CN1749403A - Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene - Google Patents

Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene Download PDF

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CN1749403A
CN1749403A CN 200410074417 CN200410074417A CN1749403A CN 1749403 A CN1749403 A CN 1749403A CN 200410074417 CN200410074417 CN 200410074417 CN 200410074417 A CN200410074417 A CN 200410074417A CN 1749403 A CN1749403 A CN 1749403A
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nucleotide sequence
dioxins
gene
recombinant vectors
recombinant
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李伟山
李心予
张简国平
黄怡玮
卢冠妤
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ZHENGXIU TECH UNIV
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ZHENGXIU TECH UNIV
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Abstract

The present invention relates to one recombinant cell strain with foreign leuciferinase gene, and is especially one DNA segment of transfected mouse liver cancer cell strain Hepalcle7-M4P2 and Hepale7-MP and with dioxin reaction segment specific nucleotide sequence ''cacgc'' and ''gcgtg''. The DNA segment is designed based on Mus musculus CYP1 A1 gene promoter area and partial fore end sequence of the structural gene. Therefore, the recombinant cell strain and the cultured descendant may be used in detecting dioxin compounds from the sample collected from natural environment by means of biological analysis process.

Description

A kind of performance according to luciferase gene detects the recombinant cell strain of dioxin compounds
Invention field
The present invention mainly relates to the recombinant cell strain (recombinant cell line) that has the plain enzyme gene of an extrinsic fluorescence (exogenousluciferase gene), particularly through rat liver cancer cell (transfected mouse hepatoma cell) Hepa1c1c7-M4P2 and the Hepa1c1c7-MP of transfection, wherein the upstream end of this luciferase gene is located to contain in the middle of one and might be the dna fragmentation of the peculiar nucleotide sequence of Dioxins fragment reaction " cacgc " with " gcgtg ".
Background technology
Because civilized over-drastic exploitation, many tangible or invisible pollution factors are over and over again attacked the human society of living in civilized world, and in all pollution factors, Dioxins (dioxins) is considered to now to be the most malicious chemical (chemicals) for the human and animal, and therefore is referred to as " poison in century ".Find that in nearly two during the last ten years researchs Dioxins not only can influence the human body fertility, cause dysplasia, destroy immunity system and disturb outside the normal internal secretion, even also have carinogenicity.Yet Dioxins is a kind of not labile material, and it can be accumulated and be present in nature and the organism constantly, and forms a serious potential hazard for entire environment.
Especially, because Dioxins is under general state, even all highly stable in the environment of heat, acid or alkali, when it is released in the environment by trace and after entering food chain, can makes concentration more and more higher along with the soaring of food chain.And the hormonal structural similitude of the chemical structure of Dioxins and human body (E.L.Gregoraszczuk, Cad Saude Publica., Mar-Apr, 2002,18 (2): 453-62), therefore, after entering human body via food chain, can form " false hormone " on the one hand and produce similar hormonal effect, can influence the intravital hormone content of body on the other hand, these two all can disturb the original endocrine mechanism of human body, and has a strong impact on the metabolism and the hormonal balance of interference of human body, causes physical function impaired.
In addition, Dioxins also is proved to be powerful carcinogenic promoting agent (tumor promoter), can cause the animal carinogenicity, comprises the incidence that increases big white mouse liver cancer, lung tumors and dermatoma.Environmental Protection Agency (US-EPA) and The World Health Organization (WHO) classify as the human carcinogens of possibility with Dioxins, and international cancer center (InternationalAgency for Research on Cancer, IARC) in 1997 with toxicity the strongest 2,3,7,8-TCDD classifies as " the one-level mankind determine to control the cancer thing " (D.B.McGregoret al. (1998), Environ Health Perspect., Apr; 106 Suppl2:755-60).Therefore, how in the shortest time, to use effective ways to detect this environmental pollution factors, promptly become correlative study personnel's an important research direction.
