CN110791502B - 人源rbp4启动子片段及其应用 - Google Patents

人源rbp4启动子片段及其应用 Download PDF

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CN110791502B
CN110791502B CN201911227955.XA CN201911227955A CN110791502B CN 110791502 B CN110791502 B CN 110791502B CN 201911227955 A CN201911227955 A CN 201911227955A CN 110791502 B CN110791502 B CN 110791502B
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程佳
王晨颖
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Abstract

本发明公开一种人源RBP4启动子片段，包含如SEQ No.1、SEQ No.2、SEQ No.3、SEQ No.4、SEQ No.5、SEQ No.6和SEQ No.7所示DNA序列的7个片段；同时，本发明还公开含有所述人源RBP4启动子片段的重组表达载体、含有所述人源RBP4启动子片段的宿主菌，以及所述人源RBP4启动子片段在启动子活性检测中的应用。本发明提供的重组表达载体pGL3‑hRBP4 promoter(1‑7)能进行RBP4启动子活性区域的鉴定，为RBP4启动子的认识和应用提供一定的理论基础。

Description

人源RBP4启动子片段及其应用
技术领域
本发明属于细胞生物技术与分子生物技术领域，具体涉及一种人源RBP4启动子片段及其应用。
背景技术
视黄醇结合蛋白(retinol binding protein, RBP)是一类疏水性转运蛋白，而视黄醇结合蛋白4（RBP4）是目前为止已经证实的唯一一类在血液中转运视黄醇的蛋白，主要在肝脏中表达。RBP4基因位于染色体10q，mRNA全长914bp，相对分子质量为21kD，由181个氨基酸组成。体内RBP4主要负责视黄醇的储存、吸收，并将视黄醇及其活性代谢产物从肝脏运输到靶组织。国内外大量研究证实RBP4在慢性炎症、代谢综合征、糖尿病血管病变等病理生理过程中发挥着重要作用，然而RBP4促使相关疾病发生、发展的分子机制仍在探索中。
在人体中，血清RBP4水平不仅与代谢综合征(metabolic syndrome, MS)的多项组分如高三酰甘油(triglyceride, TG)/高密度脂蛋白胆固醇(high density lipoproteincholesterol, HDL-C)比值、胰岛素抵抗指数(HOMA-IR)、空腹血糖(fasting bloodglucose, FBG)、收缩压(systolic blood pressure, SBP)、舒张压(diastolic bloodpressure, DBP)等密切相关。而且，现有研究已证实，肥胖导致的血清RBP4水平升高会刺激机体产生适应性免疫反应并发生组织炎症，而脂肪组织炎症引发的胰岛素作用受损被认为是导致II型糖尿病发生的主要原因。除此之外，RBP4还参与高血压、非酒精性脂肪肝甚至是癌症的发病过程。
荧光素酶(Luciferase)是生物体内催化荧光素或者脂肪醛氧化发光的一类酶的总称。荧光素酶可以从细茵、萤火虫、海星等提取而来。应用荧光素酶大大增加了生物发光免疫分析技术(bioluminescent immunoassay, BLIA)的灵敏度和应用范围，并且由于具有敏感性高、特异性好、反应迅速、操作简单、应用范围广等优点，已成为医学、生物学、环境科学等研究领域中一种新的手段。
由于RBP4在病理生理过程中发挥着重要作用，所以对于研究人源RBP4促使相关疾病发生、发展的分子机制变得十分重要。
发明内容
本发明提供一种人源RBP4启动子片段，确定RBP4启动子的活性区域，同时提供含有所述人源RBP4启动子片段的重组表达载体、所述人源RBP4启动子片段在启动子活性检测中的应用。
人源RBP4启动子片段，所述人源RBP4启动子片段包含如SEQ No.1、SEQ No.2、SEQNo.3、SEQ No.4、SEQ No.5、SEQ No.6和SEQ No.7所示DNA序列的7个片段。
一种含有所述人源RBP4启动子片段的重组表达载体，所述重组表达载体为pGL3-hRBP4 promoter(1-7)；所述pGL3-hRBP4 promoter(1-7)包含pGL3-hRBP4 promoter1、pGL3-hRBP4 promoter2、pGL3-hRBP4 promoter3、 pGL3-hRBP4 promoter4、 pGL3-hRBP4promoter5、 pGL3-hRBP4 promoter6、 pGL3-hRBP4 promoter7 7个载体，分别与SEQ No.1、SEQ No.2、SEQ No.3、SEQ No.4、SEQ No.5、SEQ No.6和SEQ No.7所示DNA序列的7个片段相对应。
优选地，所述重组表达载体是通过以下方法得到的：将所述人源RBP4启动子片段经Xho I和HindⅢ双酶切，连接至表达载体pGL3-basic，得到重组表达载体pGL3-hRBP4promoter(1-7)。
一种含有所述人源RBP4启动子片段的宿主菌，所述宿主菌为大肠杆菌。
一种所述重组表达载体的宿主菌，所述宿主菌为大肠杆菌。
所述人源RBP4启动子片段在启动子活性检测中的应用。
优选地，所述人源RBP4启动子片段在启动子活性检测中的应用为：将所述人源RBP4启动子片段连接于重组表达载体中，然后将所述重组表达载体转化到真核细胞中进行启动子活性区域的鉴定。
本发明的优点：
