pEF1α-IRES-DsRed-Express2哺乳动物表达载体说明

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真核细胞常见表达载体

真核细胞常见表达载体真核细胞, 表达载体1、pCMVp-NEO-BAN载体特点:该真核细胞表达载体分子量为6600碱基对，主要由CMVp启动子、兔β-球蛋白基因内含子、聚腺嘌呤、氨青霉素抗性基因和抗neo基因以及pBR322骨架构成，在大多数真核细胞内都能高水平稳定地表达外源目的基因。

更重要的是，由于该真核细胞表达载体中抗neo 基因存在，转染细胞后，用G418筛选，可建立稳定的、高表达目的基因的细胞株。

插入外源基因的克隆位点包括Sal1、BamH1和EcoR1位点。

注意在此载体中有二个EcoR1位点存在。

2、pEGFP, 增强型绦色荧光蛋白表达载体(Enhanced Fluorecent Protein Vector)特点: pEGFP表达载体中含有绿色荧光蛋白，在PCMV启动子驱动下，在真核细胞中高水平表达。

载体骨架中的SV40origin使该载体在任何表达SV40 T抗原的真核细胞内进行复制。

Neo抗性盒由SV40早期启动子、Tn5的neomycin/kanamycin抗性基因以及HSV-TK基因的聚腺嘌呤信号组成，能应用G418筛选稳定转染的真核细胞株。

此外，载体中的pUC origin 能保证该载体在大肠杆菌中的复制，而位于此表达盒上游的细菌启动子能驱动kanamycin抗性基因在大肠杆菌中的表达。

用途: 该表达载体EGFP上游有Nde1、Eco47111和Age1克隆位点，将外源基因扦入这些位点，将合成外源基因和EGFP的融合基因。

借此可确定外源基因在细胞内的表达和/或组织中的定位。

亦可用于检测克隆的启动子活性(取代CMV启动子,Acet1-Nhe1)。

3、pEGFT-Actin, 增强型绿色荧光蛋白/人肌动蛋白表达载体特点:pEGFP-Actin表达载体中含有绿色荧光蛋白和人胞浆β-肌动蛋白基因，在PCMV启动子驱动下，在真核细胞中高水平表达。

载体骨架中的SV40origin使该载体在任何表达SV40 T 抗原的真核细胞内进行复制。

真核细胞常见表达载体

真核细胞常见表达载体1、pCMVp-NEO-BAN载体特点: 该真核细胞表达载体分子量为6600碱基对，主要由CMVp启动子、兔β-球蛋白基因内含子、聚腺嘌呤、氨青霉素抗性基因和抗neo基因以及pBR322骨架构成，在大多数真核细胞内都能高水平稳定地表达外源目的基因。

更重要的是，由于该真核细胞表达载体中抗neo基因存在，转染细胞后，用G41 8筛选，可建立稳定的、高表达目的基因的细胞株。

插入外源基因的克隆位点包括Sal1、BamH1和EcoR1位点。

注意在此载体中有二个EcoR1位点存在。

载体骨架中的SV40 origin使该载体在任何表达SV40 T 抗原的真核细胞内进行复制。

Neo抗性盒由SV4 0早期启动子、Tn5的neomycin/kanamycin抗性基因以及HSV-TK基因的聚腺嘌呤信号组成，能应用G4 18筛选稳定转染的真核细胞株。

此外，载体中的pUC origin 能保证该载体在大肠杆菌中的复制，而位于此表达盒上游的细菌启动子能驱动kanamycin抗性基因在大肠杆菌中的表达。

用途: 该表达载体EGFP上游有Nde1、Eco47111和Age1克隆位点，将外源基因扦入这些位点，将合成外源基因和EGFP的融合基因。

借此可确定外源基因在细胞内的表达和/或组织中的定位。

亦可用于检测克隆的启动子活性(取代CMV启动子,Acet1-Nhe1)。

3、pEGFT-Actin, 增强型绿色荧光蛋白/人肌动蛋白表达载体特点: pEGFP-Actin表达载体中含有绿色荧光蛋白和人胞浆β-肌动蛋白基因，在PCMV启动子驱动下，在真核细胞中高水平表达。

载体骨架中的SV40 origin使该载体在任何表达SV40 T 抗原的真核细胞内进行复制。

哺乳动物细胞表达系统

哺乳动物细胞表达系统按照宿主细胞的类型，可将基因表达系统大致分为原核、酵母、植物、昆虫和哺乳动物细胞表达系统。

与其它系统相比，哺乳动物细胞表达系统的优势在于能够指导蛋白质的正确折叠，提供复杂的N型糖基化和准确的O型糖基化等多种翻译后加工功能，因而表达产物在分子结构、理化特性和生物学功能方面最接近于天然的高等生物蛋白质分子。

从最开始以裸露DNA直接转染哺乳动物细胞至今的30余年间，哺乳动物细胞表达系统不仅已成为多种基因工程药物的生产平台，在新基因的发现、蛋白质的结构和功能研究中亦起了极为重要的作用。