To be a group linked and the general designation of the compound that is formed by one or two Sauerstoffatom and two phenyl ring Dioxins (dioxins), and this compounds is to belong to halogenated aromatic hydrocarbon polymer (halogenated aromatic hydrocarbons, HAHs) Dang Zhong a class.Dioxins can be divided into the copline tricyclic compound of two series, is respectively: (polychlorinated dibenzo-p-dioxins, PCDDs), it is C that this compounds has a chemical general formula to (1) many chlorine biphenyl Dioxins 12H 8-nO 2Cl n, n=1～8 wherein; And (2) many chlorine biphenyl furans (polychlorinated dibenzofurans, PCDFs), it is C that this compounds has a chemical general formula 12H 8-nOCl n, n=1～8 wherein.After connecting the chlorine atom of different numbers on eight the position of substitution of phenyl ring, PCDDs has 75 kinds of isomers (isomers), and PCDFs has 135 kinds of isomers.In this Dioxins isomer of 210 kinds, with 2,3,7,8-tetrachloro dibenzo-ρ-Dioxins (2,3,7,8-tetrachloro-dibenzo-ρ-dioxin, 2,3,7, toxicity 8-TCDD) is the strongest, " poison in century " therefore is otherwise known as.
PCDDs is the aromatics that almost becomes two dimensional structure with PCDFs, the rerum natura of this two compounds is similar to voltinism, has highly stable organic molecular structure equally, and have high-melting-point, high boiling point and not labile characteristic, (the W.Parzefall (2002) that exists that therefore these compounds all can be arranged in various environmental medias (such as air, soil, water and food), Food Chem Toxicol., 40 (8): 1185-9).With 2,3,7,8-TCDD is an example, and its molecular weight is 322, and high-temp combustion need be just can be destroyed more than 850 ℃.With regard to its volatility, its vapour pressure under 25 ℃ is 7.4 * 10 -10MmHg.In addition, Dioxins is 19.3ng/L to the solubleness of water, belongs to lipotropy, and is very stable under normal circumstances.Its chemical property can be changed when being exposed to octane-iso and UV-light, Dioxins was very high for the stability of heat, acid, alkali.
Based on the above characteristic of carrying, all very slow no matter Dioxins is a evaporation rate in water or in the soil.With the environment decomposability, in the ecological test of Simulated Water, in 100 kinds of microorganisms, only there are 5 kinds can decompose TCDD.Therefore, in a single day Dioxins just is difficult to be decomposed after nature produces, and can accumulate at occurring in nature constantly.
The measure unit of Dioxins is represented to contain how many toxic equivalents in the medium usually.Because Dioxins has 75 isomers, the toxicity of each isomer differs, and experimental animal also has the variation of a wide scope to their reaction, and is exposed to dioxin compounds in the environment normally by the many kinds of mixtures that compound constituted but not the simplification compound.Therefore, mensuration mode about Dioxins, many scholars propose the viewpoint of the equivalent number of so-called toxicity, and according to aromatic hydrocarbons susceptor (arylhydrocarbon receptor, be called for short AHR) for the impression degree of each simplification compound, and stipulate out the international toxic equivalent factor (International ToxicityEquivalency Factor for the relative toxicity of material influence (relative toxicityinfluence) according to these compounds, I-TEF), and with toxicity the strongest 2,3,7,8-TCDD is decided to be 1, and becomes European Environmental Health center (the WHO EuropeanCenter for Environment ﹠amp of the World Health Organization; Health) and international chemical safety program (Interntional Programme on Chemical Safety, IPCS) the common stdn toxic equivalent factor (S.H.Safe (1990), Crit.Rev.Toxicol., 21 (1): 51-88) that propose.