本发明提供的重组表达载体pGL3-hRBP4 promoter(1-7)能进行RBP4启动子活性区域的鉴定，为RBP4启动子的认识和应用提供一定的理论基础，具体如下：
1. 本发明提供的重组表达载体pGL3-hRBP4 promoter(1-7)，一方面提供了 人源RBP4启动子片段序列，另一方面能用于鉴定RBP4启动子的活性区域，在此基础上，有助于对RBP4基因的改造研究以及调控RBP4的表达与分泌；
2. 本发明提供的重组表达载体pGL3-hRBP4 promoter(1-7)，能用于研究与RBP4启动子相结合的转录因子，并验证其激活RBP4基因表达的有效性，有助于进一步阐述在转录水平上参与调控RBP4表达的分子机制。
附图说明
图1 重组表达载体示意图。
图2 人源RBP4启动子片段扩增图； Marker DS2000；1~7泳道分别为RBP4启动子片段-1968/+98、-1795/+98、-1367/+98、-721/+98、-416/+98、-210/+98、-92/+98。
图3 人源RBP4启动子片段pGL3-hRBP4-promoter(1-7)的双酶切鉴定； MarkerD5000; 1~7泳道分别为RBP4启动子片段-1968/+98、-1795/+98、-1367/+98、-721/+98、-416/+98、-210/+98、-92/+98。
图4 pGL3-hRBP4-promoter(1-7)荧光素酶活性分析。
图5 人源RBP4启动子活性区域示意图。
具体实施方式
本发明实施例中，如无特殊说明，均为常规方法；所用的材料，如无特殊说明，均为市售购买产品。
步骤一. 引物的设计
根据NCBI人基因组序列（GeneBank No. NC_000010.11），用Primer Premier5.0软件设计人源RBP4基因启动子引物及其启动子缺失片段引物，并加入特异性酶切位点Hind Ⅲ和Xho I。引物序列（如表1所示）。
表1 人源RBP4启动子片段的克隆引物
Figure 706341DEST_PATH_IMAGE001
注：F，正向引物；R，反向引物；下划线为酶切位点。
步骤二. 人源RBP4启动子片段插入载体pGL3-basic中方向的确定
使用SnapGene软件打开pGL3-basic载体图谱，分别选择Xho I和Hind Ⅲ做为上游和下游酶切位点，并用相应的上、下游引物确定RBP4启动子的方向性。
步骤三. RBP4基因启动子扩增
人胚肾HEK293T细胞基因组的提取：取约2×106个HEK293T细胞，参照DNA提取剂盒操作说明书提取DNA，分装并保存于-20℃备用；然后，以HEK293T细胞基因组DNA为模板，进行PCR，得到RBP4启动子各个片段(如图2所示)。其中，PCR反应体系如表2，PCR反应条件如表3；
表2 PCR反应体系
Figure DEST_PATH_IMAGE003A
表3 PCR反应条件
Figure DEST_PATH_IMAGE004
。
步骤四. 重组表达载体的构建
以人胚肾HEK293T细胞基因组DNA为模板，经PCR分别获得RBP4基因启动子区长度不同的7个片段(如图2所示)，经Hind Ⅲ和Xho I双酶切使其产生粘性末端，再依次连接至pGL3-basic载体，得到重组表达载体pGL3-hRBP4 promoter(1-7)。经宿主菌的转化与扩繁，获得相应质粒后，用Hind Ⅲ和Xho I双酶切进行鉴定，出现两条特异性条带(如图3所示)，说明RBP4启动子片段成功插入pGL3-basic载体。经测序和比对显示序列正确，表明成功构建含RBP4基因启动子片段的重组报告基因载体。
步骤五. 转染细胞和荧光素酶活性分析
将含有7个RBP4启动子5`端系统性缺失片段的荧光素酶报告载体pGL3-hRBP4-promoter(1-7)（即重组表达载体），分别与海肾萤光素酶报告基因载体瞬时转染HEK293T细胞，48 h后收集细胞，通过酶标仪检测其荧光素酶相对活性；
按照荧光素酶报告基因的工作原理，荧光素酶的表达越多，说明外接基因启动子的活性越强。图4的结果表明，RBP4启动子的活性在不同片段中存在较大的差别，其中pGL3-hRBP4-promoter4的荧光素酶相对活性最强，而其他重组载体的活性均与对照组pGL3-basic没有显著性差异；
接下来，通过分析RBP4启动子荧光素酶相对活性的结果，说明在RBP4启动子上-721~+98之间的序列中含有RBP4启动子的活性中心。然而 pGL3-hRBP4-promoter5的荧光素酶相对活性又有所下降，说明在RBP4启动子上-416~+98之间的序列中已经不再包含RBP4启动子的活性中心。继而推测RBP4启动子的活性中心是主要位于RBP4启动子上-721~-416之间的片段 (如图5所示)。
SEQUENCE LISTING
<110> 陕西理工大学
<120> 人源RBP4启动子片段及其应用
<130>
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 2065
<212> DNA
<213> RBP4-1968/+98
<400> 1
gcacctacta tgaacaagtc tcttgatata cccagtggaa aagtggggag aatggggaca 60
ggtacaaaga tgaatgaaag aaacatggtc tgtctctcca tgaagcatgg caaggaaatg 120
aatgaaattt tgtgtacatt atatacgatc tataagatgt aatttaaaac ataattttac 180
ctacaggaaa aggagcaaga aaaatatttg taaaggaaat atttgcttta ttttctgatt 240
cacgttttca tcatagaaaa ttttaaaaat acaaaaaggc gcaaagaaaa aatctttcag 300