本文主要从表达系统及其两个组成部分一一表达载体和宿主细胞等方面，简要介绍哺乳动物细胞表达系统和相关的研究进展。

研究现状①部分蛋白在哺乳动物细胞中的表达已从实验室研究迈向生产或中试生产阶段。

②已有许多重要的蛋白及糖蛋白利用哺乳动物细胞系统表达和大量制备、生产。

如人组织型血纤蛋白酶原激活因子、凝血因子皿、干扰素、乙肝表面抗原、红血球生成激素、人生长激素、人抗凝血素出，集落刺激因子等。

有些产品已投入临床应用或试用。

③虽然经过多年努力，哺乳动物细胞表达系统的表达水平有大幅度增高，但从整个水平上看仍偏低，一般处在杂交瘤细胞单克隆抗体蛋白产率的下限，即1-30^g/l08细胞/24小时。

有人认为其限速步骤可嚣是在工程细胞中（对于重组蛋白来讲，常是异源的），重组蛋白的分泌效率较低。

1表达载体1. 1表达栽体的类型哺乳动物细胞表达外源重组蛋白可利用质粒转染和病毒载体的感染。

利用质粒转染获得稳定的转染细胞需几周甚至几个月时间，而利用病毒表达系统则可快速感染细胞，在几天内使外源基因整合到病毒载体中，尤其适用于从大量表达产物中检测出目的蛋白。

根据进入宿主细胞的方式，可将表达载体分为病毒载体与质粒载体。

病毒载体是以病毒颗粒的方式，通过病毒包膜蛋白与宿主细胞膜的相互作用使外源基因进入到细胞内。

常用的病毒载体有腺病毒、腺相关病毒、逆转录病毒、semliki森林病毒（sFv）载体等。

pcDNA4 myc-His C哺乳动物表达载体说明

pcDNA4/myc-His C编号载体名称北京华越洋生物VECT6123 pcDNA4/myc-‐His C pcDNA4/myc-‐His C载体基本信息载体名称: pcDNA4/myc-His C质粒类型: 哺乳动物表达载体；cDNA表达载体高拷贝/低拷贝: 高拷贝克隆方法: 多克隆位点，限制性内切酶启动子: CMV载体大小: 5071 bp5' 测序引物及序列: T7 Forward: 5’-TAATACGACTCACTATAGGG-3’ 3' 测序引物及序列: BGH Reverse: 5-TAGAAGGCACAGTCGAGG-3 载体标签: His Tag (C-端）, c-Myc Epitope Tag（C-端）载体抗性: 氨苄青霉素筛选标记: Zeocin克隆菌株: TOP10F´, DH5a, JM109, TOP10宿主细胞（系）: 常规细胞系，如293、Hela等备注: pcDNA4/myc-His C 载体是哺乳动物表达载体，适用于cDNA的表达与克隆；CMV启动子驱动目的基因的高水平表达；pcDNA4/myc-His A,B,C的区别仅在于多克隆位点处。

