CN103421831B - For improving mosaic gene and the construction process thereof of the hepatocellular physiological CYP3A4 expression of vitro culture of human - Google Patents

Abstract

The present invention discloses a kind of for improving the mosaic gene and construction process thereof that the hepatocellular physiological CYP3A4 of vitro culture of human expresses; Described mosaic gene, comprises the activation domain sequence of people's nf Pregnane X Receptor full length gene sequence and wild type P53 gene.Also has a kind of recombinant eukaryon expression vector, it mosaic gene described before comprising; A kind of construction process of foregoing recombinant eukaryon expression vector, wherein, it comprises the following steps: the activation domain sequence of cloning described people's nf Pregnane X Receptor full length gene sequence and wild type P53 gene from the cDNA in normal people's primary hepatocyte source respectively; Two sections of sequence directed clonings abovementioned steps obtained, between the β-globin/IgG chimeric intron sequence and SV40Late poly (A) signal sequence of described PCI-neo mammalian expression vector, obtain recombinant eukaryon expression vector.The present invention can significantly improve the hepatocellular physiological CYP3A4 of vitro culture of human and express.

Description

For improving mosaic gene and the construction process thereof of the hepatocellular physiological CYP3A4 expression of vitro culture of human

Technical field

The present invention relates to a kind of construction of eukaryotic expression vector technology, particularly relating to a kind of for improving the mosaic gene and construction process thereof that the hepatocellular physiological CYP3A4 of vitro culture of human expresses.

Background technology

Major part medicine enters the metabolic process after in body and completes primarily of liver cell.Liver cell contains the enzyme of various participation inside and outsides material bio-transformation.Cytochrome P 450 enzymes (CYPs, P450s) is the monooxygenase superfamily protein of a class containing hemochrome sulfhydryl structure, has been the important drugs metabolic enzyme of the detoxification/bioactivation of related substrates.Wherein, CYP3A subfamily content in human liver organization is the highest, accounts for 30%, is main clinical medicine metabolic enzymes.CYP3A4 take part in the metabolic process of the clinical application of 50%, and drug metabolism spectrum widely.In view of the vital role of CYP3A4 in drug metabolism processes, drug development process needs one can reproduce the metabolic external model of CYP3A4 in body, be used for concentrating and evaluate CYP3A4 to drug candidate metabolism and level, and drug candidate is on the impact of CYP3A4 active function.

Primary human liver cell is best suited for the CYP3A4 In vitro cell model for drug metabolism, its expressed intact I phase, II phase metabolic enzyme and other participate in molecule, can the metabolism of aids drug in human body more exactly, be the gold standard of external drug metabolism.In primary human liver cell, the substance of CYP3A4 expression and regulation mechanism is as follows: nf Pregnane X Receptor (PXR) is the most important transcription factor that regulation and control CYP3A4 transcribes, PXR and retinoic acid receptor X (RXR) α forms heterodimer, combine with the element such as nearly Pregnane X Receptor response element (prPXRE) and exogenous increased response module (XREM), the physiological of regulation and control CYP3A4 expresses (constitutive character and inducibility).

But due to primary human liver cell source shortage, difference in crowds is large, and cultured cell in vitro phenotype maintains the shortcomings such as difficulty, therefore the primary human liver cell of widespread use is more difficult.By contrast, hepatoma cell line then do not exist primary hepatocyte limited source, vitro culture difficulty etc. shortcoming, they have in-vitro multiplication ability, heredity and function phenotype relatively stable, and culture condition fairly simple and be easy to laboratory monitoring seek unity of standard.The method mainly transfection codes for tumor virogene of primary hepatocyte immortalization, the plasmid of proto-oncogene or Telomerase.Along with progress of research, the clone having occurred hybrid cell, Conditional immortalization liver cell and be separated from transgenic animal.But after primary hepatocyte immortalization, cell phenotype changes, particularly CYP3A4 expresses low, so and be not suitable for CYP3A4 related drugs metabolism research.

Liver source sexual cell is ubiquitous subject matter, is in and dedifferentes or low differentiation state, and all kinds of metabolic enzyme expression level lowly or incomplete.Therefore, the core objective of relevant transformation concentrates on how to recover and improve liver feature functionality phenotype.The method of another kind of engineered cells function is, by the plasmid of transfection coding CYP3A4, process LAN CYP3A4 in clone, improves the application potential quality of clone in the metabolism of CYP3A4 related drugs.Various non-liver sources property host cell is as bacterium, and yeast, insect cell, mammalian cell etc., by successful modification.1991, bibliographical information is successful expression in bone-marrow-derived lymphocyte sample stem cell.1998, Chen Q et al success expressed CYP3A4 in CHL cells, and for aflatoxin B1, the metabolism research of endoxan and sterigmatocystin.Hep G2 is as most widely used hepatoma cell line, and lot of documents is reported in successful process LAN CYP3A4 in Hep G2, and for the relevant drug metabolism of CYP3A4 and toxicity research.But the reforming mode of this process LAN CYP3A4 can not reproduce the physiological reaction characteristics of drug metabolism enzyme, the major issue predicting the clinical drug therapies such as drug interaction therefore can not be used for.