cgtccctaat cctatctcac agagatattc actatgaaca tttttccttt tgactttaca 360
tatacacata tttgaaaagt taatgtatga ggattttttc caaagtactg aagttaagaa 420
catcagttga cctgaattaa cttatttgta atcatgaaga aatcctagaa agaatgccaa 480
agtaaactgt tttcctctgc tggtatctgg agcactaagg tccatgattt atcattaaaa 540
gaggaggggc agaggaatgg ttacaccaac tttctttttt cttttttcac ctcaaatttt 600
tactctgaaa caacctttaa acacccaaca aataaatgaa aacttgttgc tgcatcaaaa 660
ccatcatcga aatgaactga aatacccttc attattcaac ctacagtgtg tgcaagggcc 720
accagaatgt gttctaggag aaatggctgc aaggccaggg ggtattagga gaacgtttct 780
ggcaggaata actgtccaaa ggcaactggg ctaagctagg agctctctat cactaaagaa 840
cctggagcaa aaactggaag acctctcctc tggaatgccg aagaggggat ccaagtattg 900
ggcaggagat agagaggtgc cctaaaatcc tttcccaccc agggtgcata gatatatacc 960
ccatagggtc ctgcaggaga cgatctgaag cagaacttat ttgagctgtt tgggattaca 1020
cagtcttcta taaaactggc ccaatcagaa gatttcctag tcagcttgtt gtccagaaac 1080
aggctttcct agctctgcgg cggttaggag ttaaatgcat caccgttggc tccccagaca 1140
ctgctgggac tcaaatgggt gcaagagtga aggggaaggt gggaaaaaga tggagaagca 1200
ggggggaagg aagccacaga tgtcatcttt tcaggatgct cagaattagc cctaatgccc 1260
tatagataaa ggattggtct gagaaccaat caactcctta tttctgtttg gcagccacct 1320
tcctcttccc cgatctcctc ttatttcagg tcgggctgca gcaaaatggc ccaaattgat 1380
ctcgaatctt tccaatttag gccatggaga cacaatcgat attgtatgga aagaatctca 1440
agaagagata cttctttgcc acatcatgaa ttctcattcc gtcattctca tcctttgatt 1500
ggctgctttg atcaaacgag tggaactcta acttcgaaca gaaagagaaa aacagggtca 1560
gtaaattgtg ccatcacaca ggaaaatacc taaactagtc acactgtttt gattcaattg 1620
gctactgaag ttatagaatg ttgtttactc ttctctcctt tgtctactcc ccagccaaca 1680
aaacaaccga ccttagctgt tttgaaaata aatgaaaatt ccaacatggg tttgaaataa 1740
aattgcatca taaacaatcg gtaggtgttt ttcaaagtgg tttcagggaa gtgccacgga 1800
gtaagcaggc gaccaccgag gctgctaaaa tatttcctgt cctgaccagg gttgcgtttc 1860
tggagaatat ttaacaggga gggttttaac gcttttaaag atgttgaaac taaagaacaa 1920
atattgacca gagggcacca caacgctcct gaaagagagt aaaatacatc ctttataaaa 1980
tgaaaaacta cttggatgaa ttattccaaa attcctgcac aagtggacct cagaaggcag 2040
acggaggcgc caatttggca tggcc 2065
<210> 2
<211> 1892
<212> DNA
<213> RBP4-1795/+98
<400> 2
attttaccta caggaaaagg agcaagaaaa atatttgtaa aggaaatatt tgctttattt 60
tctgattcac gttttcatca tagaaaattt taaaaataca aaaaggcgca aagaaaaaat 120
ctttcagcgt ccctaatcct atctcacaga gatattcact atgaacattt ttccttttga 180
ctttacatat acacatattt gaaaagttaa tgtatgagga ttttttccaa agtactgaag 240
ttaagaacat cagttgacct gaattaactt atttgtaatc atgaagaaat cctagaaaga 300
atgccaaagt aaactgtttt cctctgctgg tatctggagc actaaggtcc atgatttatc 360
