum (B).Lane 1,SIN/beclin ;lane 2,SIN/beclin stop;lane 3,SIN/beclin ⌬Bcl-2BD;lane 4,empty Sindbis virus vector.(C to E)Immunoperoxidase staining using anti-flag epitope antibody M2of mouse brains 2days after infection with SIN/beclin (C),SIN/beclin stop (D),or SIN/beclin ⌬Bcl-2BD (E).Magnification,ϫ111.8590LIANG ET AL.J.V IROL .。
实时荧光定量PCR操作步骤
实时荧光定量PCR操作步骤以下实验步骤仅供参考：1 样品RNA的抽提①取冻存已裂解的细胞，室温放臵5分钟使其完全溶解。
②两相分离每1ml的TRIZOL试剂裂解的样品中加入0.2ml的氯仿，盖紧管盖。
手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。
4℃下12000rpm离心15分钟。
离心后混合液体将分为下层的红色酚氯仿相，中间层以及无色水相上层。
RNA全部被分配于水相中。
水相上层的体积大约是匀浆时加入的TRIZOL试剂的60%。
③RNA沉淀将水相上层转移到一干净无RNA酶的离心管中。
加等体积异丙醇混合以沉淀其中的RNA，混匀后15到30℃孵育10分钟后，于4℃下12000rpm 离心10分钟。
此时离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块。
④RNA清洗移去上清液，每1mlTRIZOL试剂裂解的样品中加入至少1ml的75%O配制），清洗RNA沉淀。
混匀后，4℃下7000rpm离心乙醇（75%乙醇用DEPCH25分钟。
⑤RNA干燥小心吸去大部分乙醇溶液，使RNA沉淀在室温空气中干燥5-10分钟。
⑥溶解RNA沉淀溶解RNA时，先加入无RNA酶的水40μl用枪反复吹打几次，使其完全溶解，获得的RNA溶液保存于-80℃待用。
2 RNA质量检测1）紫外吸收法测定先用稀释用的TE溶液将分光光度计调零。
然后取少量RNA溶液用TE稀释（1:100）后，读取其在分光光度计260nm和280nm处的吸收值，测定RNA溶液浓度和纯度。
①浓度测定A260下读值为1表示40 µg RNA/ml。
样品RNA浓度(µg/ml)计算公式为：A260 ×稀释倍数× 40 µg/ml。
具体计算如下：RNA溶于40 µl DEPC水中，取5ul，1:100稀释至495µl的TE中，测得A260 = 0.21RNA 浓度= 0.21 ×100 ×40 µg/ml = 840 µg/ml 或 0.84 µg/µl取5ul用来测量以后，剩余样品RNA为35 µl，剩余RNA总量为：35 µl × 0.84 µg/µl = 29.4 µg②纯度检测RNA溶液的A260/A280的比值即为RNA纯度，比值范围1.8到2.1。
斑马鱼原位杂交实验方案
斑马鱼全胚胎原位杂交技术Form Dr. Kevin第一节原位杂交技术的历史Joseph G. Gall被誉为现代细胞生物学的一位奠基人，他在染色体结构和功能领域作出了杰出贡献，并发明了原位杂交技术（in situ hybridization，ISH）。
自Gall同他的研究生Mary Lou Paudue和Susan Gerbi在1969年利用放射性标记DNA在爪蟾组织切片中检测基因表达后，原位杂交技术逐渐发展为一种能使研究人员在一个染色体上定位并确定出基因和特殊的DNA序列的强大方法，推动了分子生物学的巨大进步。
Kathleen H. Cox于1984年发明了用单链RNA探针进行原位杂交的技术。
ISH技术在斑马鱼中的应用始于Westerfield等利用该技术在斑马鱼切片中检测基因的表达。
同年, Nusslein-Volhard 等将Cox 建立的RNA 原位杂交技术进行了改进, 用地高辛（digoxin）标记的RNA 在斑马鱼胚胎中检测基因表达。
2008 年, Bernard Thisse 等将该技术进一步优化, 使之更敏感, 也提高了基因表达检测的分辨率，我们在这里介绍的整胚原位杂交技术主要参照Thisse实验室2008年版本（Thisse, C. and Thisse, B., 2008, High resolution in situ hybridization on whole-mount zebrafish embryo. Nat. protoc. 3 : 59 –69）。