For realizing the object of simulation physiological regulation, people to conduct in-depth analysis research to CYP3A4 expression and regulation mechanism.Contrast the related gene expression of primary human liver cell, regulation and control and enzymic activity basic parameter, CYP3A4 expresses the reduction transcription factor relevant to CYP3A4 in clone and cofactor expression minimizing has Close relation to have scholar to find.If Hep G2 is compared with primary hepatocyte, 15%, the HNF3 α that C/EBP α mRNA only reaches primary hepatocyte is 25%, HNF1 α is 40%.Have scholar by the plasmid of the encode specialized transcription factor of transfection (C/EBP α, HNF3 γ, PXR), success improves the expression of CYP3A4 at Hep G2.Jenni K ü blbeck et al.(2010) transform out a kind of chimeric transcription factor, the P65 activation domain by NF-κ B is connected to PXR and forms mosaic gene, and transformation C3A cell, improves the expression of its CYP3A4.Although process LAN associated transcription factor can strengthen the expression of CYP3A4, still do not reach the expression level of primary hepatocyte far away.Because CYP3A4 is the regulated and control network of the complexity that a multiplefactor participates at intracellular expression regulation, expression that is single or several transcription factors improves, the expression of CYP3A4 can not be made to return to normal level, so still need to do CYP3A4 expression regulation further to attempt research.

P53 gene, the P53 albumen of its coding is core tumor suppression phosphorylated protein, and coerce when cell meets with in normal cell, when especially cell DNA sustains damage, P53 albumen sharply increases, and is mainly used in arresting cell cycle or cell death inducing; Once P53 gene is undergone mutation, P53 protein inactivation, cell fission loses restraining, may cause canceration.Half is about had to be to undergo mutation inactivation due to this gene in human cancer.P53 albumen comprise activation domain (TAD) that 4 function point analysis region: N-hold, DNA binding domain (DBD), C-end reset regulate territory and four multimerisation domain (4D), wherein the dynamic change of the activation domain (P53-AD) of the N-end of P53 albumen imparts the multifunctionality in this region.

There is some corners and spiral in P53-AD, these corners and spiral are generally the binding site of P53 albumen and other albumen (as mdm2) or chaperone, and the position of these corners and spiral and quantity can also change to some extent, which imply P53 albumen can combine with multiple protein companion thus perform various kinds of cell regulatory function.Researchist has found the protein (IUP) of its inside non-structured in P53-AD, IUP can be combined with other chaperones by changing himself structure, the IUP of this P53-AD has similar structure to other activation domains of albumen many, namely may the multi-functional, hydrophobic zonule of basis of formation structural motif, which imply P53-AD has similar structure and corresponding function to other transcription activating domain or transcription factor.P53 gene is tumor suppressor gene, it plays a significant role in cell cycle regulating, and phosphorylation has played most important functions in the Active Regulation of P53 albumen, can be controlled by the activity of phosphorylation degree to P53-AD of part important amino acid residue in control P53-AD.

Summary of the invention

The object of the invention is, with regulatory mechanism in CYP3A4 cell and based on the theoretical investigation of the above function about P53-AD for theoretical basis, according to PXR and P53 protein structure feature, P53-AD is selected to build the mosaic gene meeting the expectation of contriver's technology, the mosaic gene namely providing a kind of physiological CYP3A4 for improving Cultured Hepatocytes in vitro to express and construction process thereof.

For reaching above technical purpose, the technical solution used in the present invention is as follows:

For improving the mosaic gene that the hepatocellular physiological CYP3A4 of vitro culture of human expresses, wherein, its sequence is for shown in SEQ ID No.1 or SEQ ID No.3.

A kind of recombinant eukaryon expression vector, is characterized in that, its mosaic gene described before comprising.

Described carrier for expression of eukaryon is mammalian cell expression vector; Particularly, be PCI-neo mammalian expression vector.Described PCI-neo mammalian expression vector comprises β-globin/IgG chimeric intron sequence successively and SV40Late poly (A) signal sequence in the early stage enhancers/promoters sequence of human cytomegalovirus and downstream thereof.

Particularly, following restriction enzyme site is comprised between the β-globin/IgG chimeric intron sequence of described PCI-neo mammalian expression vector and SV40Late poly (A) signal sequence: Nhe I, Xho I, EcoR I, Mlu I, Xba I, Sal I, Acc I, Sma I and Not I.

A construction process for foregoing recombinant eukaryon expression vector, wherein, it comprises the following steps:

(1) the cDNA human cloning nf Pregnane X Receptor full length gene sequence that normal people's primary hepatocyte of including according to GenBank database is originated;

(2) the activation domain sequence of the cDNA sequence clone wild-type P53 gene of including according to GenBank database;

(3) two sections of sequence construct orderings that described step (1) and (2) obtain are classified as the mosaic gene shown in SEQ IDNo.1 or SEQ ID No.3, and between directed cloning to the β-globin/IgG chimeric intron sequence and SV40Late poly (A) signal sequence of PCI-neo mammalian expression vector, obtain recombinant eukaryon expression vector.

Particularly, the activation domain sequence orientation of described people's nf Pregnane X Receptor full length gene sequence and wild type P53 gene is inserted on Nhe I, the Xho I of described PCI-neo mammalian expression vector, EcoR I, Mlu I, Xba I, Sal I, Acc I, Sma I and any one restriction enzyme site of Not I.

Further, the recombinant eukaryon expression vector of described acquisition is by restriction map method and/or measure its nucleotide sequence and carry out screening and identification.