attaaaagag gaggggcaga ggaatggtta caccaacttt cttttttctt ttttcacctc 420
aaatttttac tctgaaacaa cctttaaaca cccaacaaat aaatgaaaac ttgttgctgc 480
atcaaaacca tcatcgaaat gaactgaaat acccttcatt attcaaccta cagtgtgtgc 540
aagggccacc agaatgtgtt ctaggagaaa tggctgcaag gccagggggt attaggagaa 600
cgtttctggc aggaataact gtccaaaggc aactgggcta agctaggagc tctctatcac 660
taaagaacct ggagcaaaaa ctggaagacc tctcctctgg aatgccgaag aggggatcca 720
agtattgggc aggagataga gaggtgccct aaaatccttt cccacccagg gtgcatagat 780
atatacccca tagggtcctg caggagacga tctgaagcag aacttatttg agctgtttgg 840
gattacacag tcttctataa aactggccca atcagaagat ttcctagtca gcttgttgtc 900
cagaaacagg ctttcctagc tctgcggcgg ttaggagtta aatgcatcac cgttggctcc 960
ccagacactg ctgggactca aatgggtgca agagtgaagg ggaaggtggg aaaaagatgg 1020
agaagcaggg gggaaggaag ccacagatgt catcttttca ggatgctcag aattagccct 1080
aatgccctat agataaagga ttggtctgag aaccaatcaa ctccttattt ctgtttggca 1140
gccaccttcc tcttccccga tctcctctta tttcaggtcg ggctgcagca aaatggccca 1200
aattgatctc gaatctttcc aatttaggcc atggagacac aatcgatatt gtatggaaag 1260
aatctcaaga agagatactt ctttgccaca tcatgaattc tcattccgtc attctcatcc 1320
tttgattggc tgctttgatc aaacgagtgg aactctaact tcgaacagaa agagaaaaac 1380
agggtcagta aattgtgcca tcacacagga aaatacctaa actagtcaca ctgttttgat 1440
tcaattggct actgaagtta tagaatgttg tttactcttc tctcctttgt ctactcccca 1500
gccaacaaaa caaccgacct tagctgtttt gaaaataaat gaaaattcca acatgggttt 1560
gaaataaaat tgcatcataa acaatcggta ggtgtttttc aaagtggttt cagggaagtg 1620
ccacggagta agcaggcgac caccgaggct gctaaaatat ttcctgtcct gaccagggtt 1680
gcgtttctgg agaatattta acagggaggg ttttaacgct tttaaagatg ttgaaactaa 1740
agaacaaata ttgaccagag ggcaccacaa cgctcctgaa agagagtaaa atacatcctt 1800
tataaaatga aaaactactt ggatgaatta ttccaaaatt cctgcacaag tggacctcag 1860
aaggcagacg gaggcgccaa tttggcatgg cc 1892
<210> 3
<211> 1464
<212> DNA
<213> RBP4-1367/+98
<400> 3
actctgaaac aacctttaaa cacccaacaa ataaatgaaa acttgttgct gcatcaaaac 60
catcatcgaa atgaactgaa atacccttca ttattcaacc tacagtgtgt gcaagggcca 120
ccagaatgtg ttctaggaga aatggctgca aggccagggg gtattaggag aacgtttctg 180
gcaggaataa ctgtccaaag gcaactgggc taagctagga gctctctatc actaaagaac 240
ctggagcaaa aactggaaga cctctcctct ggaatgccga agaggggatc caagtattgg 300
gcaggagata gagaggtgcc ctaaaatcct ttcccaccca gggtgcatag atatataccc 360
catagggtcc tgcaggagac gatctgaagc agaacttatt tgagctgttt gggattacac 420
agtcttctat aaaactggcc caatcagaag atttcctagt cagcttgttg tccagaaaca 480
ggctttccta gctctgcggc ggttaggagt taaatgcatc accgttggct ccccagacac 540
tgctgggact caaatgggtg caagagtgaa ggggaaggtg ggaaaaagat ggagaagcag 600