稳定性: 瞬表达或稳表达组成型/诱导型: 组成型病毒/非病毒: 非病毒pcDNA4/myc-‐His C载体质粒图谱和多克隆位点信息pcDNA4/myc-‐His C载体描述pcDNA4/myc-His A, B, and C are 5.1 kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins (see pages 11-12 for more information). High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cellsThree reading frames to facilitate in-frame cloning with a C-terminal peptide encoding the myc (c-myc) epitope and a polyhistidine (6xHis) metal-binding tagZeocin resistance gene for selection of stable cell lines (Mulsant et al., 1988) (see page 14 for more information).Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7).The control plasmid, pcDNA4/myc-His/lacZ is included for use as a positive control for transfection, expression, and detection in the cell line of choice.实验流程：Use the following outline to clone and express your gene of interest in pcDNA4/myc-His:1.Consult the multiple cloning sites described on pages 3-4 to determine which vector (A, B, or C) to use for cloning your gene in frame with the C-terminal myc epitope and the polyhistidine tag.2.Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/mL ampicillin or 25 to 50g/mL Zeocin in Low Salt LB. For more information.3.Analyze your transformants for the presence of insert by restriction digestion.4.Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in-frame with the C-terminal peptide.5.Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.6.Test for expression of your recombinant gene by western blot analysis or functional assay. For antibodies to the myc epitope or the C-terminal polyhistidine tag.7.To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond resin is available separatelypcDNA4/myc-‐His C载体序列ORIGIN1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG 121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC 181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA 301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC 361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC 421 ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT 541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA 601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG 661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG 781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA 841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT 901 TAAGCTTGGT ACCGAGCTCG GATCCACTAG TCCAGTGTGG TGGAATTCTG CAGATATCCA 961 GCACAGTGGC GGCCGCTCGA GGTCACCCAT TCGAACAAAA ACTCATCTCA GAAGAGGATC 1021 TGAATATGCA TACCGGTCAT CATCACCATC ACCATTGAGT TTAAACCCGC TGATCAGCCT 1081 CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA 1141 CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT 1201 GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG 1261 ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG 1321 AAAGAACCAG CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG 1381 CGGCGGGTGT GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG 1441 CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC 1501 TAAATCGGGG CATCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA 1561 AACTTGATTA GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC 1621 CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC 1681 TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGGGGATT TCGGCCTATT 1741 GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG 1801 TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGGCAGG CAGAAGTATG CAAAGCATGC 1861 ATCTCAATTA GTCAGCAACC AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA 1921 TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC 1981 CGCCCCTAAC TCCGCCCAGT TCCGCCCATT CTCCGCCCCA TGGCTGACTA ATTTTTTTTA 2041 TTTATGCAGA GGCCGAGGCC GCCTCTGCCT CTGAGCTATT CCAGAAGTAG TGAGGAGGCT 2101 TTTTTGGAGG CCTAGGCTTT TGCAAAAAGC TCCCGGGAGC TTGTATATCC ATTTTCGGAT 2161 CTGATCAGCA CGTGTTGACA ATTAATCATC GGCATAGTAT ATCGGCATAG TATAATACGA 2221 CAAGGTGAGG AACTAAACCA TGGCCAAGTT GACCAGTGCC GTTCCGGTGC TCACCGCGCG 2281 CGACGTCGCC GGAGCGGTCG AGTTCTGGAC CGACCGGCTC GGGTTCTCCC GGGACTTCGT 2341 GGAGGACGAC TTCGCCGGTG TGGTCCGGGA CGACGTGACC CTGTTCATCA GCGCGGTCCA 2401 GGACCAGGTG GTGCCGGACA ACACCCTGGC CTGGGTGTGG GTGCGCGGCC TGGACGAGCT 2461 GTACGCCGAG TGGTCGGAGG TCGTGTCCAC GAACTTCCGG GACGCCTCCG GGCCGGCCAT 2521 GACCGAGATC GGCGAGCAGC CGTGGGGGCG GGAGTTCGCC CTGCGCGACC CGGCCGGCAA 2581 CTGCGTGCAC TTCGTGGCCG AGGAGCAGGA CTGACACGTG CTACGAGATT TCGATTCCAC 2641 CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT2701 CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC 2761 TTATAATGGT TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC 2821 ACTGCATTCT AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC 2881 GTCGACCTCT AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG 2941 TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG 3001 TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC 