CN111808878B - 胆红素代谢功能基因片段及改造的HepG2细胞 - Google Patents
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Abstract
本发明公开了一种胆红素代谢功能基因片段及改造的HepG2细胞，其中胆红素代谢功能基因片段，由UGT1A1、GS、OATP1B1三种基因片段顺序构成。以该基因片段改造的HepG2细胞，UGT1A1蛋白表达提高了120倍，相对于正常肝细胞提高了15倍；OATP1B1蛋白表达提高了接近10倍，基本接近正常肝细胞水平；反应到胆红素代谢酶活性的提高了15倍；凝血因子Ⅴ分泌提高了70％；凝血因子Ⅹ分泌提高了10倍，达到正常肝细胞的五分之一。
Description
技术领域
本发明涉及基因工程领域，具体地指一种胆红素代谢功能基因片段及改造的HepG2细胞。
背景技术
HepG2细胞是一种肝癌细胞。该细胞分泌多种血浆蛋白：清蛋白、α2-巨球蛋白、血纤维蛋白溶酶原、铁传递蛋白等。
现有HepG2细胞用做血液净化时存在胆红素代谢，凝血分泌等缺陷。
发明内容
本发明的目的在于克服现有HepG2细胞的缺陷，提供一种胆红素代谢功能基因片段及改造的HepG2细胞。
为实现上述目的，本发明首先提供了一种胆红素代谢功能基因片段，由UGT1A1、GS、OATP1B1三种基因片段顺序构成，其中，各基因所对应分泌的蛋白如下：
UGT1A1为尿苷二磷酸葡萄糖醛酸基转移酶1A1；
GS为谷氨酰胺合成酶；
OATP1B1为有机阴离子转运多肽1B1；
所述胆红素代谢功能基因片段用于HepG2细胞基因改造。
优选地，所述UGT1A1-GS-OATP1B1的序列如SEQ No.1所示。
本发明还提供了一种胆红素代谢功能基因表达质粒，包含上述胆红素代谢功能基因片段。
优选地，所述质粒还包括嘌呤霉素筛选压力基因片段，所述嘌呤霉素筛选压力基因片段连接于所述胆红素代谢功能基因片段的3’端。
进一步优选地，所述质粒还包括启动子片段pTBSCH1，位于述胆红素代谢功能基因片段的5’端。
更进一步优选地，所述质粒的序列如SEQ No.2所示。
本发明还提供了一种包含胆红素代谢功能基因的HepG2细胞，包含上述功能片段，或利用上述质粒改造HepG2细胞后获得。
本发明中改造HepG2细胞采用的质粒包含的功能基因为：pTBSCH1-UGT1A1-GS-OATP1B1-Puro，序列如SEQ No.2所示，由多基因顺序连而成，其中UGT1A1在HepG2细胞的胆红素代谢通路中表达量最低；GS为谷氨酰胺合成酶，与细胞控制氮代谢及能量代谢调节有关；通过全基因组测序分析，OATP1B1为跨膜转运蛋白家族的重要成员；Puro为嘌呤霉素筛选压力。
通过基因工程改造获得了HepG2-a细胞，相对于未改造的HepG2细胞，UGT1A1蛋白表达提高了120倍，相对于正常肝细胞提高了15倍；OATP1B1蛋白表达提高了接近10倍，基本接近正常肝细胞水平；反应到胆红素代谢酶活性的提高了15倍；凝血因子Ⅴ分泌提高了70％；凝血因子Ⅹ分泌提高了10倍，达到正常肝细胞的五分之一。其中凝血因子V和凝血因子Ⅹ分泌量的提高是由前述改造使细胞功能的综合提升所带来的效果。
从现有研究文献我们可以看到，没有证据显示针对UGT1A1、OATP1B1、GS的改造直接能获得本发明的结果。参考文献如下：
Induction of Human UGT1A1 by Bilirubin Through AhR Dependent[PathwayDrug Metabolism Letters,2008,2,231-237]
Accurate identification of UDP-glucuronosyltransferase 1A1(UGT1A1)inhibitors using UGT1A1-overexpressing HeLa cells[Xenobiotica,Early Online:1–9]
In vitro UGT1A1 inhibition by tyrosine kinase inhibitors andassociation with drug-induced hyperbilirubinemia[Cancer Chemotherapy andPharmacology，3 August 2018]
Age-Dependent Oxidative Stress Elevates Arginase 1 and UncoupledNitric Oxide Synthesis in Skeletal Muscle of Aged Mice[Oxidative Medicine andCellular Longevity olume 2019,Article ID 1704650,9pages]
UGT1A1 mutations and psychoses:towards understanding the relationshipwith unconjugated bilirubin[CNS Spectrums(2019),page1–3.
