CN1152029A - P450 series of human cytochrome transgenosis cell strain - Google Patents
P450 series of human cytochrome transgenosis cell strain Download PDFInfo
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- CN1152029A CN1152029A CN 95120603 CN95120603A CN1152029A CN 1152029 A CN1152029 A CN 1152029A CN 95120603 CN95120603 CN 95120603 CN 95120603 A CN95120603 A CN 95120603A CN 1152029 A CN1152029 A CN 1152029A
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Abstract
By means of DNA recombination techn, through RT-PCR amplification of CYP1A1, 1A2, 2A6, 2B6, 2E1 and 3A4 cDNA in human liver tissue, recombination with eucaryotic expression vector pREP9, and stable expression in mammal cells, CYP transgenic cell line is set up. It is used in the study of genetic toxicology and makes it possible to determine the metabolic activation and toxicologic terminal of carcinogen inside the identical cell, and this results in high experiment sensitivity and provides useful means for the study of metabolic function of different CYP isozyme.
Description
The invention belongs to molecular biology, cytology category.
The metabolism and the genetic toxicology that are applicable to environmental carcinogens such as medicine, makeup, foodstuff additive, agricultural chemicals, Chemicals, toxin detect and study.
Main both at home and abroad at present mutagenicity, the carinogenicity of using genetic toxicity short-time test technology for detection chemical substance, because most of chemical mutagens/carcinogens needs could produce the genetic toxicity effect through cells in vivo cytochrome p 450 (being called for short CYP) metabolism activation, build the strain cell overwhelming majority and can not express CYP and tradition is used, therefore must add the hepatomicrosome enzyme system in rodent or other source or helper in the extracellular to reach the ideal activation.To carry out the activatory process in cell just in time opposite for procarcinogen in this design and the body, because ultimate carcinogens meets with the endochylema envelope barrier when entering cell and the ultimate carcinogens half life is generally all very short, in addition, because used rodentine CYP and human CYP have bigger different, thereby influenced the confidence level of result of study.
The current external main DNA recombinant technology that adopts, set up the cDNA library, after obtaining people CYP expression of gene clone, importing is built in the strain cell and is expressed, and be applied to the detection and the research of pharmacology, genetic toxicology, but it is long to exist experimental period, workload is big, cell expressing state labile, used target cell are not suitable for the pharmacology of China, toxicology detects shortcomings such as regulation, and commercial abroad product price is also higher, is difficult to apply at home.
The transgenosis cell strain of the series of stable expressing human CYP that the present invention sets up is applied to the genetic toxicity short-time test, and relevant carcinogenic metabolism activation and toxicity endpoint determination are carried out in same cell, has further improved the sensitivity and the confidence level of testing; And make the genetoxic of selecting for use than low dosage, long period exposure, detection chemical carcinogen become possibility; Set up a series of people CYP transgenic cell lines, the metabolic function research that can be different CYP isozyme provides very useful instrument.
The present invention adopts following technical proposals:
(1) clone of people CYP cDNA and evaluation:
Human liver cell sample and people's amnion FL cell with excision are parent material, extracted total RNA, after the specific primer RT-PCR of design obtains cDNA, SouthernBlot hybridization, tentatively this cDNA fragment is analyzed and identified, this cDNA fragment cloning is carried out sequential analysis and restriction enzyme mapping analysis to plasmid pGEM-3Z, errorless to confirm the gene that obtains.
(2) structure of eukaryotic cell expression recombinant chou and evaluation:
The CYP cDNA fragment that is cloned in the recombinant plasmid is downcut with restriction enzyme, Separation and Recovery on running gel, subclone is gone among the eukaryotic expression vector pREP-9, and enzyme is cut the orientation of identifying cloned sequence on the recombinant expressed body in case of necessity.