Compared with prior art, the present invention has following advantage:

(1) mosaic gene of the present invention is realized by the main upstream passages of Design & reform based on this gene physiological regulation, has the specific aim of CYP3A4 physiological ability to express;

(2) mosaic gene of the present invention is by selecting P53 gene and specific function territory segment (P53-AD) thereof, makes this mosaic gene have the ability of stronger regulation and control CYP3A4 expression.

Accompanying drawing explanation

Fig. 1 is the structural representation of mammalian expression vector PCI-neo used in the present invention.

Fig. 2 is the part-structure schematic diagram of the first recombinant eukaryon expression vector PCI-hPXR-P53 of the present invention, it illustrates the activation domain sequence connecting people's nf Pregnane X Receptor full length gene sequence and wild type P53 gene in β-globin/IgG chimeric intron sequence downstream successively.

Fig. 3 is the part-structure schematic diagram of the second recombinant eukaryon expression vector PCI-P53-hPXR of the present invention, it illustrates the activation domain sequence and people's nf Pregnane X Receptor full length gene sequence that connect wild type P53 gene in β-globin/IgG chimeric intron sequence downstream successively.

Fig. 4 is that recombinant eukaryon expression vector PCI-hPXR-P53 of the present invention and PCI-P53-hPXR transfection are to the two fluorescent reporter gene detected results to CYP3A4-XREM.luc in 293T cell.

Fig. 5 is that recombinant eukaryon expression vector PCI-hPXR-P53 of the present invention and PCI-P53-hPXR transfection are to the two fluorescent reporter gene detected results to CYP3A4-XREM.luc in C3A cell.

Fig. 6 is that recombinant eukaryon expression vector PCI-hPXR-P53 of the present invention and PCI-P53-hPXR transfection are to the CYP3A4mRNA fluorescence quantitative PCR detection result in C3A cell.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.

Embodiment 1

Build recombinant eukaryon expression vector PCI-hPXR-P53 and PCI-P53-hPXR, its step is as follows:

1. the PXR included according to GenBank database and the gene order of wild-type P53-AD, design special primer, the cDNA clone PXR adopting PCR method originate from normal people's primary hepatocyte and wild-type P53-AD gene order;

2. pass through restriction endonuclease sites, by PXR and wild-type P53-AD gene directed cloning to carrier for expression of eukaryon PCI-neo(as shown in Figure 1), adopt the method screening and identification recombinant eukaryon expression vector PCI-hPXR-P53 such as enzyme is cut, order-checking, as shown in Figure 2;

3. pass through restriction endonuclease sites, by PXR and wild-type P53-AD gene directed cloning to carrier for expression of eukaryon PCI-neo(as shown in Figure 1), adopt the method screening and identification recombinant eukaryon expression vector PCI-P53-hPXR such as enzyme is cut, order-checking, as shown in Figure 3;

Embodiment 2

Dual-luciferase reporter system is for substrate is to detect the active a kind of reporting system of Photinus pyralis LUC (fireflyluciferase) with fluorescein (luciferin).Luciferase can be oxidized to oxyluciferin by catalysis luciferin, in the process of luciferin oxidation, bioluminescence (bioluminescence) can be sent, in conjunction with ocean coelenteron luciferase reporter gene altogether, when expressing with Photinus pyralis LUC quantitate gene, ocean coelenteron luciferase is adopted to reduce the changing factor of experiment.Then Chemiluminescence Apparatus (luminometer) or liquid flashing determining instrument also can be claimed to measure the bioluminescence discharged in luciferin oxidising process by fluor tester.Fluorescein and this noclilucence system of luciferase, can detect the expression of gene extremely sensitive, efficiently.

Transcription factor is a kind of protein molecule with special construction, exerts regulatory genetic expression function, also referred to as trans-acting factor.The distinguished sequence of some transcription factor only in its target promotor is combined, these specific sequences are called as cis-acting elements, the DNA binding domain of transcription factor and cis-acting elements realize covalent attachment, thus play a part to suppress or strengthen to the expression of gene.Luciferase reporter gene experiment (luciferase assay) is the important means that the special order detected in this kind of transcription factor and its target promotor combines.Its principle is summarized as follows:

A. the reporter plasmid that the specific fragment of target promotor is inserted into luciferase expression sequence front by is built, as pGL3-basic etc.

The transcription factor expression plasmid that b. will detect and reporter plasmid cotransfection 293T cell or other relevant clone.If this transcription factor can activate target promotor, then luciferase gene will be expressed, and the expression amount of luciferase is directly proportional to the action intensity of transcription factor.

C. add specific luciferase substrate, luciferase and substrate reactions, produce fluorescence, the activity of luciferase can be measured by the intensity detecting fluorescence, thus judge whether transcription factor can have effect by target promoter fragment therewith.

Quantitative fluorescent PCR is used for the result of the two fluoroscopic examination of checking further.