The use of TEF value is based on following hypothesis: (1) supposes that all dioxin compounds all only cause toxic action via AHR; (2) the toxicity summation of dioxin compounds is the result that toxicity of compound out of the ordinary is summed into; And (3) only use TEF to infer most toxic action.So, 2,3,7, the account form of the toxic equivalent of 8-TCDD is as follows:
TEQ=TEF * actual concentration (true concentration)
In the world, the toxic equivalent of dioxin compounds is to represent that with I-TEQ (International Toxic Equivalency) wherein airborne Dioxins concentration can ng-TEQ/Nm 3Represent that the Dioxins concentration in the soil is then represented with pg-TEQ/g.If the Dioxins quantity that is contained in simple calculation medium can adopt the weight representation, commonly used have pg/g, ppt, a ppb etc.For example, pg/kg-bw is meant that per kilogram of body weight contains the Dioxins of how many pg.The World Health Organization advises that the Dioxins of everyone every day allows that intake is 1～4pg/kg-bw, if with the grownup of 60 kilograms of body weight, the intake of allowing that every day is the highest is the Dioxins of 240pg.
In the past over 20 years, the Chemical Analysis method of Dioxins has been developed quite perfectly, mainly be to utilize gas chromatography (gas chromatography) to add that electron capture detects (electron capture detection) or mass spectrograph (massspectrometry) in the middle of this, and analytical procedure consists predominantly of: sample collecting, extraction, purification, separation and quantitative assay (C.Rappe (1991), IARC Sci Publ., 108:1-426; De Jong APJM and AKD.Liem (1993), TrandsAnal Chem., 12:115-124).The chemical detection method can be analyzed out with all the components concentration of Dioxins accurately, but needs to expend many time and money costs in whole testing process, and is unfavorable for being used in the daily routine check.
Then, in this area, develop and another kind of Enzyme Linked Immunoadsorbent Assay (enzyme-linked immunosorbent assay based on immuno-chemical method, ELISA) (Zajicek JL, Application of enzyme-linkedimmunosorbent assay (ELISA) for measurement ofpolychlorinated biphenyls from hydrophobic solutions:extracts of fish and dialysates of semipermeable membranedevices.In:Van Emon JM et al., editors.Environmentalinnunochemical methods, chapter 26.American ChemicalSociety, 1996, pp.307-325).This kind technology is according to having a splendid specificity and an affinity between the antibody antigen corresponding with it, therefore can utilize haptens (hapten) to produce Dioxins-specificity antibody (Y.Sugawara et al. (1998) at the Dioxins chemical structure, Anal Chem., 70 (6): 1092-1099), or can specificity the antibody of the AHR that engages with Dioxins of ground identification meeting, and come concentration (the A.Poland et al. (1991) of the external Dioxins of identification by the degree of the AHR that measure to transform, Mol Pharmacol., 39 (4): 435).Yet this type of detection method is quite complicated on experimental implementation.
In recent years there is the people to set up biological inductor (biosensor) technology (S.Bender et al. (1998) based on this immunoassay, Environ Sci Technol., 32:788-797), wherein cooperate the suitable detection system to constitute the immunologic pattern biological inductor with antibody, and by the antigen concentration in the next quantitative sample of the quality change in the detection reaction process.For example, via chemical bond antibody effectively is fixed on the gold electrode surfaces, the antibody that is present in the antigen in the sample and is fixed produces the affinity combination, detect via optics (optical fiber and surface plasma resonant) or oscillating crystal signal converting systems such as (quartz oscillation crystal and surface elasticity wave apparatus) again, thus, can be effectively the quantitative antigen concentration in the sample.In addition, also can be by on antibody, dealing with, make its with Dioxins in conjunction with after can emit electrochemistry signal (electrochemical signal), come interpretation Dioxins quantity by signal again.But the technology of this respect also is not very ripe, is not widely used at present yet.
In order to develop quicker and detection mode easily,, come into one's own gradually based on the biological detecting method (bioassays) of biological respinse also along with people day by day understand Dioxins intoxicating mechanism in vivo.
In the time of in an organism is exposed to the environment that contains Dioxins, it can produce many biochemistry, physiological variation, and can judge the existence of Dioxins by observing these variations.In the intoxicating mechanism of Dioxins by broad research the most be AHR bang path (AHR pathway) (O.Hankinson (1995), Annu Rev PharmacolToxicol., 35:307-40).