3061 GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT 3121 GCGTATTGGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT 3181 GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA 3241 TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC 3301 CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG 3361 CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG 3421 AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT 3481 TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA ATGCTCACGC TGTAGGTATC TCAGTTCGGT 3541 GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG 3601 CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT 3661 GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT 3721 CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT 3781 GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC 3841 CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC 3901 TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG 3961 TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA 4021 AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA 4081 ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC 4141 CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC 4201 TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC 4261 AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT 4321 TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT 4381 TGCCATTGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC 4441 CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG 4501 CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT 4561 TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC 4621 TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG 4681 CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT 4741 TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC 4801 GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC 4861 TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA 4921 ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG 4981 TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG 5041 CACATTTCCC CGAAAAGTGC CACCTGACGT C//其他哺乳动物表达载体：pCHO1.0 pBApo-CMV-Pur pOPRSVIpcDNA3.1/His C pcDNA5/FRT/V5-His-TOPO pREP4pcDNA3.1/His B pcDNA5/FRT/TO-TOPO pDual-GCpcDNA3.1/His A pcDNA5/TO pBK-RSVpIRESpuro3 pcDNA5/FRT/TO pBK-CMVpIRES2-EGFP pcDNA5/FRT pBI-CMV4pTT5 pFLAG-CMV2 pcDNA4/TO/Myc-His/LacZ pNFkB-DD-tdTomato pcDNA3.1/CT-GFP-TOPO pOPI3CATpBI-CMV5 pcDNA3.1/NT-GFP-TOPO pGene/V5-His B pSEAP2-Basic pOptiVEC-TOPO pSwitchpSEAP2-Control pCMV-MEKK1 pCMVLacIpBI-CMV3 pCMV-MEK1 pVgRxRpBI-CMV2 pCMV-PKA pINDpBI-CMV1 pcDNA6.2/nTC-Tag-DEST pTRE3G-LucpNFκB-MetLuc2-Reporter pcDNA6.2/cTC-Tag-DEST pTRE3GpCRE-MetLuc2-Reporter pcDNA3.2/V5/GW/D-TOPO pTRE2-hygropAcGFP1-Actin pcDNA6.2/V5/GW/D-TOPO pTRE-TightpAcGFP1-N In-Fusion Ready pcDNA6.2/nGeneBLAzer-GW/D-TOPO pTK-hygpAcGFP1-C3 pcDNA6.2/C-YFP-DEST pTet-OnpAcGFP1-C pcDNA6.2/cGeneBLAzer-DEST pTet-OffpAcGFP1-p53 pcDNA6/V5-His A pTet on advanced pAcGFP1-Mito pcDNA6/V5-His B pRevTREpAcGFP1-Mem pcDNA6/V5-His C pRevTet-OnpAcGFP1-Lam pcDNA6/myc-His C pRevTet-OffpAcGFP1-Golgi pcDNA6/myc-His A pCMV-Tet3GpAcGFP1-F pcDNA6/myc-His B pTRE2pAcGFP1-Hyg-C1 pcDNA6.2/nGeneBLAzer-DEST pBD-NF-κBpAsRed2-N1 pcDNA4/HisMax-TOPO pCMV-ADptdTomato-N1 pcDNA6.2/nLumio-DEST pCMV-BDpCMV-tdTomato pcDNA6.2/cLumio-DEST pBIND-Id ControlpCRE-DD-tdTomato pcDNA4/myc-His C pBINDpCMV-DsRed-Express2 pcDNA4/HisMax C pG5 luciferasepEF1α-tdTomato pcDNA4/HisMax A pACT-MyoDpCRE-hrGFP c-Flag pcDNA3 pACTptdTomato-C1 pcDNA4/HisMax B pCMV-SPORT6 pAsRed2-C1 pcDNA4/myc-His B pGL4.13pGL3-Promoter pcDNA4/myc-His A pGL4.19pGL3 basic pcDNA4/His C pGL4.26pAcGFP1-C2 pcDNA4/His B pGL4.20pAcGFP1-C1 pcDNA4/His A pGL4.29pAcGFP1-N3 pcDNA6/TR pGL4.30pAcGFP1-N2 pcDNA4/TO/Myc-His A pGL4.27pAcGFP1-N1 pcDNA4/TO pGL4.75pAcGFP1-C In-Fusion Ready pcDNA4/TO/Myc-His B pGL4.10pCRE-DD-AmCyan1 pcDNA4/TO/Myc-His C pGRN145pNFkB-DD-AmCyan1 pcDNA3.3-TOPO pSecTag2 A pDsRed2-Bid pBudCE4.1 pEBVHis B pDsRED2-Mito pFLAG-CMV-4 pEBVHis ApDD-AmCyan1 Reporter pFLAG-CMV-3 pCMV-Tag 3C pAmCyan1-N1 pFLAG-CMV-2 pCMV-Tag 3A pAmCyan1-C1 pFLAG-CMV-5a pCMV-Tag 3BpEF1α-IRES-DsRed-Express2 p3XFLAG-CMV-9 pCMV-Tag 5CpEF1α-DsRed-Monomer-N1 p3xFLAG-CMV-10 pCMV-Tag 5A pDsRED-Monomer-N1 p3XFLAG-CMV-8 pCMV-Tag 4A pDsRed-Express-N1 p3XFLAG-CMV-7.1 pCMV-Tag 5Bp3XFLAG-CMV-7 pDsRed-Monomer-N In-Fusion Ready pCMV-Tag 4B pDsRed-Express-C1 p3XFLAG-CMV-13 pCMV-Tag 2C pIRES2-ZsGreen1 p3XFLAG-CMV-14 pCMV-Tag 2B pDsRed-Express2-C1 plRES2-ZsGreen1 pCMV-Tag 2A pDsRed-Express2-N1 pBApo-EF1α-pur pCMV-LacZpEF1α-DsRed-Express2 pBApo-EF1α-neo pCMV-MycpIRES2-DsRed-Express pBApo-CMV pEF1α-IRES-AcGFP1 pIRES2-DsRed-Express2 pBApo-CMV-neo pEF1α-IRES-ZsGreen1 pIRES-hrGFP-1a pIRES-EGFP pEF1α-AcGFP1-N1 pIRESneo2 pIRESneo3 pIRES2-DsRed2 pIRESneo pDsRed-Monomer pIRES2-AcGFP1 pIREShyg3 pIRES。