CambridgeUniversity Press 2019 doi:10.1017/S1092852919001251]
Clinical Importance of OATP1B1 and OATP1B3 in Drug-Drug Interactions[Drug Metab.Pharmacokinet.26(3):220227(2011).]
Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndromeby interrupting conjugated bilirubin reuptake into the liver[The Journal ofClinical Investigation http://www.jci.org Volume 122 Number 2 February 2012]
“Interaction of Sulfonylureas with Liver Uptake Transporters OATP1B1and OATP1B3[Basic Clin Pharmacol Toxicol,2018 Aug,123(2):147-154.]
Encapsulation of glutamine synthetase in mouse erythrocytes:a newprocedure for ammonia dethoxification.Biochem.Cell Biol.86:469-476(2008).]
Pivotal role of glutamine synthetase in ammonia detoxificationHepatology[Hepatology.2017 Jan,65(1):281-293.]
本发明的有益效果：所获得的HepG2-a细胞在胆红素代谢能力和关键凝血因子的分泌方面均有很大提升；HepG2-a为生物人工肝和肝组织工程构建提供新的种源细胞选择的可能。
附图说明
图1为本发明用于HepG2细胞改造的质粒结构图。
图2为HepG2和HepG2-a细胞的胆红素降解率比较图。
图3为HepG2和HepG2-a细胞梯度实验的胆红素降解率比较图。
图4为HepG2和HepG2-a细胞多个细胞因子表达量比较图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步的详细描述。以下实施例中采用的试剂或产品，未标明出处的均为市售，不用于限制本发明。
本发明中核心的功能基因片段由UGT1A1-GS-OATP1B1三个基因构成，各个基因为人源，公开序列可以在(NCBI GenBank)中查询到。本发明中合成质粒中的该基因片段序列如SEQ No.1所示。
实施例：HepG2细胞系改造的方案：
1)全基因组合成pTBSCH1-UGT1A1-GS-OATP1B1-Puro，序列如SEQ No.2。
2)当天晚上消化好HepG2细胞(美国模式菌种收集中心ATCC)，其序列在(NCBIGenBank)中可查，以40％-50％的密度种到T25中，过夜贴壁后，第二天上午进行转染。
3)转染试剂及材料
(质粒：转座子＝10:1)
混匀，静置5min,加入T25中，根据细胞状态进行换液。
4)细胞转染五天后，消化，并计数，每孔1000～2000个左右的细胞种96孔板。
5)待细胞长够一定数量后，继续种板筛选。经前期摸索阴性细胞(HepG2)后，以5μg/ml的筛选浓度进行筛选，得到改造后的HepG2-a细胞。
临床验证：
对改造后的HepG2-a-2-6(HepG2-a型工程改造细胞，第二批次，传的第六代)，细胞密度75％，直接用于实验。
A)胆红素降解实验
主要步骤及结果如下：
1.消化好细胞，计数，每个T25种500万个细胞，12h待其贴壁；
2.细胞贴壁后外源添加病人血浆，病人血浆用DMEM培养基稀释(含量为70％)，最后终体积为4.8ml添加；
3.每次取0.8ml上清，1200rpm，5min，之后样品送同济(华中科技大学同济医学院附属同济医院)生化分析仪测定胆红素降解量；
4.分别在6h和12h取样，如图2所示：
HepG2细胞胆红素降解率为：6h：11.6％ 12h：17.3％
HepG2-a细胞胆红素降解率为：6h：43.9％ 12h：53.5％
B)细胞梯度实验
将HepG2细胞，HepG2-a细胞按比例和血浆混合，其中改造细胞以梯度比例混合血浆，进行细胞梯度实验，结果如图3，我们发现细胞数量与血浆比例至少需要达到100万细胞/1ml。在HepG2-a细胞50万/1ml血浆情况下，6h血浆共培养实验中，细胞的胆红素代谢不明显，并且在12小时，细胞出现形态改变、脱水、细胞核扩散现象，细胞存活率接近70％。在HepG2-a细胞200万/1ml血浆情况下，6h血浆共培养实验中，胆红素代谢率达到44.5％。按人体肝细胞与血浆比例推算，在500万细胞/1ml血浆情况下，实验效果最佳。
细胞基因组分析：
第三方对HepG2细胞基因测序，结果和HepG2细胞比较显示两者不同：HepG2细胞存在插入缺失变异和单核苷酸多态性(SNP)。外显子区的缺失插入变异有216个，SNP有16936个。对于氨代谢和胆红素代谢途径的酶，有一些SNP变异，基因组测序结果总结如表1：
表1 HepG2细胞基因组分析
HepG2-a细胞的蛋白组测序的分析：
iTRAQ蛋白质组学的分析：HepG2-a细胞相比HepG2细胞，有162个蛋白表达发生上调，相对于正常肝细胞仍有318个蛋白发生下调。
如图4所示，相对于未改造的HepG2细胞，HepG2-a细胞中UGT1A1蛋白表达提高了120倍，相对于正常肝细胞提高了15倍；OATP1B1蛋白表达提高了接近10倍，基本接近正常肝细胞水平；反应到胆红素代谢酶活性的提高远高于15倍；凝血因子Ⅴ分泌提高了70％；凝血因子Ⅹ分泌提高了10倍，达到正常肝细胞的五分之一。
序列表
<110> 武汉仝干医疗科技股份有限公司
<120> 胆红素代谢功能基因片段及改造的HepG2细胞
<130> 200602-1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4799