(3) the eukaryotic cell expression recombinant chou imports and expresses and build strain in the culturing cell:
Utilize the electroporation transfer techniques that the recombined eukaryotic cell expression vector is imported in the suitable culturing cell, filter out resistant cell with G418 after, adopt the determination of activity technical evaluation of Northern Blot and CYP isozyme, to obtain cell strain of expressing steady in a long-term.
(4) this clone is combined with genetic toxicology short-time test technology, set up the preceding mutagen of new detection/carcinogenic method, be used for genetic toxicology research.
The people CYP gene Selection of clonal expression be present in liver and three extended familys that in medicine and preceding mutagen/carcinogens metabolism, play a significant role in genes of individuals: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1 and CYP3A4.The mammals target cell of genetic expression is selected the cell of the relevant rules and regulations of country such as Chinese hamster CHL, V79 cell for use, people's amnion FL cell and be suitable for other all cells that this gene line tabulation reaches.
Compared with prior art, the present invention has further improved the confidence level and the sensitivity of result of study, for the metabolic function research of the individual isozyme of people CYP provides very useful instrument, promotes and improve the monitoring and the research level of China's environmental carcinogen.This serial people CYP transgenosis cell strain can be applicable to environment protection, monitoring, medicine, makeup, foodstuff additive, and the production monitoring and the management of aspects such as daily chemical product and agricultural chemicals, thereby have certain economic benefits.
Technically, the present invention at first adopts RT-PCR technology human cloning CYP gene, has overcome the difficulty in the process of construction cDNA library, alleviated workload greatly, saved cost, shortened experimental period, and a few strain cells of the relevant rules and regulations of application country are as genetically modified target cell.Experimental result shows: the gene energy stably express of importing is more than half a year.
The present invention is described further in conjunction with the embodiments:
Embodiment 1: clone and the evaluation of people CYP cDNA
According to the CYP cDNA sequence of having delivered, select 3 of its 5 ' end and coding region sequence ' hold the initiating terminal be respectively the both sides primer, 5 ' end of two PCR primers contains the restriction enzyme enzyme recognition site respectively.
Human liver tissue is taken from the cancer beside organism of excision, and total RNA presses the AGPC method and extracts, and measures and definite RNA purity, concentration and the integrity that is obtained of denaturing formaldehyde gel electrophoresis through ultraviolet spectrophotometer.
The total RNA of RT-PCR amplification CYP cDNA:lug is through 65 ℃ of sex change 5min, put and add 4ul 5 * reversed transcriptive enzyme damping fluid on ice successively, 2ul 10mM dNTP, 0.5ulRNA enzyme inhibitor (RNAsin, 40u/ul), lul 100mM random primer, 2ul reversed transcriptive enzyme (AMV, 7.5u/ul), add distilled water to final volume 20ul, room temperature 10min, 42 ℃ of blue or green 1.5h of temperature, add each 30pmoles of both sides primer then, lul Taq enzyme (2u/ul) adds distilled water to final volume 100ul, loop parameter: 94 ℃ of 1min, 55 ℃ of 2min, 72 ℃ of 1min are totally 35 back 72 ℃ of 7min of circulation.After pcr amplification finishes, get 10ul reactant electrophoresis detection on 1% sepharose.
With intrinsic corresponding restriction endonuclease respectively enzyme cut pcr amplification product and pGEM-3Z plasmid (2.74kb).1% agarose gel electrophoresis separates, and electrophoresis reclaims in dialysis tubing, connects the screening of transformation receptor bacterium JM 109 and positive colony by the method for document description.
The restriction endonuclease map of cloned sequence and partial sequence Analysis and Identification, by Gen Bank software analysis on the computer relatively, select a suitable restriction enzyme, make restricted enzyme cutting spectrum analysis, the polymorphism and the pseudogene of difference gene are applied to this aim sequence analysis and are undertaken by the test kit process specifications.