第二节原位杂交技术的原理原位杂交能在成分复杂的组织中进行单一细胞的研究而不受同一组织中其他成分的影响，因此对于那些细胞数量少且散在于其他组织中的细胞内DNA或RNA研究更为方便；同时由于原位杂交不需要从组织中提取核酸，对于组织中含量极低的靶序列有极高的敏感性，并可完整地保持组织与细胞的形态，更能准确地反映出组织细胞的相互关系及功能状态。
细胞生物学考研真题
2006年中科院细胞生物学考研一、是非题：20题，每题1.5分，共30分。
答“是”写“+”，答“非”写“-”。
1.成熟的哺乳动物红细胞没有细胞核，但有转录活性。
2.细胞质膜一侧面向细胞外环境而另一侧朝向细胞内环境，所以内外两侧的磷脂是不同的。
而细胞器的膜处于细胞内，因此两侧的磷脂时相同的。
3.酶偶连受体的下游是腺苷酸环化酶。
4.直径约为15nm的核糖体的亚基是通过直径约为9nm的核孔运输到胞质中的。
5.B细胞分化成熟过程中，抗体分子的重链基因重排早于轻链基因重排。
6.病毒基因组可以是单链或双链DNA，也可以是单链或双链RNA.7.原位杂交技术可以将特异核酸序列和蛋白质在细胞内定位。
8.胚胎干细胞是在体内一直维持不分化的细胞群体。
9.负责纤毛运动和内膜细胞器运输的动力蛋白（dynein）是不同的。
10.脂双层内的脂质能围绕其长轴旋转。
11.细胞膜上载体协同运输系统可对分子进行同向转移和异向转移。
同向转移的是相同的分子而异向转移的是不同的分子。
12.直接来自有机体组织的细胞培养称为原代培养。
13.与微管和微丝相似，中等纤维蛋白也在各种组织中广泛表达，与细胞的分化状态无关。
14.接头蛋白中的SH2（Src homolog region 2）结构域可选择性地识别磷酸化的酪氨酸残基，并与之结合。
15.超速离心机可用来分离细胞器和生物大分子。
16.线粒体的分裂有以下几种形式：间壁分离，收缩后分离和出芽。
17.多数类型的动物细胞在离体培养时都能产生迁移运动。
18.姐妹染色单体存在于整个细胞周期中。
19.Bcl-2蛋白对细胞调亡有抑制作用。
20.激光扫描共焦显微术利用激光切割细胞产生光切片（optical section），从而得到该层的清晰图像。
二、选择题：30题，每题1.5分，共45分。
1.导致非整倍体的不正常染色体分离发生在细胞分裂的_________.A）前期B）中期C）后期D）末期2.光学显微镜最容易观察到的细胞器或细胞内结构是________.A）线粒体B）内质网C）细胞核D）微管3.细胞核不是哪一生命活动的场所：A）DNA合成B）蛋白质合成C）RNA合成D）核糖体亚基的装配4.桥粒与胞内的_________相连。
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大鼠δ阿片受体基因小发卡RNA及人前脑啡肽原基因双表达慢病毒载体的构建与鉴定逄坤芳;陈鹤翔;杨辉;卜慧莲;刘希江【摘要】目的构建大鼠δ阿片受体(delta opioid receptor,DOR)基因小发卡RNA(shRNA)及人前脑啡肽原基因(human preproenkephalin gene,hPPE)双表达慢病毒载体并鉴定.方法根据大鼠DOR mRNA序列设计shRNA并合成包含正、反义靶序列的互补DNA链,退火后插入至shRNA表达载体pENTR/U6 vector中,构建shRNA表达质粒,并转化至感受态细胞DH5α,抽提质粒后进行测序.合成hPPE基因序列并插入到表达载体pcDNA3.1(+)中,转化至感受态细胞DH5α,挑取多个单克隆进行测序验证.将hPPE插入已构建成功的pENTR/U6-shRNA载体中,再与慢病毒载体pLenti7.3/V5-DEST重组,构建慢病毒载体pLenti-CMV-hPPE-U6-shRNA并转化DH5α感受态细胞,筛选阳性克隆并测序验证,保留测序验证正确的LR重组质粒.用构建的慢病毒表达载体pLenti-CMV-hPPE-U6-shRNA和包装质粒共转染293T细胞,包装病毒,并测定滴度.结果经测序验证重组质粒pENTR/U6-shRNA、pcDNA3.1(+)-hPPE和慢病毒载体pLenti-CMV-hPPE-U6-shRNA均构建成功.大鼠δ阿片受体基因小发卡RNA及人前脑啡肽原基因双表达慢病毒包装成功,病毒滴度为1×107 TU/mL.