<212> DNA
<213> UGT1A1-GS-OATP1B1(Artificial Sequence)
<400> 1
gttcgttgca acaaattgat gagcaatgct tttttataat gccaactttg tacaaaaaag 60
ttggcaccat ggctgtggag tcccagggcg gacgcccact tgtcctgtgt cccatgctgg 120
gaagatactg ttgatcccag tggatggcag ccactggctg agcatgcttg gggccatcca 180
gcagctgcag cagaggggac atgaaatagt tgtcctagca cctgacgcct cgttgtacat 240
cagagacgga gcattttaca ccttgaagac gtaccctgtg ccattccaaa gggaggatgt 300
gaaagagtct tttgttagtc tcgggcataa tgtttttgag aatgattctt tcctgcagcg 360
tgtgatcaaa acatacaaga aaataaaaaa ggactctgct cccacttact gcacaacaag 420
gagctcatgg cctccctggc agaaagcagc tttgatgtca tgctgacgga ccctttcctt 480
ccttgcagcc ccatcgtggc ccagtacctg tctctgccca ctgtattctt cttgcatgca 540
ctgccatgca gcctggaatt tgaggctacc cagtgcccca agcctctctc ctctcattca 600
gatcacatga ccttcctgca gcgggtgaag aacatgctca ttgccttttc acagaacttt 660
ctgtgcgacg tggtttattc cccgtatgca gagaggtgac tgtccaggac ctattgagct 720
ctgcatctgt ctggctgttt agaagtgact ttgtgaagga ttaccctagg cccatcatgc 780
ccaataactg ccttcaccaa aatccactat cccaggaatt tgaagcctac attaatgctt 840
ctggagaaca tggaattgtg gttttctctt tgggatcaat ggtctcagaa atggcaattg 900
ctgatgcttt gggcaaaatc ccggtacact ggaacccgac catcgaatct tgcgaacaac 960
acgatacttg ttaagtggct accccaaaac gatctgcttg gtcacccgat gacccgtgcc 1020
tttagaaagc atatgcaatg gcgttcccat ggtgatgatg cccttgtttg gtgatcagat 1080
ggacaatgca aagcgcatgg agactagttc tggaaatgac ttctgaagat ttagaaaatg 1140
ctctaaaagc agtcatcaat gacaaaagtt aagccttcac aaggaccgcc cggtggagcc 1200
gctggacctg gccgtgttct gggtggagtt tgtgatgagg cacaaggccc acgacctcac 1260
ctggtaccag taccattcct tggacgtgat tggtttcctc ttggccgtcg tgctgacagt 1320
ggccttcatc acctttaaat gttgtgctta tggctaccgg gcgagttaag aaagcccaca 1380
aatccaagac ccatttgcca actttcttgt acaaagttgg cattataaga aagcattgct 1440
tatcaatttg ttgcaacgaa catggccacc tcagcaagtt cccactcaaa tgtacttgtg 1500
cctgccccag ggtgagaaag tccaagccat gtatatctgg gttgatggta ctggagaagg 1560
actgcgctgc aaaacccgca ccctggactg tgagcccaag tgtgtagaag agttacccct 1620
ttcagtctga gggctccaac agtgacatgt atctcagccc tgttgccatg cccaacaagc 1680
tggtgttctg tgaagttttc aagtacaacc ggaagcctgc agagaccaat ttaaggcact 1740
cgtgtaaacg gatatggaat ggaacaggag tatactctga tgggaacaga tgggcaccca 1800
aggtccgtat tactgtggtg tgggcgcaga caaagcctat ggcagggata tcgtggaggc 1860
tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1920
gcctgcccag tgggagttcc aatcatctct gggtggcccg tttcatcttg catcgagtat 1980
gtgaagactt tggggtaata gcaacctttg accccaagcc cattcctggg aactggaatg 2040
gtgcaggctg ccataccaac tttagcacca aggccatgcg ggagcgagaa actaagcaag 2100
cggcaccggt accacattcg agcctacgat cccaaggggg gcctggacaa tgcccgtcgt 2160