The structure and the evaluation of embodiment 2 eukaryotic cell expression recombinant chous:
From the recombinant plasmid of amplification through enzyme cut, Separation and Recovery CYP cDNA fragment on the running gel, directly be connected with the eukaryotic expression vector pREP-9 that cuts through enzyme, calf intestine alkaline phosphatase (CIP) is handled, transformed competence colibacillus intestinal bacteria Top10, extract plasmid, carry out length analysis and restriction enzyme mapping analysis, filter out positive colony.To single endonuclease digestion site clone's fragment, still need and insert the evaluation of direction of fragments.
Amplification forward eukaryotic cell expression body utilizes electroporation technology that recombinant expressed body is imported in the suitable culturing cell, and the method for electroporation and program are undertaken by the Bio-Rad specification sheets, behind the electroporation, cell is resuspended in the 20ml DMEM nutrient solution, moves into 2 10cm culture dish, put 37 ℃ of 5%CO
2Cultivate 48h, add G418 then, changed liquid once, screened for two weeks altogether, the cell clone growth occurred in 10 days every 3 days to final concentration 500mg/ml, with the clone cell enlarged culturing, in time frozen.Press the described method of document, from the transgenic cell cultivated and maternal cell thereof, extract total RNA, with
32P-CYP cDNA respective segments is a probe, make Northern Blot and analyze, and with β-actin as internal reference, detect the CYP rna expression level of transgenic cell.The CYP isozyme determination of activity of transgenic cell, in culturing cell, extract S9 or microsome after, the specific enzymes reaction substrate of getting separately reacts, reaction result with fluorescent spectrophotometer assay as calculated the specific activity of enzyme represent.
Application on the embodiment 4 genetic toxicology short-time test
Adopt the test of double-core micronucleus method.5 * 10
5Cell inoculation is in 25cm
2Spend the night in the Tissue Culture Flask (every kind is tried 2 bottles of concentration inoculations), use 5ml instead and be added with the serum-free MEM substratum that is tried mutagen, behind the 4h, wash attached cell 2 times with PBS, add the MEM 5ml that contains 10% calf serum and 3ug/ml CYB (B cytochalasin B), cultivate 16h through 36.5 ℃, the trysinization collecting cell, be suspended among the 2ml PBS, slowly add stationary liquid (1: 3 the glacial acetic acid: methyl alcohol), pre-fix 30min of 1/5Vol, 1000rpm * 10min is centrifugal, sedimentary cell is fixed 2 times with the 4ml stationary liquid again, each 30min, and the cell after fixing drips sheet with air drying, the 10%Giemsa 15min that dyes, calculate 1000 dikaryotic micronucleated cell frequencies under powerful microscope, the judgement of micronucleated cell is undertaken by the revised standard of Ciaravino, and experimental result is carried out statistical study with the Kastenbaum-Bowman method.
The transgenosis cell strain that the stably express CYP that we set up is used in this research carries out the double-core micronucleus test to mutagen/carcinogens before a group, is contrast with parent cell simultaneously.
Description of drawings:
Fig. 1. the design of graphics of recombinant plasmid and the recombinant expressed body of eukaryotic cell
The last figure of Fig. 2 .Northern Blot result: Lane 1,3,5 be the total RNA of transgenic cell with
32P-CYP cDNA is hybridized knot
Really
Figure below as a result: the wash-out caudacoria with
32The result of P-β-actin hybridization
Fig. 3. transgenic cell (CHL-1A1) and control cells EROD enzymic activity
Fig. 4., Fig. 5. shown in: transgenosis cell strain CHL-2B6, the CHL-1A1 that uses CYP2B6 of stably express respectively that we set up and CYP 1A1 carries out the double-core micronucleus test to causing projecture/carcinogens before one group, be that CHL is contrast with parent cell simultaneously, the result shows, mutagen NNK, DEN, NM, AFBl and CP all can induce the micronuclear rates of CHL-2B6 cell significantly to increase before five kinds, NNK wherein, CP and AFBl have dose-response relationship, increase by 3～8 times approximately at the highest concentration micronuclear rates that tried.Mutagen before five kinds, benzo [α] pyrene, 3-MECA, benzanthrene, dimethylbenzene anthracene and anthracene all can induce the micronuclear rates of CHL-1A1 cell significantly to increase, and have dose-response relationship.The above results shows that transgenosis cell strain has important use value aspect the genetic toxicology research of chemical carcinogen.