结论大鼠DOR基因小发卡RNA及人前脑啡肽原基因双表达慢病毒载体构建成功,为研究DOR和hPPE在吗啡耐受中的作用机制及探求更加合理的镇痛方式奠定了基础.%Objective To construct lentivector encoding rat S opioid receptor shRNA and human preproenkephalin gene and identify its titer. Methods According to the sequence of rat DOR mRNA,we designed its shRNA sequence and synthesized the sense and antisense oligonucleotides. Afterannealing,these double DNA strands were cloned to the pKNTR/U6 vector and then transformed into competent cell DH5a. The plasmids were extracted and sequenced. hPPE gene sequence was synthesized and inserted into the expression vector pcDNA3. L(~r~) , then the recombinant plasmid was transformed into competent cell DH5a, and several positive monoclones were picked for sequencing. hPPE sequence was inserted into the built pENTR/U6 shRNA vector. And then this vector re-combined with lentiviral vector pLenti7. 3/V5-DEST to construct lentiviral vector pLenti-CMV-hPPE-U6-shRNA. Lentiviral vector pLenti-CMV-hPPE-U6-shRNA was transformed into DH5a competent cell, positive mono-clones were selected and sequenced, and the correct sequencing LR recombinant plasmids were retained. The constructed lentiviral vector pLenti-CMV-hPPE-U6-shRNA and packaging plasmids were co-transfected into 293T cells for viral packaging, virus stock solution was collected and concentrated by ultracentrifugation,and the titer was determined. Results Verified by sequencing, the recombinant plasmid pENTR/U6-shRNA,pcDNA3. L( + )-hPPE and lentiviral vector pLenti-CMV-hPPE-U6-shRNA were all constructed successfully. Lentivirus encoding rat 5 opioid receptor shRNA and human preproenkephalin gene was successfully packaged with the titer of 1 X 107 TU/mL. Conclusion Lentivirus encoding rat DOR shRNA and human preproenkephalin gene was successfully packaged, which laid the foundation for the study of DOR and hPPE in morphine tolerance mechanism and exploring a more rational way for analgesia.