ctgactgggt tccacgaaac gtccaacatc aacgactttt ctgctggtgt cgccaatcgc 2220
agtgccagca tccgcattcc ccggactgtc ggccaggaga agaaaggtta ctttgcccct 2280
ttgcagtgac agaagccatc gtccgcacat gccttctcaa tgagactggc gacgagccct 2340
tccaatacaa aaactaaaaa gggtggactt gttgcagttg ctgtaggatt ctaaatccag 2400
gtgattgttt caaactgagc atcatctata tttcaatcat ggaccaaaat caacatttga 2460
ataaaacagc agaggcacaa ccttcagaga ataagaaaac aagatactgc aatggattga 2520
agatgttctt ggcagctctg tcactcagct tattatgaaa agttccatca ttcatataga 2580
acggagattt gagatatcct cttctcttgt tggggaaatt tgcttgtgat tgtatttgtg 2640
agttactttg gatccaaact acatagacca aagttaattg gaatcggttg tttcattatg 2700
ggaattggag gttcatggga tattacaggt attctaaaga aactaatatc aattcatcag 2760
aaaattcaac atcgacctta tccacttgtt taattaatca aattttatca ctcaatagag 2820
catcacctga gatagtggga aaaggttgtt taaaggaatc tgggtcatac atgtggatat 2880
atgtgttcat gggtaatatg cttcgtggaa taggggagac tcccatagta ccattggggc 2940
tgaaggacat tcttctttgt atttaggtat attgaatgca atagcaatga ttggtccaat 3000
cattggcttt accctgggat ctctgttttc taaaatgtac gtggatattg gatatgtaga 3060
tctaagcact atcaggataa ctcctactga ttctcgatgg gttggagctt ggtggcttaa 3120
tttccttgtg tctggactat tctccattat ttcttccata ccattctttt tcttgcccca 3180
aactccaaca ctgtctttgc atgtgctgga aacaaatgat gaaaaggatc aaacagctaa 3240
tttgaccaat caaggaaaaa atattaccaa aaatgtgact ggttgtatgt tatgtttgtg 3300
cttttgacgt tgttacaagt aagcagctat attggtgctt ttacttatgt cttcaaatac 3360
gtagagcaac agtatggtca gccttcatct aaggctaaca tcttattggg agtcataacc 3420
atacctattt ttgcaagtgg aatgttttta ggaggatata tcattaaaaa attcaaactg 3480
aacaccgttg gaattgccaa attctcatgt tttactgctg tgatgtcatt gtccttttac 3540
ctattatatt ttttcatact ctgtgaaaac aaatcagttg ccggactaac catgacctat 3600
gatggaaata atccagtgac atctcataga gatgtaccat tgtgatgaaa gtcaatggga 3660
accagtctgt ggaaacaatg gaataactta catctcaccc tgtctagcag gttgcaaatc 3720
ttcaagtggc aataaaaagc ctatagtgtt ttacaactgc agttgtttgg aagtaactgg 3780
tctccagaac agaaattact cagcccattt gggtgaatgc ccaagagatg atgcttgtac 3840
aaggaaattt tacttttttg ttgcaataca agtcttgaat tacatgtcat gctgattgtt 3900
aaaattgttc aacctgaatt gaaatcactt gcactgggtt tccactcaat ggttatacga 3960
gcactaggag gaattctagc tccaatatat tttggggctc tgattgatac aacgtgtata 4020
aagtggtcca ccaacaactg tggcacacgt gggtcatgta ggacatataa ttccacatca 4080
ttttcaaggg tctacttggg cttgtcttca atccatgaag aaaaaatatc aagagaaaga 4140
tatcaatgca tcagaaaatg gaagtgtcat ggatgaagca aacttagaat ccttaaataa 4200
aaataaacat tttgtccctt ctgctggggc agatagtgaa acacattgtt aaggggagaa 4260
aaaaagccac ttctgcttct gtgtttccaa acagcattgc attgattcag taagatgtta 4320