After Dioxins enters cell, it can enter to nucleus via a series of message transmission in the AHR bang path, can pass through and AHR nuclear translocation albumen (AHRnuclear translocator at last, ARNT) combination and be brought to Dioxins fragment reaction (dioxin response elements on the DNA, DREs) locate, and then the regulatory gene effect.In this respect, Cytochrome P450 1A (cytochrome P450 1A, CYP1A) gene is one famous " an in vivo biomarker (in vivo biomarker) ", no matter be in the cell of the mankind or mouse, the CYP1A gene can be initiated a large amount of performances (M.Merchant et al. (1992) via the AHR bang path, Arch Biochem Biophys., 298 (2): 389-94; J.P.Vanden-Heuvel et al. (1993), Carcinogenesis., 14:2003-2006; Denison MS, Withlock JP Jr. (1995), Journal of Biological Chemistry, 1995; 270 (31): 18175-8).
According to reports, the DREs that is present in the gene of many different cells is found out (Z.-W.Lai et al. (1996), Chemico-Biological Interaction, 100:97-112), and in these DREs, have some specific nucleotide sequences (such as cacgc or gcgtg), they in the identification of Dioxins, play the part of a key player (J.Corchero et al. (2001), Pharmacogenetics., 11:1-6).
In biological detecting method, CALUX[chemical-activatory luciferase performance (chemical-activated luciferase expression)] analytical method is very valued in recent years a kind of method, its major technique notion is to connect reporter gene (reporter gene) in the back of DREs.
Hans Postlind in 1993 construction 2 human CYP1-luciferase plasmids, he intercept out from the mankind's CYP1 gene front promotor (promoter) and 5 '-flanking sequence (flanking sequence) fragment, and put into the plasmid that has luciferase (luciferase) to respectively, again that construction is good recombinant type plasmid is fed through in the human 101L cell, the TCDD that adds different concns afterwards stimulates transfected cell and measures the activity of luciferase, by this, the concentration of Dioxins can show and tested making (H.Postlind et al. (1993) by detected fluorescence, Toxicol Appl Pharmacol., 118 (2): 255-262).
In gross, the most accurate with chemical analysis method in present Dioxins detection method, long and required cost is high but shortcoming is complicated operating process, proving time.As for the enzyme linked immunosorbent assay based on immuno-chemical method, it has the specificity and the sensitivity advantage of bioassay method, but still quite loaded down with trivial details in operation.And be the biological inductor that the basis is set up out with the immunoassay, its maturation and not being widely used not enough on the whole technique aspect.With regard to bioassay method, the specificity, it more saves time than chemical process and is with low cost on having toxicity again, and can delicately, specificity detect the TEQ of dioxin compounds, and is applicable to the detection of a large amount of environmental samples.Therefore, develop one and be applicable to of the effective detection of the recombinant cell strain of bioassay method, become the research direction that the correlative study personnel in this area are endeavoured for dioxin compounds.
Summary of the invention
So, aspect first, it is right to the invention provides a kind of primer of cloning for the reactive dna fragmentation of Dioxins tool of being used to, it comprises a forward primer (forwardprimer) and a reverse primer (reverse primer), this forward primer has just like the sequence identification numbers: 1 or sequence identification numbering: the nucleotide sequence shown in 3, and this reverse primer has just like the sequence identification and number: 2 or the sequence identification number: the nucleotide sequence shown in 4:
Forward primer 1
5 '-cagagagcacctgcaaaaca-3 ' (sequence identification numbering: 1)
Reverse primer 1
5 '-ggctacaaagggtgatgctt-3 ' (sequence identification numbering: 2)
Forward primer 2
5 '- CtcgagAgggcaggtgaaggtgttag-3 ' (sequence identification numbering: 3)
Reverse primer 2
5 '- AagcttAagtgaagagtgttctctagg-3 ' (sequence identification numbering: 4).
So, when using the CYP1A1 gene order as template (template) and use according to primer of the present invention when carrying out the PCR reaction, can obtain one and have and to be the dna fragmentation of the peculiar nucleotide sequence of Dioxins fragment reaction (DREs) (cacgc and gcgtg).