gggggaagga agccacagat gtcatctttt caggatgctc agaattagcc ctaatgccct 660
atagataaag gattggtctg agaaccaatc aactccttat ttctgtttgg cagccacctt 720
cctcttcccc gatctcctct tatttcaggt cgggctgcag caaaatggcc caaattgatc 780
tcgaatcttt ccaatttagg ccatggagac acaatcgata ttgtatggaa agaatctcaa 840
gaagagatac ttctttgcca catcatgaat tctcattccg tcattctcat cctttgattg 900
gctgctttga tcaaacgagt ggaactctaa cttcgaacag aaagagaaaa acagggtcag 960
taaattgtgc catcacacag gaaaatacct aaactagtca cactgttttg attcaattgg 1020
ctactgaagt tatagaatgt tgtttactct tctctccttt gtctactccc cagccaacaa 1080
aacaaccgac cttagctgtt ttgaaaataa atgaaaattc caacatgggt ttgaaataaa 1140
attgcatcat aaacaatcgg taggtgtttt tcaaagtggt ttcagggaag tgccacggag 1200
taagcaggcg accaccgagg ctgctaaaat atttcctgtc ctgaccaggg ttgcgtttct 1260
ggagaatatt taacagggag ggttttaacg cttttaaaga tgttgaaact aaagaacaaa 1320
tattgaccag agggcaccac aacgctcctg aaagagagta aaatacatcc tttataaaat 1380
gaaaaactac ttggatgaat tattccaaaa ttcctgcaca agtggacctc agaaggcaga 1440
cggaggcgcc aatttggcat ggcc 1464
<210> 4
<211> 818
<212> DNA
<213> RBP4-721/+98
<400> 4
agccctaatg ccctatagat aaaggattgg tctgagaacc aatcaactcc ttatttctgt 60
ttggcagcca ccttcctctt ccccgatctc ctcttatttc aggtcgggct gcagcaaaat 120
ggcccaaatt gatctcgaat ctttccaatt taggccatgg agacacaatc gatattgtat 180
ggaaagaatc tcaagaagag atacttcttt gccacatcat gaattctcat tccgtcattc 240
tcatcctttg attggctgct ttgatcaaac gagtggaact ctaacttcga acagaaagag 300
aaaaacaggg tcagtaaatt gtgccatcac acaggaaaat acctaaacta gtcacactgt 360
tttgattcaa ttggctactg aagttataga atgttgttta ctcttctctc ctttgtctac 420
tccccagcca acaaaacaac cgaccttagc tgttttgaaa ataaatgaaa attccaacat 480
gggtttgaaa taaaattgca tcataaacaa tcggtaggtg tttttcaaag tggtttcagg 540
gaagtgccac ggagtaagca ggcgaccacc gaggctgcta aaatatttcc tgtcctgacc 600
agggttgcgt ttctggagaa tatttaacag ggagggtttt aacgctttta aagatgttga 660
aactaaagaa caaatattga ccagagggca ccacaacgct cctgaaagag agtaaaatac 720
atcctttata aaatgaaaaa ctacttggat gaattattcc aaaattcctg cacaagtgga 780
cctcagaagg cagacggagg cgccaatttg gcatggcc 818
<210> 5
<211> 513
<212> DNA
<213> RBP4-416/+98
<400> 5
cagggtcagt aaattgtgcc atcacacagg aaaataccta aactagtcac actgttttga 60
ttcaattggc tactgaagtt atagaatgtt gtttactctt ctctcctttg tctactcccc 120
agccaacaaa acaaccgacc ttagctgttt tgaaaataaa tgaaaattcc aacatgggtt 180
tgaaataaaa ttgcatcata aacaatcggt aggtgttttt caaagtggtt tcagggaagt 240
gccacggagt aagcaggcga ccaccgaggc tgctaaaata tttcctgtcc tgaccagggt 300
tgcgtttctg gagaatattt aacagggagg gttttaacgc ttttaaagat gttgaaacta 360