tttttgagga gttcctggtc ctttcactaa gaatttccac atcttttatg gtggaagtat 4380
aaataagcct atgaacttat aataaaacaa actgtaggta gaaaaaatga gagtactcat 4440
tgttacatta tagctacatc catacaaatt aaagtgagag acatggttac tgtgtaataa 4500
aagaaaaaat acttgttcag gtaattctaa ttcttaataa aacaaatgag tatcatacag 4560
gtagaggtta aaaaggagga gctagattca tatcctaagt aaagagaaat gcctagtgtc 4620
tattttatta aacaaacaaa cacagagttt gaactataat actaaggcct gaagtctagc 4680
ttggatatat gctacaataa tatctgttac tcacataaaa ttatatattt cacagacttt 4740
atcaatgtat aatacattta agtattgtgg aagaaataaa gacattccaa tatttgcaa 4799
<210> 2
<211> 7994
<212> DNA
<213> pTBSCH1-UGT1A1-GS-OATP1B1-Puro(Artificial Sequence)
<400> 2
ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca 60
ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca 120
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 180
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 240
acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420
tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 480
gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 540
gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 600
ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 660
ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 720
caccgggacc gatccagcct ccatcggctc gcatctctcc ttcacgcgcc cgccgcccta 780
cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct 840
cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc 900
cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc 960
ttgctcaact ctagttaacg gtggagggca gtgtagtctg agcagtactc gttgctgccg 1020
cgcgcgccac cagacataat agctgacaga ctaacagact gttcctttcc atgggtcttt 1080
tctgcagtca ccgtcgttcg ttgcaacaaa ttgatgagca atgctttttt ataatgccaa 1140
ctttgtacaa aaaagttggc accatggctg tggagtccca gggcggacgc ccacttgtcc 1200
tgtgtcccat gctgggaaga tactgttgat cccagtggat ggcagccact ggctgagcat 1260
gcttggggcc atccagcagc tgcagcagag gggacatgaa atagttgtcc tagcacctga 1320
cgcctcgttg tacatcagag acggagcatt ttacaccttg aagacgtacc ctgtgccatt 1380
ccaaagggag gatgtgaaag agtcttttgt tagtctcggg cataatgttt ttgagaatga 1440
ttctttcctg cagcgtgtga tcaaaacata caagaaaata aaaaaggact ctgctcccac 1500
ttactgcaca acaaggagct catggcctcc ctggcagaaa gcagctttga tgtcatgctg 1560
acggaccctt tccttccttg cagccccatc gtggcccagt acctgtctct gcccactgta 1620
ttcttcttgc atgcactgcc atgcagcctg gaatttgagg ctacccagtg ccccaagcct 1680
ctctcctctc attcagatca catgaccttc ctgcagcggg tgaagaacat gctcattgcc 1740
ttttcacaga actttctgtg cgacgtggtt tattccccgt atgcagagag gtgactgtcc 1800
aggacctatt gagctctgca tctgtctggc tgtttagaag tgactttgtg aaggattacc 1860
ctaggcccat catgcccaat aactgccttc accaaaatcc actatcccag gaatttgaag 1920