Above-mentioned dna fragmentation, its nucleotide sequence that has are one of following:
(1) reacts the nucleotide sequence that dna fragmentation had that be amplified out according to above-mentioned primer to carrying out PCR as template and use by use CYP1A1 gene order corresponding to one in fact;
(2) in fact corresponding to numbering: the nucleotide sequence shown in 5 just like the sequence identification;
(3) in fact corresponding to numbering: the nucleotide sequence shown in 6 just like the sequence identification;
(4) in fact corresponding to one with the complementary nucleotide sequence mutually of the nucleotide sequence described in the nucleotide sequence of the dna fragmentation described in (1) or (2) or (3).
This dna fragmentation connects and can be incorporated in the carrier with a reporter gene (for example luciferase gene).Therefore, aspect second, the invention provides a kind of recombinant vectors, it has a reporter gene, and as mentioned above a dna fragmentation is positioned at the upstream end of this report gene; This report gene is a luciferase gene.
Above-mentioned corresponding to numbering just like the sequence identification: the recombinant vectors of the nucleotide sequence shown in 5 is recombinant vectors M4P2.
Above-mentioned corresponding to numbering just like the sequence identification: the recombinant vectors of the nucleotide sequence shown in 6 is recombinant vectors MP.
This recombinant vectors and then can be used to the selected host cell of transfection one.Therefore, aspect the 3rd, the invention provides a kind of recombinant cell strain, it is by coming the transfection one host cell person of being formed with as mentioned above a recombinant vectors.
This host cell that is used to transfection is rat liver cancer cell Hepa-1C1C7.
This recombinant cell strain is to be deposited at the rat liver cancer transformant strain Hepa1c1c7-MP of American type culture collection or secondly to cultivate the offspring to deposit numbering PTA-6089.
Be deposited at the rat liver cancer transformant strain Hepa1c1c7-M4P2 of American type culture collection or secondly cultivate the offspring to deposit numbering PTA-6090.
According to recombinant cell strain of the present invention and secondly cultivate the offspring and can be applied to a kind of bioanalysis that is used for detecting the dioxin compounds of an environmental sample, wherein a sample that contains dioxin compounds is touched with this recombinant cell strain in cultivating and lasts for some time, and cause the reporter gene that this recombinant vectors had of position in this recombinant cell strain to be showed, the gene product that is generated thus (for example luciferase) can produce a detectable phenomenon (for example luminous intensity), and this detectable phenomenon promptly can be used as the index of the existence of qualitative and quantitative dioxin compounds.
Description of drawings
Come below in conjunction with drawings and Examples that the present invention is described in detail, thus the present invention in above-mentioned and other purpose and feature, can by the reference following description, with the attached claims of literary composition inspection and follow graphicly become more obvious, in the accompanying drawing:
Fig. 1 shows plasmid yT﹠amp; The framework of A cloning vector (2728bp), wherein have ampicillin resistance gene (ampicillin resistance gene, AP), T 7Promotor (T 7Promoter), beta-galactosidase gene (β-galactosidase gene, lacZ) and multiple clone site (multiple cloning site, MCS);
Fig. 2 shows pGL2-promoter vector (pGL2-promoter vector) framework (5790bp), wherein has ampicillin resistance gene (Amp r), SV40 promotor, luciferase gene (luc) and multiple clone site etc.;
Fig. 3 shows pGL3-underlying carrier (pGL3-basic vector) framework (4818bp), wherein has ampicillin resistance gene (Amp r), luciferase gene (luc +) and multiple clone site etc.;
Fig. 4 shows one by using the chromosomal DNA (chromosomal DNA) that goes out from mouse macrophage J774A.1 isolation and purification to be used as template, and use according to forward primer 1 of the present invention and reverse primer 1 and carry out the nucleotide sequence that PCR reacts the PCR product that is amplified, wherein this PCR product total length is 479bp, and in the middle of contain 7 and may be the peculiar nucleotide sequence cacga of Dioxins fragment reaction (DREs) and gcgtg (part of being got up by frame);