Claims (3)
1. the serial transgenosis cell strain of a human-cytochrome P450 (being called for short CYP), comprising that the structure of the clone of people CYP cDNA and evaluation, eukaryotic cell expression recombinant chou and evaluation, eukaryotic cell expression recombinant chou import expresses in the culturing cell and builds strain etc., it is characterized in that:
(1) clone of people CYP cDNA and evaluation:
Human liver cell sample and people's amnion FL cell with excision are parent material, extracted total RNA, after the specific primer RT-PCR of design obtains cDNA, SouthernBlot hybridization, tentatively this cDNA fragment is analyzed and identified, this cDNA fragment cloning is carried out sequential analysis and restriction enzyme mapping analysis to plasmid pGEM-3Z, errorless to confirm the gene that obtains.
(2) structure of eukaryotic cell expression recombinant chou and evaluation:
The CYP cDNA fragment that is cloned in the recombinant plasmid is downcut with restriction enzyme, Separation and Recovery on running gel, subclone is gone among the eukaryotic expression vector pREP-9, and enzyme is cut the orientation of identifying cloned sequence on the recombinant expressed body in case of necessity.
(3) the eukaryotic cell expression recombinant chou imports and expresses and build strain in the culturing cell:
Utilize the electroporation transfer techniques that the recombined eukaryotic cell expression vector is imported in the suitable culturing cell, filter out resistant cell with G418 after, adopt the determination of activity technical evaluation of Northern Blot and CYP isozyme, to obtain cell strain of expressing steady in a long-term.
2. P 450 series of human cytochrome transgenosis cell strain as claimed in claim 1 is characterized in that: people CYP gene Selection CYP1A1, CYP1A2, CYP2A6, CYP 2B6, CYP2E1 and the CYP3A4 etc. of clonal expression.
3. P 450 series of human cytochrome transgenosis cell strain as claimed in claim 1 is characterized in that: the mammals target cell of genetic expression is selected for use, Chinese hamster CHL, V
79Cell, people's amnion FL cell and be suitable for other all cells that the tabulation of this gene line reaches.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95120603 CN1152029A (en) | 1995-12-11 | 1995-12-11 | P450 series of human cytochrome transgenosis cell strain |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 95120603 CN1152029A (en) | 1995-12-11 | 1995-12-11 | P450 series of human cytochrome transgenosis cell strain |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8049063B2 (en) | 2005-06-28 | 2011-11-01 | Zhejiang University | Rice bentazon and sulfonylurea herbicide resistant gene Cyp81a6 |
| CN102321641A (en) * | 2003-10-16 | 2012-01-18 | 美国无烟烟草有限责任公司 | From the tobacco cloning of cytochrome P 450 genes |
| CN105238731A (en) * | 2015-09-28 | 2016-01-13 | 中南民族大学 | Expression system of rat cytochrome P4501A1 enzyme and building method and application thereof |
-
1995
- 1995-12-11 CN CN 95120603 patent/CN1152029A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321641A (en) * | 2003-10-16 | 2012-01-18 | 美国无烟烟草有限责任公司 | From the tobacco cloning of cytochrome P 450 genes |
| US8049063B2 (en) | 2005-06-28 | 2011-11-01 | Zhejiang University | Rice bentazon and sulfonylurea herbicide resistant gene Cyp81a6 |
| CN105238731A (en) * | 2015-09-28 | 2016-01-13 | 中南民族大学 | Expression system of rat cytochrome P4501A1 enzyme and building method and application thereof |
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