【期刊名称】《华中科技大学学报（医学版）》【年(卷),期】2012(041)003【总页数】5页(P329-332,352)【关键词】δ阿片受体;人前脑啡肽原基因;RNA干扰;慢病毒;吗啡耐受【作者】逄坤芳;陈鹤翔;杨辉;卜慧莲;刘希江【作者单位】华中科技大学同济医学院附属同济医院麻醉学教研室,武汉,430030;华中科技大学同济医学院附属同济医院麻醉学教研室,武汉,430030;华中科技大学同济医学院附属同济医院麻醉学教研室,武汉,430030;华中科技大学同济医学院附属同济医院麻醉学教研室,武汉,430030;华中科技大学同济医学院附属同济医院麻醉学教研室,武汉,430030【正文语种】中文【中图分类】R749.611阿片类药物长期应用于慢性疼痛患者将导致药物耐受，影响治疗效果并增加副作用，其发生机制可能与μ 受体（mu opioid receptor，MOR）和δ受体（DOR）形成异源二聚体，经过细胞内吞作用，泛素化并在溶酶体中降解有关［1］。
有文献证实应用小剂量的DOR 拮抗剂能抑制吗啡耐受的产生并能逆转已产生的耐受［2］，表明拮抗DOR 对于抑制吗啡耐受可能有一定作用。
脑啡肽作为一种内源性阿片肽，在许多动物实验中已经被证实具有一定的镇痛作用［3］。
由于慢病毒载体对分裂细胞和非分裂细胞均具有较强的转染能力且转染效率高，可在细胞中稳定持久地表达［4］，因此本实验拟构建具有双表达功能的pLenti－CMV－hPPE－U6－shRNA 慢病毒载体，此病毒载体含人前脑啡肽原基因（hPPE），能表达脑啡肽；同时构建的病毒中还含有特异性抑制DOR 受体表达的小发夹RNA 片段，以期将此病毒载体运用于慢性疼痛的研究，探讨其在镇痛及吗啡耐受中的作用，为找到更合理的镇痛方法提供新的思路。
1 材料与方法1.1 主要材料pENTR／U6vector 质粒载体、platinum pfx polymerase、pcDNA3.1（＋）质粒、LR clonase Ⅱenzyme mix、pLenti7.3／V5－DEST、包装质粒、293T细胞、DMEM ＋10% 胎牛血清、lipofectamine 2000、Opti－MEM 培养液以及0.05% 胰蛋白酶均购自Invitrogen公司；oligo DNA 购自上海英骏生物工程技术有限公司；HindⅢ、KpnⅠ、SaⅡ、BamHⅠ以及T4DNA 连接酶均购自New England Biolabs公司；DNA 凝胶回收试剂盒和小量质粒提取试剂盒购自Axygen 公司；感受态大肠埃希菌DH5α购自天根生化公司。
1.2 DOR－shRNA干扰载体构建1.2.1 靶向DOR 基因的shRNA 序列的合成根据本实验室先前已筛选出的大鼠DOR 基因序列GACGAGAACTTCAAGCGCT，合成1 对shRNA寡聚单链DNA。
上游：5′－CACCGACGAGAACTTCAAGCGCTCGAAAGCGCTTGAAGTTCTCGTC －3′，下游：5′－AAAAGACGAGAACTTCAAGCGCTTTCGAGCGCTTGAAGTTCTCGTC－3′。
1.2.2 DOR－shRNA 表达载体pENTR／U6vector的构建将2对寡聚单链DNA退火成双链，重组克隆后将退火而成的双链shRNA oligo插入shRNA 表达载体pENTR／U6vector中，构建shRNA表达质粒，并转化至感受态细胞DH5α，于37℃培养箱过夜培养。
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序。
1.3 合成hPPE基因序列及hPPE－pcDNA3.1（＋）表达载体的构建根据hPPE基因序列（大小约为830bp），设计并合成若干条oligo进行基因合成，在序列的5′端和3′端分别加上HindⅢ和KpnⅠ酶切位点，以便亚克隆至pcDNA3.1（＋）中。
将合成好的oligo配成oligo mix，进行PCR 反应，电泳并回收PCR 产物。
用HindⅢ和KpnⅠ对PCR 产物和pcDNA3.1（＋）同时进行酶切，37℃酶切2h。
电泳并回收酶切的PCR产物和载体，用T4DNA 连接酶连接回收的片段与载体，室温连接2h。
取5μL连接产物转化感受态细胞DH5α。
挑取多个单克隆进行测序。
1.4 pLenti－CMV－hPPE－U6－shRNA慢病毒载体构建1.4.1 构建带hPPE 的pENTR／U6shRNA 表达载体设计合成引物，扩增hPPE－BGHpA 片段，上游引物加Sal Ⅰ位点，下游引物加BamH Ⅰ位点。