pDsRed2-Bid哺乳动物表达载体说明

pDsRed2-Bid编号载体名称北京华越洋生物VECT6108 pDsRed2-‐BidpDsRed2-‐Bid载体基本信息载体名称: pDsRed2-Bid质粒类型: 哺乳动物细胞表达载体；信号通路报告载体高拷贝/低拷贝: 高拷贝克隆方法: 限制性内切酶，多克隆位点启动子: CMV IE载体大小: 5.3 kb5' 测序引物及序列: --3' 测序引物及序列: --载体标签: --载体抗性: 卡那霉素筛选标记: 新霉素（Neomycin）克隆菌株: DH5α, HB101宿主细胞（系）: 常规细胞系，293、CV-1、CHO等备注: pDsRed2-Bid载体是红色荧光报告载体，用于研究依赖Bid的细胞凋亡信号通路。

稳定性: 瞬表达或稳表达组成型/诱导型: 组成型病毒/非病毒: 非病毒pDsRed2-‐Bid载体质粒图谱和多克隆位点信息pDsRed2-Bid载体描述pDsRed2-Bid is a mammalian expression vector that encodes a fusion of Discosoma sp. red fluorescent protein (DsRed2; 1, 2) and Bid, a member of the Bcl-2 “pro-apoptosis” family (3). Developed for our ApoAlert line, pDsRed2-Bid is designed to help researchers study Bid-dependent apoptosis pathways. Because of its fluorescent label, the Bid-DsRed2 fusion is easily detected by microscopy, allowing researchers to track its movements in response to certain apoptosis inducing agents (e.g., TNF-α).Bid’s activity is closely tied to its location in the cell. In healthy, non-apoptotic cells, Bid normally resides in the cytosol. But soon after the induction of apoptosis, it translocates to mitochondria, where it stimulates the release of cytochrome c—a key amplification step in the apoptotic cascade (4–7). The translocation is triggered by caspase-8, which, when activated by a death signal, cleaves Bid to produce a 15-kDa C-terminal fragment. The fragment, often referred to as truncated Bid or tBid, transmits the death signal further by translocating to the mitochondria. You can monitor this event visually by expressing Bid as a DsRed2 fusion. Bid-DsRed2, thefusion expressed by this vector, for example, emits red fluorescence—even after truncation—so it can be followed as it moves from the cytosol to the mitochondria. To drive expression of Bid-DsRed2, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE), positioned just upstream of the Bid sequence. A short linker joins the Bid coding sequence to the 5'-end of DsRed2. Farther downstream, the vector contains a pair of SV40 polyadenylation signals, which direct proper processingof the 3'-end of the Bid-DsRed2 mRNA. The vector also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418 (8). A bacterial promoter upstream of the cassette confers kanamycin resistance (Kanr) to E. coli.Cells transfected with pDsRed2-Bid constitutively express Bid as an N-terminal fusionto DsRed2. pDsRed2-Bid can be introduced into a variety of mammalian cell lines using any standard transfection method. If required, stable transfectants can be selected using G418 (8).Propagation in E. coliSuitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requiresa host containing an F plasmid such as JM109 or XL1-Blue.Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.E. coli replication origin: pUCCopy number: ~500Plasmid incompatibility group: pMB1/ColE1Red Fluorescent Protein (DsRed2)Excitation/Emission Maxima: 558 nm / 583 nm其他哺乳动物表达载体：pCHO1.0 pBApo-CMV-Pur pOPRSVIpcDNA3.1/His C pcDNA5/FRT/V5-His-TOPO pREP4pcDNA3.1/His B pcDNA5/FRT/TO-TOPO pDual-GCpcDNA3.1/His A pcDNA5/TO pBK-RSVpIRESpuro3 pcDNA5/FRT/TO pBK-CMVpIRES2-EGFP pcDNA5/FRT pBI-CMV4pTT5 pFLAG-CMV2 pcDNA4/TO/Myc-His/LacZ pNFkB-DD-tdTomato pcDNA3.1/CT-GFP-TOPO pOPI3CATpBI-CMV5 pcDNA3.1/NT-GFP-TOPO pGene/V5-His B pSEAP2-Basic pOptiVEC-TOPO pSwitchpSEAP2-Control pCMV-MEKK1 pCMVLacIpBI-CMV3 pCMV-MEK1 pVgRxRpBI-CMV2 pCMV-PKA pINDpBI-CMV1 pcDNA6.2/nTC-Tag-DEST pTRE3G-Luc pNFκB-MetLuc2-Reporter pcDNA6.2/cTC-Tag-DEST pTRE3GpCRE-MetLuc2-Reporter pcDNA3.2/V5/GW/D-TOPO pTRE2-hygro pAcGFP1-Actin pcDNA6.2/V5/GW/D-TOPO pTRE-Tight pAcGFP1-N In-Fusion Ready pcDNA6.2/nGeneBLAzer-GW/D-TOPO pTK-hyg pAcGFP1-C3 pcDNA6.2/C-YFP-DEST pTet-On pAcGFP1-C pcDNA6.2/cGeneBLAzer-DEST pTet-Off pAcGFP1-p53 pcDNA6/V5-His A pTet on advanced pAcGFP1-Mito pcDNA6/V5-His B pRevTRE pAcGFP1-Mem pcDNA6/V5-His C pRevTet-On pAcGFP1-Lam pcDNA6/myc-His C pRevTet-Off pAcGFP1-Golgi pcDNA6/myc-His A pCMV-Tet3G pAcGFP1-F pcDNA6/myc-His B pTRE2 pAcGFP1-Hyg-C1 pcDNA6.2/nGeneBLAzer-DEST pBD-NF-κB pAsRed2-N1 pcDNA4/HisMax-TOPO pCMV-AD ptdTomato-N1 pcDNA6.2/nLumio-DEST pCMV-BD pCMV-tdTomato pcDNA6.2/cLumio-DEST pBIND-Id Control pCRE-DD-tdTomato pcDNA4/myc-His C pBINDpCMV-DsRed-Express2 pcDNA4/HisMax C pG5 luciferase pEF1α-tdTomato pcDNA4/HisMax A pACT-MyoD pCRE-hrGFP c-Flag pcDNA3 pACT ptdTomato-C1 pcDNA4/HisMax B pCMV-SPORT6 pAsRed2-C1 pcDNA4/myc-His B pGL4.13pGL3-Promoter pcDNA4/myc-His A pGL4.19pGL3 basic pcDNA4/His C pGL4.26 pAcGFP1-C2 pcDNA4/His B pGL4.20 pAcGFP1-C1 pcDNA4/His A pGL4.29 pAcGFP1-N3 pcDNA6/TR pGL4.30 pAcGFP1-N2 pcDNA4/TO/Myc-His A pGL4.27 pAcGFP1-N1 pcDNA4/TO pGL4.75 pAcGFP1-C In-Fusion Ready pcDNA4/TO/Myc-His B pGL4.10pCRE-DD-AmCyan1 pcDNA4/TO/Myc-His C pGRN145 pNFkB-DD-AmCyan1 pcDNA3.3-TOPO pSecTag2 A pDsRed2-Bid pBudCE4.1 pEBVHis B pDsRED2-Mito pFLAG-CMV-4 pEBVHis ApDD-AmCyan1 Reporter pFLAG-CMV-3 pCMV-Tag 3C pAmCyan1-N1 pFLAG-CMV-2 pCMV-Tag 3A pAmCyan1-C1 pFLAG-CMV-5a pCMV-Tag 3B pEF1α-IRES-DsRed-Express2 p3XFLAG-CMV-9 pCMV-Tag 5C pEF1α-DsRed-Monomer-N1 p3xFLAG-CMV-10 pCMV-Tag 5A pDsRED-Monomer-N1 p3XFLAG-CMV-8 pCMV-Tag 4ApDsRed-Express-N1 p3XFLAG-CMV-7.1 pCMV-Tag 5Bp3XFLAG-CMV-7 pDsRed-Monomer-N In-Fusion Ready pCMV-Tag 4B pDsRed-Express-C1 p3XFLAG-CMV-13 pCMV-Tag 2C pIRES2-ZsGreen1 p3XFLAG-CMV-14 pCMV-Tag 2B pDsRed-Express2-C1 plRES2-ZsGreen1 pCMV-Tag 2A pDsRed-Express2-N1 pBApo-EF1α-pur pCMV-LacZpEF1α-DsRed-Express2 pBApo-EF1α-neo pCMV-MycpIRES2-DsRed-Express pBApo-CMV pEF1α-IRES-AcGFP1 pIRES2-DsRed-Express2 pBApo-CMV-neo pEF1α-IRES-ZsGreen1 pIRES-hrGFP-1a pIRES-EGFP pEF1α-AcGFP1-N1 pIRESneo2 pIRESneo3 pIRES2-DsRed2 pIRESneo pDsRed-Monomer pIRES2-AcGFP1 pIREShyg3 pIRES。