Fig. 5 shows one by using the karyomit(e) D NA that goes out from mouse macrophage J774A.1 isolation and purification to be used as template, and use according to forward primer 2 of the present invention and reverse primer 2 and carry out the nucleotide sequence that PCR reacts the PCR product that is amplified, wherein this PCR product total length is 1503bp, and in the middle of contain 12 and may be the peculiar nucleotide sequence cacga of Dioxins fragment reaction (DREs) and gcgtg (part of being got up by frame);
Fig. 6 show according to rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention with the Dioxins of 100pM (2,3,7,8-TCDD) handled and lasted afterwards measured light intensity result of one different period (5,10,15 and 20 hours);
Fig. 7 show according to rat liver cancer transformant strain Hepa1c1c7-MP of the present invention with the Dioxins of 100pM (2,3,7,8-TCDD) handled and lasted afterwards measured light intensity result of one different period (5,10,15 and 20 hours);
Fig. 8 show according to rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention with the Dioxins of different concns (2,3,7,8-TCDD) handled last 15 hours after measured light intensity result;
Fig. 9 show according to rat liver cancer transformant strain Hepa1c1c7-MP of the present invention with the Dioxins of different concns (2,3,7,8-TCDD) handled last 15 hours after measured light intensity result;
Figure 10 show according to rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention with the Dioxins of different concns (2,3,7,8-TCDD), the standard model S of two kinds of concentration 1With S 2And flying dust sample 29 handled last 15 hours after measured light intensity result; And
Figure 11 show according to rat liver cancer transformant strain Hepa1c1c7-MP of the present invention with the Dioxins of different concns (2,3,7,8-TCDD), the standard model S of two kinds of concentration 1With S 2And flying dust sample 29 is handled measured light intensity result after 15 hours.
Rat liver cancer transformant strain Hepa1c1c7-MP and Hepa1c1c7-M4P2 have been deposited at American type culture collection (ATCC June 18 2004 Christian era, P.O.Box1549, Manassas, VA 20108, USA), deposit numbering and be respectively PTA-6089 and PTA-6090.
Embodiment
Previous studies show that, meeting and have two heat shock protein 90s (heat shock protein 90 after Dioxins enters cell, HSP90) aromatic hydrocarbon acceptor (aromatichydrocarbon receptor, AHR) in conjunction with and form mixture, and this mixture can separate after entering nucleus, and toss about in bed to be passed on the DNA after the effect by a series of downstream molecules, and go up existing Dioxins fragment reaction (DRE) with DNA and combine, and make that the gene that regulated and control by this Dioxins fragment reaction is showed in large quantities, and then have influence on the physiological system of whole organism.Characteristic at this bang path, the applicant attempts making up the recombinant vectors (recombinant vector) that contains Dioxins fragment reaction (DRE) and reporter gene, and formed recombinant vectors sent in the competent host cell (competent cell), to set up out the recombinant cell strain that has this recombinant vectors.
Resulting thus recombinant cell strain can be applied to a kind of bioanalysis of dioxin compounds that is taken from the sample of physical environment that is used for detecting.At first, use the Dioxins standard substance of different concns to handle this recombinant cell strain, to set up out a relevant Dioxins concentration with respect to the cognation between the expressive ability of the reporter gene that is had on the recombinant vectors that this recombinant cell strain was had.
Then, from a physical environment (for example: soil, the water source, edible meat, fish, vegetables and fruits or the like, the flue of factory or incinerator, bottom ash, bed mud or the like) obtain a primary sample (original sample) that may contain dioxin compounds (dioxin-like compounds), and do suitable processing according to hereinafter described various pre-treatment (preliminarytreatment), want underproof testing sample (analyteunder test) and obtain one.
Afterwards, make this testing sample contact this recombinant cell strain and last for some time, and measure the performance results of the reporter gene that is had on the recombinant vectors that this recombinant cell strain has.Resulting measurement result is thus brought and the top Dioxins concentration of setting up out is done a comparison with respect to the cognation between the expressive ability of this report gene, just can be extrapolated the Dioxins quantity that is contained in this primary sample.
