| 出品公司: | ZYbscience |
|---|---|
| 试剂名称: | pCL-Eco |
| 质粒类型: | 哺乳动物载体;逆病毒包装;单质粒包装系统 |
| 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 |
| 载体大小: | 12341 bp |
| 5' 测序引物及序列: | CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’ |
| 3' 测序引物及序列: | -- |
| 载体标签: | 无 |
| 载体抗性: | Ampicillin |
| 筛选标记: | 无 |
| 克隆菌株: | DH5α等 |
| 宿主细胞(系): | 包装细胞系如293T |
| 备注: | 逆病毒包装载体pCL-Eco是单质粒包装系统,快速产生Helper-free、毒力高的逆病毒颗粒。 |
| 产品目录号: | ZY12371 |
| 稳定性: | 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件:
-20℃低温保存
- 保质期:
三年
- 英文名:
pCL-Eco
- 库存:
20
- 供应商:
泽叶生物
载体基本信息
载体质粒图谱和多克隆位点信息

载体简介
逆病毒包装载体pCL-Eco的使用方法
293T Growth Medium:
450 ml DMEM (JRH Biosciences)
50 ml FBS (JRH Sciences)
5 ml Glutamine (200 mM in saline, JRH Sciences)
2.5 ml Gentamycin (10 mg/ml in water, Sigma)
HC11 Growth Medium:
5 ml L-Glutamine (200 mM in saline, JRH Sciences)
2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)
2.5 ml Gentamycin (10 mg/ml in water, Sigma)
0.5 ml EGF (10 μg/ml in water, Gibco)
50 ml FBS (JRH Sciences)
440 ml RPMI (JRH Sciences)
Day 0: Split 293T cells for transfection:
Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube
Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes
Spin at 1000 rpm in clinical centrifuge for 1-2 minutes
Resuspend cells in 10 ml growth medium
Count cells
Plate cells at 1X106 cells per 100 mm dish
Day 2: Transfect 293T cells:
Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total
volume per 100 mm dish
Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of
200 μl
Mix DNA plasmids in polypropelene tubes (Falcon #2063):
1 μg DNA of interest
1 μg pCL-Eco
Add 200 μl diluted FuGene dropwise to each DNA tube
Incubate RT 15 min
Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media
Day 3: Change medium:
• Remove viral medium and CAREFULLY feed with 6 ml growth medium
This step may reduce a cytostatic factor (produced after transfections) that
can inhibit growth and infection of your target cells.
Split HC11 cells for infection
Seed HC11 cells at 300,000 cells per 100 mm dish
If using coverslips, incubate with FBS for at least 1 hour prior to plating
Day 4: Infect target (HC11) cells:
• Collect virus-containing media off 293T cells
• Syringe filter media through .45 μm filter onto target cells
(Evaporation O/N and loss in filter leaves about 5 ml per plate)
• Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)
(Store polybrene in aliquots to decrease freeze/thaw cycles)
• Wrap plates in parafilm
• Spin cells in clinical centrifuge 10 min at 1800 rpm
• Rotate plate 1/3. Spin 10 min at 1800 rpm
• Rotate plate 1/3. Spin 10 min at 1800 rpm
• Remove viral media and replace with fresh growth media
Day 6+: Harvest HC11 cells:
• Wash cells with HBSS
• Scrape remaining cells, pellet and flash freeze for protein/RNA analysis
Stain cells for beta-gal expression:
Fixing Solution :
1.35 ml 37% Formaldehyde
0.2 ml Gluteraldehyde
1X PBS to 25 mls
Staining Solution:
625μl 40 mg/ml X-gal in DMF
50 μl 1M MgCL2
750 μl 100 mM Potassium Ferricyanide
750 μl 100 mM Potassium Ferrocyanide
1X PBS to 25 mls
[100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]
[100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]
Store at room temperature, protect from light.
1. Make -gal solution and warm in 37°C water bath to prevent crystallization
2. Aspirate off media
3. Wash cells 1X PBS
4. Fix cells 5 min at RT in fixing solution
5. Wash 3X PBS
6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces
crystallization)
7. Stain cells with staining solution at 37°C for 1-24 hrs
8. Store stained cells in 70% EtOH at 4°C
(EMBO J. 5: pg 3133, 1986)
Note:
HC11 and MEC primary culture cells are very adherent and can withstand
the force of spinning the plates at 1800 rpm. We found that HC11 cells died at
2200 rpm. However, this is cell type-dependent, and spinning speed should be
optimized accordingly.
Expect to see -gal staining at the periphery of the plate. During the spin
infection the media is pushed to the outside. The cells in the center of the plate
will not be exposed to virus and will not be efficiently infected with retrovirus.