Based on above principle, build the reporter plasmid CYP3A4-XREM.luc of recombinant eukaryon expression vector PCI-hPXR-P53 and PCI-P53-hPXR and the subcloning vector PCI-hPXR of contrast, its step is as follows:

1. the gene order of the CYP3A4 included according to GenBank database, design special primer, the regulating and controlling sequence gene order between the cDNA clone CYP3A4-362 to+53 parts and-7838 to-7208 adopting PCR method originate from normal people's primary hepatocyte;

2. pass through restriction endonuclease sites, by the regulating and controlling sequence directed cloning between CYP3A4-362 to+53 parts and-7838 to-7208 in carrier for expression of eukaryon PGL3-basic, adopt the method screening and identification recombinant eukaryon expression vector CYP3A4-XREM.luc such as enzyme is cut, order-checking;

3. the carrier PCI-hPXR-P53 Nhe I/BamH I enzyme built is scaled off, be connected with the Nhe I/BamH I digestion products of pcDNA3.1 (+), obtain an intermediate carrier pcDNA3.1-hPXR.Intermediate carrier obtains EcoR I restriction enzyme site.The Nhe I/EcoR I digestion products of pcDNA3.1-hPXR is connected with the Nhe I/EcoR I digestion products of PCI-neo, adopts enzyme to cut qualification subcloning vector PCI-hPXR.

The functional verification of recombinant eukaryon expression vector PCI-hPXR-P53 and PCI-P53-hPXR, its step is as follows:

1. object plasmid (PCI-hPXR-P53, PCI-P53-hPXR, PCI-hPXR or PCI-neo), reporter plasmid (CYP3A4-XREM.luc) and internal reference plasmid (TK) are pressed 46ng:140ng:14ng transient transfection 293T cell respectively.After transfection 24 hours, Rifampin group (RIF+) added the Rifampin solution of 0.2 μ L10mM, and control group (RIF-) adds 0.2 μ L DMSO, and medicine irritation, after 48 hours, carries out Dual-Luciferase detection, the results are shown in Figure 4.

2. object plasmid (PCI-hPXR-P53, PCI-P53-hPXR, PCI-hPXR or PCI-neo), reporter plasmid (CYP3A4-XREM.luc) and internal reference plasmid (TK) are pressed 46ng:140ng:14ng transient transfection C3A cell respectively.After transfection 24 hours, Rifampin group (RIF+) added the Rifampin solution of 0.2 μ L10mM, and control group (RIF-) adds 0.2 μ L DMSO, and medicine irritation, after 48 hours, carries out Dual-Luciferase detection, the results are shown in Figure 5.

3. in 24 orifice plates, by 1 × 10 ⁵the density inoculation C3A cell of individual/ml, every hole 1ml, 37 DEG C, 5%CO ₂quiescent culture in incubator, then distinguishes transfection PCI-hPXR-P53, PCI-P53-hPXR, PCI-hPXR and PCI-neo.After transfection 24 hours, Rifampin group (RIF+) added the Rifampin solution of 1 μ L10mM, and control group (RIF-) adds 1 μ L DMSO.After thing stimulates 48 hours, collecting cell carries out fluorescence quantitative PCR detection, the results are shown in Figure 6.

Please refer to Fig. 4, by plasmid PCI-PXR-P53, PCI-P53-hPXR, PCI-hPXR and PCI-neo in two fluoroscopic examination 293T cell to the effect of reporter plasmid.Rif(+) organize Average normalized luciferase activity and be respectively 464 ± 9.5,2899 ± 385,320 ± 23,254 ± 25, difference has statistical significance (P < 0.05); Rif(-) organize Average normalized uciferase activity and be respectively 344 ± 11,2560 ± 247,263 ± 22,229 ± 23, difference has statistical significance (P < 0.05).

Please refer to Fig. 5, by plasmid PCI-hPXR-P53, PCI-P53-hPXR, PCI-hPXR and PCI-neo in two fluoroscopic examination C3A cell to the effect of reporter plasmid.Rif(+) organize Average normalized uciferase activity and be respectively 6633 ± 443,20046 ± 2806,1539 ± 137,626 ± 94, difference has statistical significance (P < 0.05); Rif(-) organize Average normalized uciferase activity and be respectively 4098 ± 920,16391 ± 2412,435 ± 168,732 ± 93, difference has statistical significance (P < 0.05).

Please refer to Fig. 6, changed by fluorescence quantitative PCR detection CYP3A4mRNA at C3A cell transient transfection plasmid PCI-hPXR-P53, PCI-P53-hPXR, PCI-hPXR and PCI-neo.Rif(+) organize CYP3A4mRNA2-△ △ ct value and be respectively 9.52 ± 2.61,6.01 ± 0.27,3.82 ± 0.15,1.57 ± 0.11, difference has statistical significance (P < 0.05); Rif(-) organize CYP3A4mRNA2-△ △ ct value and be respectively 8.07 ± 0.11,6.36 ± 0.94,2.37 ± 0.22,1.02 ± 0.02, difference has statistical significance (P < 0.05).

From above result, after 293T transit cell dye PCI-hPXR-P53 and PCI-P53-hPXR, uciferase activity will apparently higher than PCI-hPXR, prove that PCI-hPXR-P53 and PCI-P53-hPXR is stronger to the activation of CYP3A4 promotor, this also points out P53-AD to play certain positive effect in this process indirectly.After adding Rifampin induction, the uciferase activity of each group all improves, and illustrates that the activation of PCI-hPXR-P53 and PCI-P53-hPXR to CYP3A4 promotor is adjustable, meets normal physiological conditions.In C3A cell, because C3A cell is tumor cell of liver source, self can express the atopen of a series of participation such as PXR CYP3A4 regulating and expressings, so the uciferase activity detected all is higher than 293T cell, but overall trend and 293T cell basically identical.By two fluoroscopic examination, from gene level prove chimeric vector PCI-hPXR-P53 and PCI-P53-hPXR can in conjunction with and activate CYP3A4 promotor.