cctacattaa tgcttctgga gaacatggaa ttgtggtttt ctctttggga tcaatggtct 1980
cagaaatggc aattgctgat gctttgggca aaatcccggt acactggaac ccgaccatcg 2040
aatcttgcga acaacacgat acttgttaag tggctacccc aaaacgatct gcttggtcac 2100
ccgatgaccc gtgcctttag aaagcatatg caatggcgtt cccatggtga tgatgccctt 2160
gtttggtgat cagatggaca atgcaaagcg catggagact agttctggaa atgacttctg 2220
aagatttaga aaatgctcta aaagcagtca tcaatgacaa aagttaagcc ttcacaagga 2280
ccgcccggtg gagccgctgg acctggccgt gttctgggtg gagtttgtga tgaggcacaa 2340
ggcccacgac ctcacctggt accagtacca ttccttggac gtgattggtt tcctcttggc 2400
cgtcgtgctg acagtggcct tcatcacctt taaatgttgt gcttatggct accgggcgag 2460
ttaagaaagc ccacaaatcc aagacccatt tgccaacttt cttgtacaaa gttggcatta 2520
taagaaagca ttgcttatca atttgttgca acgaacgatg gcaaggctga ggacgggagg 2580
ctgattgaga ggcgaaggta caccctaatc tcaatacaac ctttggagct aagccagcaa 2640
tggtagaggg aagattctgc acgtcccttc caggcggcct ccccgtcacc acccccccca 2700
acccgccccg accggagctg agagtaattc atacaaaagg actcgcccct gccttgggga 2760
atcccaggga ccgtcgttaa actcccacta acgtagaacc cagagatcgc tgcgttcccg 2820
ccccctcacc cgcccgctct cgtcatcact gaggtggaga agagcatgcg tgaggctccg 2880
gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 2940
tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 3000
tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 3060
ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg 3120
gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca 3180
cgcccctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag 3240
agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct 3300
gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc 3360
gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa 3420
gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg 3480
ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg 3540
cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc 3600
ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt 3660
gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg 3720
cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc 3780
tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag 3840
ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag 3900
tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc 3960
tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg 4020
gttcaaagtt tttttcttcc atttcaggtg tcgtgaatgg ccacctcagc aagttcccac 4080
tcaaatgtac ttgtgcctgc cccagggtga gaaagtccaa gccatgtata tctgggttga 4140
tggtactgga gaaggactgc gctgcaaaac ccgcaccctg gactgtgagc ccaagtgtgt 4200
agaagagtta cccctttcag tctgagggct ccaacagtga catgtatctc agccctgttg 4260