pBApo-EF1α-neo哺乳动物表达载体说明

pBApo-EF1α-neo哺乳动物表达载体说明pBApo-EF1α-neo编号载体名称北京华越洋生物VECT6066 pBApo--‐EF1α--‐neo pBApo--‐EF1α--‐neo载体基本信息载体名称: pBApo-EF1α-neo质粒类型: 哺乳动物细胞表达载体高拷贝/低拷贝: 低拷贝克隆方法: 限制性内切酶，多克隆位点启动子: EF1α载体大小: 5167 bp5' 测序引物及序列: --3' 测序引物及序列: --载体标签: 无标签载体抗性: 氨苄青霉素筛选标记: 新霉素（Neomycin）克隆菌株: TOP10, DH5α, JM109宿主细胞（系）: 常规细胞系，293、CV-1、CHO等备注: pBApo-EF1α-neo载体可用来表达microRNA和其它转录产物；很容易将{promoter+ORF+PolyA signal}框转移到腺病毒载体上，用于构建腺病毒载体。

稳定性: 瞬表达或稳表达组成型/诱导型: 组成型病毒/非病毒: 非病毒pBApo--‐EF1α--‐neo载体质粒图谱和多克隆位点信息pBApo--‐EF1α--‐neo载体简介pBApo-EF1α-Neo DNA是一种简单的应用于哺乳动物细胞的基因表达载体。

该载体具有人多肽链延伸因子基因来源的启动子(EF1αpromoter)、单纯疱疹病毒胸苷激酶来源的polyA信号、新霉素抗性基因。

通过在MCS区域插入目的基因的开放阅读框(ORF)，构建目的基因表达载体。

除了通常的基因以外该载体可以用于pri-microRNA的转录。

此外，还可以很容易地将(promoter＋ORF＋PolyA signal) 从这个载体上转移到腺病毒载体上，尤其是转移到Adenovirus Dual Expression Kit的组分pAxcwit上。

腺病毒载体感染效率高，范围广，可以用于体外和体内的基因转导。

pBApo--‐EF1α--‐neo使用方法1. 插入基因在质粒载体的克隆位点处插入目的基因的开放阅读框（ORF）。

pCDH-CMV-MCS-EF1-RFP使用说明

pCDH-CMV-MCS-EF1-RFPpCDH-CMV-MCS-EF1-RFP 载体基本信息：载体名称:pCDH-CMV-MCS-EF1-RFP 质粒类型: 慢病毒表达载体；cDNA 表达载体；双启动子载体克隆方法: 多克隆位点，限制性内切酶启动子:CMV 载体大小:-- 5' 测序引物及序列:CMV-F :CGCAAATGGGCGGTAGGCGTG 3' 测序引物及序列:EF1a-R 载体标签: 无载体抗性: 氨苄青霉素（Ampicillin ）筛选标记: 嘌呤霉素克隆菌株: E.coli cells(RecA-)推荐: Stbl2 ,OmniMAX 2 T1R 宿主细胞（系）: 造血干细胞、胚胎干细胞备注: pCDH-CMV-MCS-EF1-RFP 慢病毒表达载体是基于HIV 的慢病毒载体；用于cDNA 表达和克隆；高效转染细胞，建立稳定细胞系；CMV 启动子驱动目的基因的高水平表达，EF1a 启动子驱动报告基因的中等水平的表达。