Quantitative fluorescent PCR demonstrates the result of two fluoroscopic examination further, and after C3A cell transient transfection carrier PCI-hPXR-P53 and PCI-P53-hPXR, CYP3A4mRNA transcribes and is significantly higher than PCI-hPXR, and can be induced by Rifampin.

In sum, the mosaic gene of the activation domain sequence of the people's of comprising nf Pregnane X Receptor full length gene sequence of the present invention and wild type P53 gene and construction process thereof can significantly improve the hepatocellular physiological CYP3A4 of vitro culture of human and express.

Above-described embodiment is the present invention's preferably embodiment; but be not merely restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be all included within protection scope of the present invention.

SEQUENCE LISTING

<110> Zhujiang Hospital attached to Nanfang Medical Univ.

<120> is for improving mosaic gene and the construction process thereof of the hepatocellular physiological CYP3A4 expression of vitro culture of human

<160>5

<210>1

<211>7016

<212>DNA

<213> artificial sequence

<220>

<221>PCI-hPXR-P53

<223> mosaic gene

<400>1

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tattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcc

cgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttc

gaaatgaccgaccaagcgacgcccaacctgccatcacgatggccgcaataaaatatctttattttc

attacatctgtgtgttggttttttgtgtgaatcgatagcgataaggatccgcgtatggtgcactct

cagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgc

gccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctg

catgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcct

atttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaa

tgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagaca

ataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt

cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaa

agtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcgg

taagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct

atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattc

tcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag

agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgat

cggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcg

ttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaat

ggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaat

agactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtt

tattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccaga

tggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaa

tagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactc

atatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttt

tgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtaga

aaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa

accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaac

tggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccactt

caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag

tggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtc

gggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata

cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggt

aagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttta

tagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcg

gagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgc

tcacatggctcgacagatct

<210>2

<211>664

<212>PRT

<213> artificial sequence

<220>

<221>PCI-hPXR-P53

<223>143-576 is hPXR, 577-584 be linker, 585-656 is P53

<400>2

Arg Pro Val Asp Ala Asn Gly Arg Xaa Ala Cys Thr Val Gly Gly Leu

5 10 15

Tyr Lys Gln Ser Ser Phe Ser Glu Pro Ser Asp His Xaa Lys Leu Tyr

20 25 30

Cys Gly Ser Leu Ser Gln Leu Asn Cys Xaa Arg Ser Gln Cys Phe Xaa

35 40 45

His Asn Ser Leu Glu Leu Lys Leu Gln Xaa Leu Ser Xaa Gly Ser Leu

50 55 60

Ala Glu Val Gly Arg Glu Ala Leu Gly Arg Xaa Val Ser Arg Leu Gln

65 70 75 80

Asp Arg Phe Lys Glu Thr Asn Arg Asn Trp Ala Cys Arg Asp Arg Glu

85 90 95

Asp Ser Cys Val Ser Asp Arg His Leu Leu Val Leu Leu Thr Ser Thr

100 105 110

Leu Pro Phe Ser Pro Gln Val Ser Thr Pro Ser Ser Ile Thr Ala Leu

115 120 125

Lys Ala Arg Val Leu Asn Thr Thr His Tyr Arg Leu Ala Thr Met Glu

130 135 140

Val Arg Pro Lys Glu Ser Trp Asn His Ala Asp Phe Val His Cys Glu

145 150 155 160

Asp Thr Glu Ser Val Pro Gly Lys Pro Ser Val Asn Ala Asp Glu Glu

165 170 175

Val Gly Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala Thr Gly

180 185 190

Tyr His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg

195 200 205

Arg Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg Lys Gly

210 215 220

Ala Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala Cys Arg

225 230 235 240

Leu Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met Ile Met Ser

245 250 255

Asp Glu Ala Val Glu Glu Arg Arg Ala Leu Ile Lys Arg Lys Lys Ser

260 265 270

Glu Arg Thr Gly Thr Gln Pro Leu Gly Val Gln Gly Leu Thr Glu Glu

275 280 285

Gln Arg Met Met Ile Arg Glu Leu Met Asp Ala Gln Met Lys Thr Phe

290 295 300

Asp Thr Thr Phe Ser His Phe Lys Asn Phe Arg Leu Pro Gly Val Leu

305 310 315 320

Ser Ser Gly Cys Glu Leu Pro Glu Ser Leu Gln Ala Pro Ser Arg Glu

325 330 335

Glu Ala Ala Lys Trp Trp Ser Gln Val Arg Lys Asp Leu Cys Ser Leu

340 345 350

Lys Val Ser Leu Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn Tyr

355 360 365

Lys Pro Pro Ala Asp Ser Gly Gly Lys Glu Ile Phe Ser Leu Leu Pro

370 375 380

His Met Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Glu Ile Ile Ser

385 390 395 400

Phe Ala Lys Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln

405 410 415

Ile Ser Leu Leu Lys Gly Ala Ala Phe Glu Leu Cys Gln Leu Arg Phe

420 425 430

Asn Thr Val Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys Gly Arg Leu

435 440 445

Ser Tyr Cys Leu Glu Asp Thr Ala Gly Gly Phe Gln Gln Leu Leu Leu

450 455 460

Glu Pro Met Leu Lys Phe His Tyr Met Leu Lys Lys Leu Gln Leu His

465 470 475 480

Glu Glu Glu Tyr Val Leu Met Gln Ala Ile Ser Leu Phe Ser Pro Asp

485 490 495

Arg Pro Gly Val Leu Gln His Arg Val Val Asp Gln Leu Gln Glu Gln

500 505 510

Phe Ala Ile Thr Leu Lys Ser Tyr Ile Glu Cys Asn Arg Pro Gln Pro

515 520 525

Ala His Arg Phe Leu Phe Leu Lys Ile Met Ala Met Leu Thr Glu Leu

530 535 540

Arg Ser Ile Asn Ala Gln His Thr Gln Arg Leu Leu Arg Ile Gln Asp

545 550 555 560

Ile His Pro Phe Ala Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr

565 570 575

Gly Ser Gly Ser Thr Arg Tyr Gln Ala Arg Glu Glu Pro Gln Ser Asp

580 585 590

Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp

595 600 605

Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala

610 615 620

Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr

625 630 635 640

Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro

645 650 655

Pro Val Xaa Glu Phe Thr Arg Gly

660

<210>3

<211>7013

<212>DNA

<213> artificial sequence

<220>

<221>PCI-P53-hPXR

<223> mosaic gene