ccatgcccaa caagctggtg ttctgtgaag ttttcaagta caaccggaag cctgcagaga 4320
ccaatttaag gcactcgtgt aaacggatat ggaatggaac aggagtatac tctgatggga 4380
acagatgggc acccaaggtc cgtattactg tggtgtgggc gcagacaaag cctatggcag 4440
ggatatcgtg gaggctcact accgcgcctg cttgtatgct ggggtcaaga ttacaggaac 4500
aaatgctgag gtcatgcctg cccagtggga gttccaatca tctctgggtg gcccgtttca 4560
tcttgcatcg agtatgtgaa gactttgggg taatagcaac ctttgacccc aagcccattc 4620
ctgggaactg gaatggtgca ggctgccata ccaactttag caccaaggcc atgcgggagc 4680
gagaaactaa gcaagcggca ccggtaccac attcgagcct acgatcccaa ggggggcctg 4740
gacaatgccc gtcgtctgac tgggttccac gaaacgtcca acatcaacga cttttctgct 4800
ggtgtcgcca atcgcagtgc cagcatccgc attccccgga ctgtcggcca ggagaagaaa 4860
ggttactttg cccctttgca gtgacagaag ccatcgtccg cacatgcctt ctcaatgaga 4920
ctggcgacga gcccttccaa tacaaaaact aaaaagggtg gacttgttgc agttgctgta 4980
ggattctaaa tccaggtgat tgtttcaaac tgagcatcat ctatatttca atcatggacc 5040
aaaatcaaca tttgaataaa acagcagagg cacaaccttc agagaataag aaaacaagat 5100
actgcaatgg attgaagatg ttcttggcag ctctgtcact cagcttatta tgaaaagttc 5160
catcattcat atagaacgga gatttgagat atcctcttct cttgttgggg aaatttgctt 5220
gtgattgtat ttgtgagtta ctttggatcc aaactacata gaccaaagtt aattggaatc 5280
ggttgtttca ttatgggaat tggaggttca tgggatatta caggtattct aaagaaacta 5340
atatcaattc atcagaaaat tcaacatcga ccttatccac ttgtttaatt aatcaaattt 5400
tatcactcaa tagagcatca cctgagatag tgggaaaagg ttgtttaaag gaatctgggt 5460
catacatgtg gatatatgtg ttcatgggta atatgcttcg tggaataggg gagactccca 5520
tagtaccatt ggggctgaag gacattcttc tttgtattta ggtatattga atgcaatagc 5580
aatgattggt ccaatcattg gctttaccct gggatctctg ttttctaaaa tgtacgtgga 5640
tattggatat gtagatctaa gcactatcag gataactcct actgattctc gatgggttgg 5700
agcttggtgg cttaatttcc ttgtgtctgg actattctcc attatttctt ccataccatt 5760
ctttttcttg ccccaaactc caacactgtc tttgcatgtg ctggaaacaa atgatgaaaa 5820
ggatcaaaca gctaatttga ccaatcaagg aaaaaatatt accaaaaatg tgactggttg 5880
tatgttatgt ttgtgctttt gacgttgtta caagtaagca gctatattgg tgcttttact 5940
tatgtcttca aatacgtaga gcaacagtat ggtcagcctt catctaaggc taacatctta 6000
ttgggagtca taaccatacc tatttttgca agtggaatgt ttttaggagg atatatcatt 6060
aaaaaattca aactgaacac cgttggaatt gccaaattct catgttttac tgctgtgatg 6120
tcattgtcct tttacctatt atattttttc atactctgtg aaaacaaatc agttgccgga 6180
ctaaccatga cctatgatgg aaataatcca gtgacatctc atagagatgt accattgtga 6240
tgaaagtcaa tgggaaccag tctgtggaaa caatggaata acttacatct caccctgtct 6300
agcaggttgc aaatcttcaa gtggcaataa aaagcctata gtgttttaca actgcagttg 6360
tttggaagta actggtctcc agaacagaaa ttactcagcc catttgggtg aatgcccaag 6420
agatgatgct tgtacaagga aattttactt ttttgttgca atacaagtct tgaattacat 6480