稳定性: 稳表达组成型/诱导型: 组成型病毒/非病毒: 慢病毒（HIV)pCDH-CMV-MCS-EF1-RFP 载体质粒图谱和多克隆位点信息：pCDH-CMV-MCS-EF1-RFP载体简介：This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.基于HIV-1的pCDH 慢病毒载体特征：Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.WPRE element—enhances stability and translation of the CMV-driven transcripts.SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.Hybrid RSV/5L TR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.Genetic elements (cPPT, gag, env, L TRs)—necessary for packaging, transducing, and stablyintegrating the vira expression construct into genomic DNA.SV40 origin—for stable propagation of the pCDH plasmid in mammalian cells.pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.Ampicillin resistance gene—for selection in E.coli cells.pCDH 慢病毒表达载体的优势：Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.pCDH 慢病毒载体的包装载体及细胞系The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transientlyco-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.启动子的选择：SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.MSCV promoter is the 5’-L TR promoter of murine stem cell virus. When a portion of the U3 regionof the 3’ HIV L TR was replaced with the U3 region of MSCV L TR, the resulted hybrid HIV/MSCVL TR has dramatically increased the transgene expression level in human CD34+ hematopoieticcells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acce ptor sites derived fromthe lentiviral vector. Further studies found that additional CpG mutations in the MSCV L TRreduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV L TR. After integration into genomic DNA, 3’MSCV/L TR will replace the 5’L TR and provide a high level of expression of the target gene and reporter gene downstream.SBI第三代慢病毒载体SBI offers a third generation of the most popular HIV-1 based lentivector expression systemwhich consists of three maincomponents:(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)(3) A pseudoviral particle producer cell line (e.g., 293TN cells)2A Peptide-enabled dual expression systemCoexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgeneswith an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines—however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006).The 2A-like sequences exist in several viruses and are used to mediate protein cleavage froma single open reading frame. Through a ribosomal skip mechanism, the 2A peptide preventsnormal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reportergene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.pCDH-CMV-MCS-EF1-RFP载体序列：pCDH-CMV-MCS-EF1-RFP其他相关慢病毒载体：Tet-pLKO-neo Tet-pLKO-puro pPACKH1-GAG pMD2.G pCMV-dR8.2-dvpr pLKO.1-GFP-shRNA pLKO.1-TRC control pLKO.1-hygro pLKO.1-TRC pCDH-MSCV-MCS-EF1-copGFP pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro FUW-tetO-hOKMSFUW-tetO-hOCT4 FUW-tetO-hSOX2 FUW-tetO-hKLF4FUW pLVX-AcGFP1-N1 pLVX-AcGFP1-C1pLVX-AmCyan1-N1 pLVX-DsRed-Express2-C1 pLVX-DsRed-Express2-N1 pLVX-DsRed-Monomer-N1 pLVX-PAmCherry-C1 pLVX-PAmCherry-N1pLVX-ZsGreen1-N1 pLVX-IRES-ZsGreen1 pLVX-IRES-mCherrypLVX-mCherry-C1 pLVX-mCherry-N1 pLVX-tdTomato-C1pLKO.1-puro pLentilox 3.7 pLVX-Tet-On-Advanced pLVX-IRES-Puro pLVX-IRES-Neo pLVX-IRES-HygpLVX-EF1α-DsRed-Monomer-C1 pLVX-EF1α-AcGFP1-N1 pLVX-EF1α-AcGFP1-C1 pLVX-EF1α-mCherry-C1 pLVX-EF1α-IRES-mCherry pLVX-EF1α-IRES-ZsGreen1 pLVX-MetLuc Control pLVX-MetLuc pLVX-Hom-Mem1pLVX-Het-2 pLVX-DD-AcGFP1-Actin pPRIME-TET-GFP-FF3pSIH1-H1-CopGFP pCDH-EF1-MCS-T2A-Puro pCDH-CMV-MCS-EF1-Puro pCDF1-MCS2-EF1-copGFP pLOX-CWBmi1 pLOX-CW-CREpRSV-rev pMDLg-pRRE pLL3.7pLVX-DD-AmCyan1 Control pLVX-DD-AmCyan1 Reporter pLVX-DD-tdTomato Reporter pLVX-DD-tdTomato Control pLVX-PTuner-Green pLVX-CherryPicker2pLVX-TetOne-Puro-Luc pLVX-TetOne pLVX-TetOne-PuropLVX-TetOne-Luc pLVX-rHom-Nuc1 pLVX-rHom-Sec1pLVX-rHom-1 pLVX-Hom-Nuc1 pLVX-Het-Nuc1pLVX-PTuner pLVX-PTuner2 pLVX-DD-ZsGreen1 Reporter pLVX-Het-1 pLVX-CherryPicker Control pLVX-Tet3GpCDH-CMV-MCS-EF1-RFP-T2A-Puro pCDH-CMV-MCS-EF1-Hygro pCDH-CMV-MCS-EF1-Neo pCDH-MCS-T2A-Puro-MSCV pCDH1-MCS2-EF1-copGFP pCDF1-MCS2-EF1-Puro pCDH-EF1-MCS-T2A-copGFP pWPXL pLVX-TRE3G-ZsGreen1 pLVX-TRE3G-mCherry pLenti6.3-EmGFP-BveI miR pLenti6/V5-GW/lacZpLenti6.3/V5-GW/EmGFP pLenti6.3-MCS pLenti6.3-DsRed2-BveI miR pLenti6.3-MCS-IRES2-EGFP pLVX-shRNA2 psPAX2VSV-G pSico PGK Puro pcDNA6.2-DsRed2-MCS1 miR pcDNA6.3-EmGFP-NC- II pcDNA6.2-EmGFP-NC- I pcDNA6.2-EmGFP-BsaI miR pLenti6.3-BveI miR pLenti6.3-MCS-IRES2-DsRed2 pLEX-MCSpGIPZ pLP2 pLP1FUGW pFUGW pLOX-Ttag-iresTKpMDLg/pRRE pLentG-KOSM pCMV-dR8.91pLVX-TRE3G-Luc Control pLVX-TRE3G-IRES pCgpvpSico pSicoR pLVTHMpGensil-1 pLVX-EF1α-IRES-Puro pCDF1-MCS2-EF1-copGFP pPACKH1-REV pLVX-Het-Mem1 pLVX-shRNA1pLKO.1-puro-GFP-siRNA pPRIME-TREX-GFP-FF3 pcDNA6.2-DsRed2-BsmBI miR pCDH-MSCV-MCS-EF1-Puro pCDH-CMV-MCS-EF1-copGFP pLVX-TRE3GFUW-tetO-hMYC pLOX-TERT-iresTK pLP/VSVGFUW-M2rtTA pCDH-EF1-MCS-(PGK-Puro) pcDNA6.2-EmGFP-MCS1 miR pLVX-AmCyan1-C1 pLVX-Hom-1 pcDNA6.2-BsaI miRpLVX-DsRed-Monomer-C1 pLVX-mCherry-Actin pTRIPZpLVX-ZsGreen1-C1 pLVX-CherryPicker1 LeGO-iC2 pLVX-IRES-tdTomato pCDH-CMV-MCS-EF1-copGFP-T2A-Puro pLKO.3GpLVX-tdTomato-N1 pLVX-PTuner2-C pLVX-Puro pLVX-Tight-Puro pLVX-DD-ZsGreen1 Control pSicoR PGK Puro pLVX-EF1α-DsRed-Monomer-N1 pCDH-UbC-MCS-EF1-Hygro pLVTHpLVX-EF1α-mCherry-N1 pCDH-CMV-MCS-EF1-RFP。