<400>3

tcaatattggccattagccatattattcattggttatatagcataaatcaatattggctattggcc

attgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgcc

atgttggcattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccc

atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc

ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacg

tcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaag

tccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

acgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt

ttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccat

tgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactg

cgatcgcccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcaga

gctcgtttagtgaaccgtcagatcactagaagctttattgcggtagtttatcacagttaaattgct

aacgcagtcagtgcttctgacacaacagtctcgaacttaagctgcagtgactctcttaaggtagcc

ttgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggaga

ccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtct

tactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctcttaagg

ctagagtacttaatacgactcactataggctagccaccatggaggagccgcagtcagatcctagcg

tcgagccccctctgagtcaggaaacattttcagacctatggaaactacttcctgaaaacaacgttc

tgtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaacaatggt

tcactgaagacccaggtccagatgaagctcccagaatgccagaggctgctccccccgtgggatcca

cacgataccaagccgaggtgagacccaaagaaagctggaaccatgctgactttgtacactgtgagg

acacagagtctgttcctggaaagcccagtgtcaacgcagatgaggaagtcggaggtccccaaatct

gccgtgtatgtggggacaaggccactggctatcacttcaatgtcatgacatgtgaaggatgcaagg

gctttttcaggagggccatgaaacgcaacgcccggctgaggtgccccttccggaagggcgcctgcg

agatcacccggaagacccggcgacagtgccaggcctgccgcctgcgcaagtgcctggagagcggca

tgaagaaggagatgatcatgtccgacgaggccgtggaggagaggcgggccttgatcaagcggaaga

aaagtgaacggacagggactcagccactgggagtgcaggggctgacagaggagcagcggatgatga

tcagggagctgatggacgctcagatgaaaacctttgacactaccttctcccatttcaagaatttcc

ggctgccaggggtgcttagcagtggctgcgagttgccagagtctctgcaggccccatcgagggaag

aagctgccaagtggagccaggtccggaaagatctgtgctctttgaaggtctctctgcagctgcggg

gggaggatggcagtgtctggaactacaaacccccagccgacagtggcgggaaagagatcttctccc

tgctgccccacatggctgacatgtcaacctacatgttcaaaggcatcatcagctttgccaaagtca

tctcctacttcagggacttgcccatcgaggaccagatctccctgctgaagggggccgctttcgagc

tgtgtcaactgagattcaacacagtgttcaacgcggagactggaacctgggagtgtggccggctgt

cctactgcttggaagacactgcaggtggcttccagcaacttctactggagcccatgctgaaattcc

actacatgctgaagaagctgcagctgcatgaggaggagtatgtgctgatgcaggccatctccctct

tctccccagaccgcccaggtgtgctgcagcaccgcgtggtggaccagctgcaggagcaattcgcca

ttactctgaagtcctacattgaatgcaatcggccccagcctgctcataggttcttgttcctgaaga

tcatggctatgctcaccgagctccgcagcatcaatgctcagcacacccagcggctgctgcgcatcc

aggacatacacccctttgctacgcccctcatgcaggagttgttcggcatcacaggtagctgagaat

tcacgcgtggtacctctagagtcgacccgggcggccgcttccctttagtgagggttaatgcttcga

gcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgc

tttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtt

aacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggttttttaaagc

aagtaaaacctctacaaatgtggtaaaatccgataaggatcgatccgggctggcgtaatagcgaag

aggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggacgcgccctgtag

cggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccct

agcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagc

tctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaact

tgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt

ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggt

ctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattta

acaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttcctgatgcggtattttctc

cttacgcatctgtgcggtatttcacaccgcatacgcggatctgcgcagcaccatggcctgaaataa

cctctgaaagaggaacttggttaggtaccttctgaggcggaaagaaccagctgtggaatgtgtgtc

agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaatt

agtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc

tcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagtt

ccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcgg

cctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttg

attcttctgacacaacagtctcgaacttaaggctagagccaccatgattgaacaagatggattgca

cgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcgg

ctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccga

cctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacggg

cgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcga

agtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctga

tgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcg

catcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagca

tcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatct

cgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggatt

catcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatat

tgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccga

ttcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaa

atgaccgaccaagcgacgcccaacctgccatcacgatggccgcaataaaatatctttattttcatt

acatctgtgtgttggttttttgtgtgaatcgatagcgataaggatccgcgtatggtgcactctcag

tacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgcc

ctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcat

gtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatt

tttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgt

gcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaata

accctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgc

ccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt

aaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa

gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatg

tggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctca

gaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagaga

attatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgg

aggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttg

ggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggc

aacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaataga

ctggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat

tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatgg

taagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatag

acagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcata

tatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttga

taatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa

gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc

accgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactgg

cttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaa

gaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg

cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcggg

ctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct

acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag

cggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatag

tcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggag

cctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca

catggctcgacagatct

<210>4

<211>676

<212>PRT

<213> artificial sequence

<220>

<221>PCI-P53-hPXR

<223>143-215 is P53,216-222 be linker, 223-655 is hPXR.