aagaacaaat attgaccaga gggcaccaca acgctcctga aagagagtaa aatacatcct 420
ttataaaatg aaaaactact tggatgaatt attccaaaat tcctgcacaa gtggacctca 480
gaaggcagac ggaggcgcca atttggcatg gcc 513
<210> 6
<211> 307
<212> DNA
<213> RBP4-210/+98
<400> 6
cggtaggtgt ttttcaaagt ggtttcaggg aagtgccacg gagtaagcag gcgaccaccg 60
aggctgctaa aatatttcct gtcctgacca gggttgcgtt tctggagaat atttaacagg 120
gagggtttta acgcttttaa agatgttgaa actaaagaac aaatattgac cagagggcac 180
cacaacgctc ctgaaagaga gtaaaataca tcctttataa aatgaaaaac tacttggatg 240
aattattcca aaattcctgc acaagtggac ctcagaaggc agacggaggc gccaatttgg 300
catggcc 307
<210> 7
<211> 189
<212> DNA
<213> RBP4-92/+98
<400> 7
gggagggttt taacgctttt aaagatgttg aaactaaaga acaaatattg accagagggc 60
accacaacgc tcctgaaaga gagtaaaata catcctttat aaaatgaaaa actacttgga 120
tgaattattc caaaattcct gcacaagtgg acctcagaag gcagacggag gcgccaattt 180
ggcatggcc 189

Claims (6)

1.人源RBP4启动子片段，其特征在于：所述人源RBP4启动子片段为如SEQ ID No.4所示DNA序列的片段。
2.一种含有如权利要求1所述人源RBP4启动子片段的重组表达载体。
3.根据权利要求2所述重组表达载体，其特征在于：所述重组表达载体是通过以下方法得到的：将所述人源RBP4启动子片段经XhoI和HindⅢ双酶切，连接至表达载体pGL3-basic，得到重组表达载体pGL3-hRBP4 promoter4。
4.一种含有权利要求1所述人源RBP4启动子片段的宿主菌，其特征在于：所述宿主菌为大肠杆菌。
5.一种含有权利要求2或3所述重组表达载体的宿主菌，其特征在于：所述宿主菌为大肠杆菌。
6.权利要求1所述人源RBP4启动子片段在研究与RBP4启动子相结合的转录因子的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001080897A2 (en) * 2000-04-21 2001-11-01 Vical Incorporated Compositions and methods for in vivo delivery of polynucleotide-based therapeutics
CN105940109A (zh) * 2013-12-06 2016-09-14 国立健康与医学研究所 用于在受试者的视网膜色素上皮中表达目的多核苷酸的方法和药物组合物
CN112912112A (zh) * 2018-10-26 2021-06-04 Vrije布鲁塞尔大学 肝特异性核酸调节元件以及其方法及用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040208847A1 (en) * 2003-03-28 2004-10-21 Fabienne Rolling Method and vectors for selectively transducing retinal pigment epithelium cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001080897A2 (en) * 2000-04-21 2001-11-01 Vical Incorporated Compositions and methods for in vivo delivery of polynucleotide-based therapeutics
CN105940109A (zh) * 2013-12-06 2016-09-14 国立健康与医学研究所 用于在受试者的视网膜色素上皮中表达目的多核苷酸的方法和药物组合物
CN112912112A (zh) * 2018-10-26 2021-06-04 Vrije布鲁塞尔大学 肝特异性核酸调节元件以及其方法及用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Negative control of liver-specific gene expression：cloned human retinol-binding protein gene is repressed in HeLa cells;V.Colantuonil et al.;《The EMBO Journal》;19871231;第6卷(第3期);第631-636页 *
RBP4启动子克隆及VDR/VD3对RBP4表达调控作用研究;王晨颖;《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》;20201215(第12期);E059-44 *

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