载体序列
LOCUS pCL-Eco Sequencing Result 12341 bp DNA SYN
DEFINITION pCL-Eco Sequencing Result
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..12341
/organism="pCL-Eco Sequencing Result"
/mol_type="other DNA"
rep_origin 267..886
/label="pBR322_origin"
misc_feature complement(1160..1179)
/label="SV40pro_F_primer"
rep_origin complement(1164..1241)
/label="SV40_origin"
terminator complement(1709..1828)
/label="SV40_PA_terminator"
misc_feature complement(1723..1742)
/label="EBV_rev_primer"
misc_feature complement(2253..2300)
/label="SV40_3_splice"
misc_feature complement(2306..2321)
/label="SV40_int"
misc_feature 2540..2562
/label="pGEX_3_primer"
CDS complement(3033..5030)
/label="ORF frame 1"
/translation="MARSTLSKPLKNKVNPRGPLIPLILLMLRGVSTASPGSSPHQVY
NITWEVTNGDRETVWATSGNHPLWTWWPDLTPDLCMLAHHGPSYWGLEYQSPFSSPPG
PPCCSGGSSPGCSRDCEEPLTSLTPRCNTAWNRLKLDQTTHKSNEGFYVCPGPHRPRE
SKSCGGPDSFYCAYWGCETTGRAYWKPSSSWDFITVNNNLTSDQAVQVCKDNKWCNPL
VIRFTDAGRRVTSWTTGHYWGLRLYVSGQDPGLTFGIRLRYQNLGPRVPIGPNPVLAD
QQPLSKPKPVKSPSVTKPPSGTPLSPTQLPPAGTENRLLNLVDGAYQALNLTSPDKTQ
ECWLCLVAGPPYYEGVAVLGTYSNHTSAPANCSVASQHKLTLSEVTGQGLCIGAVPKT
HQALCNTTQTSSRGSYYLVAPTGTMWACSTGLTPCISTTILNLTTDYCVLVELWPRVT
YHSPSYVYGLFERSNRHKREPVSLTLALLLGGLTMGGIAAGIGTGTTALMATQQFQQL
QAAVQDDLREVEKSISNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYA
DHTGLVRDSMAKLRERLNQRQKLFESTQGWFEGLFNRSPWFTTLISTIMGPLIVLLMI
LLFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKPIEYEP*"
CDS complement(4970..8242)
/label="ORF frame 2"
/translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
AP*"
misc_feature complement(5079..5095)
/label="pBMN_5_primer"
/translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW
AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ
SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL
DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL
ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ
VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP
LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL
TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA
VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD
ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG
TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN
KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL
IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ
LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH
RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR
FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK
LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP
SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK
GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE
AP*"
CDS complement(8570..10186)
/label="ORF frame 2"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(9770..10177)
/label="gag"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(9783..9799)
/label="pBABE_5_primer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(9815..9837)
/label="MSCV_primer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(9815..9837)
/label="pLXSN_5_primer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(10319..11018)
/label="5_LTR"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
promoter complement(10497..11077)
/label="CMV_immearly_promoter"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(10524..10544)
/label="CMV_fwd_primer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature complement(10710..10997)
/label="CAG_enhancer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
misc_feature 11342..11364
/label="pGEX_3_primer"
/translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP
TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH
PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE
DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR
AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT
LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR
QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN
VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE
KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK
EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"
promoter 11523..11551
/label="AmpR_promoter"
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文献和实验物中。而D,L―PLA具有较小的结晶度,是一种完全无定形的共聚物,常用作药物控释载体。meso―PLA不常使用。由于I―PLA的降解产物L―乳酸能被人体完全代谢,因此它具有无毒、无组织反应的特殊优点,更具竞争力。 2.聚己内酯(PCL) 起初人们只认识到PCL能被微生物降解,故大量作为包装材料使用,后来发现PCL在生理条件下也能水解,因而引发出其在医学领域的应用。与PGA和PLA比较,PCL因结晶性太强而导致降解速率要慢得多,因此PCL更适合作为长效植入药物的控释载体
O,或最终通过肾排出体外。PCL的降解速度远低于PLA,因此它可用于制成长效、可植人性的药物载体。PCL无毒,具有良好的组织相容性。在欧洲,PCL在临床上已被用作骨钉。 聚原酸酯类主要用于对蛋白质,肪类等温度或溶剂敏感药物的释放;聚碳酸酯是一种表面浸蚀材料;聚酸酐片不仅可保护药物,而且使药物的有效释放达4星期,大约和聚酸酐的降解完毕时间相同。由于聚酸酐片将药物定位输送到脑部,其组织毒性也大大降低。通过大量动物实验证明这些聚酸酐是安全的。卓仁禧等根据肿瘤细胞的磷酸配酶活性高于
粥样硬化 (atherosclerosis, AS) 的形成1,2。过表达突变的Pcsk9D377Y,能加速LDLR的降解,严重影响血浆中LDL-C的清除能力,最终导致高脂血症。因此我们利用这原理,以AAV作为过表达Pcsk9D377Y的载体,使用APOE的启动子确保能在肝脏中特异表达,诱导野生型小鼠产生动脉粥样硬化,AAV结构如图1所示。 图1. AAV9-Pcsk9D377Y示意图 该模型的构建方法也十分简单,AAV病毒进入循环系统后能倾向于在肝脏中富集,因此,病毒的递送至肝脏十分简单。构建模型时,野生型C57
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