<400>4

Arg Pro Val Asp Ala Asn Gly Arg Xaa Ala Cys Thr Val Gly Gly Leu

5 10 15

Tyr Lys Gln Ser Ser Phe Ser Glu Pro Ser Asp His Xaa Lys Leu Tyr

20 25 30

Cys Gly Ser Leu Ser Gln Leu Asn Cys Xaa Arg Ser Gln Cys Phe Xaa

35 40 45

His Asn Ser Leu Glu Leu Lys Leu Gln Xaa Leu Ser Xaa Gly Ser Leu

50 55 60

Ala Glu Val Gly Arg Glu Ala Leu Gly Arg Xaa Val Ser Arg Leu Gln

65 70 75 80

Asp Arg Phe Lys Glu Thr Asn Arg Asn Trp Ala Cys Arg Asp Arg Glu

85 90 95

Asp Ser Cys Val Ser Asp Arg His Leu Leu Val Leu Leu Thr Ser Thr

100 105 110

Leu Pro Phe Ser Pro Gln Val Ser Thr Pro Ser Ser Ile Thr Ala Leu

115 120 125

Lys Ala Arg Val Leu Asn Thr Thr His Tyr Arg Leu Ala Thr Met Glu

130 135 140

Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr

145 150 155 160

Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro

165 170 175

Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile

180 185 190

Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg Met

195 200 205

Pro Glu Ala Ala Pro Pro Val Gly Ser Thr Arg Tyr Gln Ala Glu Val

210 215 220

Arg Pro Lys Glu Ser Trp Asn His Ala Asp Phe Val His Cys Glu Asp

225 230 235 240

Thr Glu Ser Val Pro Gly Lys Pro Ser Val Asn Ala Asp Glu Glu Val

245 250 255

Gly Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala Thr Gly Tyr

260 265 270

His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg

275 280 285

Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg Lys Gly Ala

290 295 300

Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala Cys Arg Leu

305 310 315 320

Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met Ile Met Ser Asp

325 330 335

Glu Ala Val Glu Glu Arg Arg Ala Leu Ile Lys Arg Lys Lys Ser Glu

340 345 350

Arg Thr Gly Thr Gln Pro Leu Gly Val Gln Gly Leu Thr Glu Glu Gln

355 360 365

Arg Met Met Ile Arg Glu Leu Met Asp Ala Gln Met Lys Thr Phe Asp

370 375 380

Thr Thr Phe Ser His Phe Lys Asn Phe Arg Leu Pro Gly Val Leu Ser

385 390 395 400

Ser Gly Cys Glu Leu Pro Glu Ser Leu Gln Ala Pro Ser Arg Glu Glu

405 410 415

Ala Ala Lys Trp Ser Gln Val Arg Lys Asp Leu Cys Ser Leu Lys Val

420 425 430

Ser Leu Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn Tyr Lys Pro

435 440 445

Pro Ala Asp Ser Gly Gly Lys Glu Ile Phe Ser Leu Leu Pro His Met

450 455 460

Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Ile Ile Ser Phe Ala Lys

465 470 475 480

Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln Ile Ser Leu

485 490 495

Leu Lys Gly Ala Ala Phe Glu Leu Cys Gln Leu Arg Phe Asn Thr Val

500 505 510

Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys Gly Arg Leu Ser Tyr Cys

515 520 525

Leu Glu Asp Thr Ala Gly Gly Phe Gln Gln Leu Leu Leu Glu Pro Met

530 535 540

Leu Lys Phe His Tyr Met Leu Lys Lys Leu Gln Leu His Glu Glu Glu

545 550 555 560

Tyr Val Leu Met Gln Ala Ile Ser Leu Phe Ser Pro Asp Arg Pro Gly

565 570 575

Val Leu Gln His Arg Val Val Asp Gln Leu Gln Glu Gln Phe Ala Ile

580 585 590

Thr Leu Lys Ser Tyr Ile Glu Cys Asn Arg Pro Gln Pro Ala His Arg

595 600 605

Phe Leu Phe Leu Lys Ile Met Ala Met Leu Thr Glu Leu Arg Ser Ile

610 615 620

Asn Ala Gln His Thr Gln Arg Leu Leu Arg Ile Gln Asp Ile His His

625 630 635 640

Pro Phe Ala Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr Gly Ser

645 650 655

Xaa Glu Phe Thr Arg Gly Thr Ser Arg Val Asp Pro Gly Gly Arg Phe

660 665 670

Pro Leu Val Arg

675

<210>5

<211>5472

<212>DNA

<213>PCI-neo Vector

<400>5

tcaatattggccattagccatattattcattggttatatagcataaatcaatattggctattggcc

attgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgcc

atgttggcattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccc

atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc

ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacg

tcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaag

tccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

acgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggtt

ttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccat

tgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactg

cgatcgcccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcaga

gctcgtttagtgaaccgtcagatcactagaagctttattgcggtagtttatcacagttaaattgct

aacgcagtcagtgcttctgacacaacagtctcgaacttaagctgcagtgactctcttaaggtagcc

ttgcagaagttggtcgtgaggcactgggcaggtaagtatcaaggttacaagacaggtttaaggaga

ccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcacctattggtct

tactgacatccactttgcctttctctccacaggtgtccactcccagttcaattacagctcttaagg

ctagagtacttaatacgactcactataggctagcctcgagaattcacgcgtggtacctctagagtc

gacccgggcggccgcttccctttagtgagggttaatgcttcgagcagacatgataagatacattga

tgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgc

tattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattt

tatgtttcaggttcagggggagatgtgggaggttttttaaagcaagtaaaacctctacaaatgtgg

taaaatccgataaggatcgatccgggctggcgtaatagcgaagaggcccgcaccgatcgcccttcc

caacagttgcgcagcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtg

tggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttct

tcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttag

ggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgta

gtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtg

gactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataaggga

ttttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttta

acaaaatattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttc

acaccgcatacgcggatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggtta

ggtaccttctgaggcggaaagaaccagctgtggaatgtgtgtcagttagggtgtggaaagtcccca

ggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaag

tccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtc

ccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggc

tgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtag

tgaggaggcttttttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcg

aacttaaggctagagccaccatgattgaacaagatggattgcacgcaggttctccggccgcttggg

tggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttcc

ggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaac

tgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcg

acgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgt

catctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgc

ttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga

tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaac

tgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcct

gcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtg

tggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaat

gggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatc

gccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaa

cctgccatcacgatggccgcaataaaatatctttattttcattacatctgtgtgttggttttttgt

gtgaatcgatagcgataaggatccgcgtatggtgcactctcagtacaatctgctctgatgccgcat

agttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccgg

catccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcat

caccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataa

taatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttat

ttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat

attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcat

tttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg

gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccg

aagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg

acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac

cagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacca

tgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt

ttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcca

taccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaa

ctggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttg

caggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtg

agcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtta

tctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcct

cactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaac

ttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctt

aacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatc

ctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtt

tgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaa

atactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacat

acctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggt

tggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacac

agcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcg

ccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagc

gcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctct

gacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacg

cggcctttttacggttcctggccttttgctggccttttgctcacatggctcgacagatct

Claims (9)

1., for improving the mosaic gene that the hepatocellular physiological CYP3A4 of vitro culture of human expresses, it is characterized in that: its sequence is for shown in SEQ ID No.1 or SEQ ID No.3.

2. a recombinant eukaryon expression vector, is characterized in that, it comprises mosaic gene as claimed in claim 1.

3. recombinant eukaryon expression vector as claimed in claim 2, is characterized in that: described carrier for expression of eukaryon is mammalian cell expression vector.

4. recombinant eukaryon expression vector as claimed in claim 3, is characterized in that: described mammalian cell expression vector is PCI-neo mammalian expression vector.

5. recombinant eukaryon expression vector as claimed in claim 4, is characterized in that: described PCI-neo mammalian expression vector comprises β-globin/IgG chimeric intron sequence successively and SV40Late poly (A) signal sequence in the early stage enhancers/promoters sequence of human cytomegalovirus and downstream thereof.

6. recombinant eukaryon expression vector as claimed in claim 5, is characterized in that: comprise following restriction enzyme site between the β-globin/IgG chimeric intron sequence of described PCI-neo mammalian expression vector and SV40Late poly (A) signal sequence: Nhe I, Xho I, EcoR I, Mlu I, Xba I, Sal I, Acc I, Sma I and Not I.

7. a construction process for the recombinant eukaryon expression vector as described in claim 2-6 any one, is characterized in that, it comprises the following steps:

(1) the cDNA human cloning nf Pregnane X Receptor full length gene sequence that normal people's primary hepatocyte of including according to GenBank database is originated;

(2) the activation domain sequence of the cDNA sequence clone wild-type P53 gene of including according to GenBank database;

(3) two sections of sequence construct orderings that described step (1) and (2) obtain are classified as the mosaic gene shown in SEQ ID No.1 or SEQ ID No.3, and between directed cloning to the β-globin/IgG chimeric intron sequence and SV40Late poly (A) signal sequence of PCI-neo mammalian expression vector, obtain recombinant eukaryon expression vector.

8. the construction process of recombinant eukaryon expression vector as claimed in claim 7, is characterized in that: the activation domain sequence orientation of described people's nf Pregnane X Receptor full length gene sequence and wild type P53 gene is inserted on Nhe I, the Xho I of described PCI-neo mammalian expression vector, EcoR I, Mlu I, Xba I, Sal I, Acc I, Sma I and any one restriction enzyme site of Not I.

9. the construction process of recombinant eukaryon expression vector as claimed in claim 7, is characterized in that: the recombinant eukaryon expression vector of described acquisition is by restriction map method and/or measure its nucleotide sequence and carry out screening and identification.