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哺乳动物蛋白表达系统.docx

哺乳动物细胞表达系统背景简介哺乳动物细胞表达系统以哺乳动物细胞作为宿主进行蛋白表达生产，具有转录后修饰、蛋白正确折叠与装配等功能，表达的蛋白更接近天然状态，并且很多蛋白可以经哺乳动物细胞分泌到细胞外，这利于蛋白的别离。

如果缺少正确的转录后修饰，可能影响蛋白质的稳定和配体结合, 进而增加免疫原性，因此正确的转录后修饰对于治疗蛋白的生产非常重要。

但是哺乳动物表达系统也具有细胞系生长缓慢、生长环境要求严格、易污染、费用高昂等缺点。

哺乳动物细胞已经成为治疗蛋白生产的主力军，生产的治疗蛋白有上百种。

非洲绿猴肾细胞（COS ）适合于小规模的单抗制备，如果要大规模地生产，最常用的是中国仓鼠卵巢细胞（CHO ）细胞。

图1、哺乳动物细胞表达系统生产重组蛋白的流程示意图。

转染哺乳动物细胞的转染有瞬时转染和稳定转染两种。

瞬时转染不会将载体整合到宿主基因组中，可以快速生产少量的蛋白，瞬时转染还可以帮助测试比拟不同的载体，这对建立稳定表达细胞系非常有必要。

瞬时转染方法有氮化钙法、磷酸钙法、DEAE —葡聚糖法、PEI 法和电穿孔法等。

瞬时转染适用的细胞系有COS （非洲绿猴肾细胞）、BHK （叙利亚仓鼠肾细胞）、HEK 293 （人胚肾细胞293）等等。

这几个细胞系能进行快速转染，可以悬浮培养，还能在不含血清的培养基中生长，这有助于表达蛋白的纯化并减少污染的可能性。

当用PEI 法对HEK293细胞系进Host cell lineTransfectionRepeat with increasing drug concentrationsExpression vector TTTTTTT lllllll©Single Cell Cloning or ILimiting Dilution fPreliminary clone evaluation and Expansion of selected clones行瞬时转染，可以得到50-80%成功表达GFP (绿色荧光蛋白)的重组子。

pEGFP-N1哺乳细胞表达载体使用说明

pEGFP-N1哺乳细胞表达载体使用说明
货号：P6460
规格：20μL
保存：-20°C
载体信息：
启动子：CMV
复制子：pUC ori，f1ori
终止子：SV40ploy（A）
载体别名：pEGFPN1，pEGFP N1
载体分类：哺乳细胞表达载体，荧光蛋白报告载体
载体大小：4733bp
载体标签：增强型荧光蛋白报告基因EGFP
原核抗性：卡那霉素（Kanamycin）
筛选标记：新霉素（Neomycin）
克隆菌株：DH5α
表达宿主：哺乳细胞
培养条件：37℃，LB培养基
5'测序引物：CMV-F:5'-CGCAAATGGGCGGTAGGCGTG-3'
3'测序引物：EGFP-N:5'-CGTCGCCGTCCAGCTCGACCAG-3'
备注：载体的MCS多克隆酶切位点，有EcoR I，BamH I等酶切位点。

载体简介：
pEGFP-N1编码野生型GFP的一个红移变体，已被优化，有更亮的荧光，在哺乳细胞有
更高的表达，（最大激发波长=488nm，最大释放波长=507nm）。

EGFP基因编码序列，含有超过190个沉默碱基的变化，这符合人类密码子使用偏好。

EGFP序列的一侧已经转变成和KozaK 一致的翻译起始位点，进一步提高了在真核细胞的翻译效率。

载体图谱：。