gtcatgctga ttgttaaaat tgttcaacct gaattgaaat cacttgcact gggtttccac 6540
tcaatggtta tacgagcact aggaggaatt ctagctccaa tatattttgg ggctctgatt 6600
gatacaacgt gtataaagtg gtccaccaac aactgtggca cacgtgggtc atgtaggaca 6660
tataattcca catcattttc aagggtctac ttgggcttgt cttcaatcca tgaagaaaaa 6720
atatcaagag aaagatatca atgcatcaga aaatggaagt gtcatggatg aagcaaactt 6780
agaatcctta aataaaaata aacattttgt cccttctgct ggggcagata gtgaaacaca 6840
ttgttaaggg gagaaaaaaa gccacttctg cttctgtgtt tccaaacagc attgcattga 6900
ttcagtaaga tgttattttt gaggagttcc tggtcctttc actaagaatt tccacatctt 6960
ttatggtgga agtataaata agcctatgaa cttataataa aacaaactgt aggtagaaaa 7020
aatgagagta ctcattgtta cattatagct acatccatac aaattaaagt gagagacatg 7080
gttactgtgt aataaaagaa aaaatacttg ttcaggtaat tctaattctt aataaaacaa 7140
atgagtatca tacaggtaga ggttaaaaag gaggagctag attcatatcc taagtaaaga 7200
gaaatgccta gtgtctattt tattaaacaa acaaacacag agtttgaact ataatactaa 7260
ggcctgaagt ctagcttgga tatatgctac aataatatct gttactcaca taaaattata 7320
tatttcacag actttatcaa tgtataatac atttaagtat tgtggaagaa ataaagacat 7380
tccaatattt gcaaatgaca gagtataaac ctaccgtgag actggctaca agagatgatg 7440
tcccaagggc cgtgagaaca ctggctgccg cttttgctga ctaccccgcc acaaggcaca 7500
ctgtggaccc tgataggcat atcgagcgcg tgactgagct gcaagaactg ttcctgaccc 7560
gcgtgggact ggacattggc aaggtctggg tcgcagacga tggagccgct gtggctgtct 7620
ggaccacacc tgagtccgtg gaagccgggg ctgtctttgc agagatcgga ccacgcatgg 7680
cagaactgag cgggtcccga ctggcagcac agcagcagat ggagggactg ctggcaccac 7740
accggcccaa ggaacctgca tggttcctgg ctacagtggg cgtctcaccc gatcatcagg 7800
gcaaagggct gggatctgct gtggtcctgc caggagtgga ggccgccgag agagctggcg 7860
tccccgcatt tctggagact tctgccccta gaaacctgcc attctacgaa aggctgggct 7920
ttactgtgac cgctgacgtg gaggtcccag aaggccccag gacctggtgc atgacacgaa 7980
agcctggggc ttaa 7994
Claims (6)
1.一种胆红素代谢功能基因片段，由UGT1A1、GS、OATP1B1三种基因片段顺序构成，其核苷酸序列如SEQ No.1所示。
2.一种胆红素代谢功能基因表达质粒，包含权利要求1所述胆红素代谢功能基因片段。
3.根据权利要求2所述的胆红素代谢功能基因表达质粒，其特征在于：还包括嘌呤霉素筛选压力基因片段，所述嘌呤霉素筛选压力基因片段连接于所述胆红素代谢功能基因片段的3’端。
4.根据权利要求3所述的胆红素代谢功能基因表达质粒，其特征在于：所述质粒的核苷酸序列如SEQ No.2所示。
5.一种包含胆红素代谢功能基因的HepG2细胞，包含权利要求1所述胆红素代谢功能基因片段。
6.一种包含胆红素代谢功能基因的HepG2细胞，利用权利要求2~4任一项所述的质粒改造HepG2细胞后获得。
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| CN109694844A (zh) * | 2017-10-24 | 2019-04-30 | 南方医科大学珠江医院 | 一种用于培养肝细胞的完全培养基 |
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| CN109694844A (zh) * | 2017-10-24 | 2019-04-30 | 南方医科大学珠江医院 | 一种用于培养肝细胞的完全培养基 |
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| "表达UGT1A1重组腺相关病毒载体的构建";陈霞等;《重庆医学》;20061130;第35卷(第21期);第1929页摘要部分和左栏第1段 * |
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