According to the present invention, the CYP1A1 gene is one to be suitable for as the source of seeking useful Dioxins fragment reaction (DRE), therefore, the applicant is deposited numbering (accession number) AF210905[house mouse (Mus musculus) CYP1A1 gene according to what login in the NCBI network address, promoter region and partial sequence] when shown in nucleotide sequence and deposit numbering X01681[house mouse (Mus musculus) CYP1A1 gene] when shown in nucleotide sequence, and design at following two groups of primers of the Dioxins fragment reaction (DREs) in house mouse (Mus musculus) the CYP1A1 gene order (primer pairs):
Forward primer 1
5 '-cagagagcacctgcaaaaca-3 ' (sequence identification numbering: 1)
Reverse primer 1
5 '-ggctacaaagggtgatgctt-3 ' (sequence identification numbering: 2)
Forward primer 2
5 '- CtcgagGagggcaggtgaaggtgttag-3 ' (sequence identification numbering: 3)
Reverse primer 2
5 '- AagcttAagtgaagagtgttctctagg-3 ' (sequence identification numbering: 4)
So, when with house mouse (Mus musculus) CYP1A1 gene order during as template (template), use is carried out polymerase chain reaction (PCR) by first primer that forward primer 1 and reverse primer 1 are constituted to (first primer pair), can obtain one and be the PCR product of 479bp (sequence identification numbering: 5) (referring to Fig. 4), and use second primer that is constituted by forward primer 2 and reverse primer 2 that (second primer pair) carried out PCR, can obtain one and be the PCR product of 1503bp (sequence identification numbering: 6) (referring to Fig. 5).These two PCR products are found and have 7 and 12 respectively and may be the peculiar nucleotide sequence of Dioxins fragment reaction (cacgc and gcgtg).So the present invention uses these two PCR products further to make up the recombinant vectors of being desired.
According to the present invention, can in clone's process of recombinant vectors, (for example use intermediary's cell (intermediate cells), Bacillus coli cells) above-mentioned two the PCR products that increase in a large number, and involved experimental technique and material are to be familiar with just implementer according to this of textbook that this technology personage knows with reference to knowing clearly in this area in the middle of this.For example, referring to, Sambrook J, Russell DW (2001) Molecular Cloning:aLaboratory Manual, 3rd ed.Cold Spring Harbor LaboratoryPress, New York.
Be applicable to that the carrier that inserts above-mentioned arbitrary PCR product comprises a confession and screens marker gene of this carrier (marker gene) or reporter gene (reportergene) under suitable condition.
Can be used for marker gene of the present invention comprises, for example, the dihydrofolate reductase gene and G418 or Xin Meisu (neomycin) resistant gene that are used for eukaryotic cell culture, and the penbritin (ampicilin), Streptomycin sulphate (streptomycine), tsiklomitsin (tetracycline) or that mycin of health (kanamycine) resistant gene that are used for intestinal bacteria and other bacterial cultures.
Be applicable to that carrier of the present invention can contain other performance control elements, a transcription initiation site (transcription starting site) for example, one Transcription Termination site (transcription termination site), one ribosome bind site (ribosomebinding site), one RNA splice site (RNA splicing site), a polyadenylation site (polyadenylation site) and one translate termination site (translation termination site), multiple clone site (multiple cloningsite, MCS).Be applicable to that carrier of the present invention also can comprise other regulation and control key element, for example enhancer sequence of transcription/translation (enhancer sequences) etc.Be applicable to that carrier of the present invention also can comprise the nucleotide sequence of a coding secretion signal (secretion signal).These sequences are that to be familiar with this operator known.
In a preferred embodiment of the present invention, one has luciferase gene (luclluc +) be used with the carrier as reporter gene, this includes, but not limited to pGL2-promoter vector and pGL3-underlying carrier.
In a preferred embodiment of the present invention, the PCR product of this 479bp is incorporated in the pGL2-promoter vector after handling through the propagation of using intermediary's cell, and obtains a recombinant vectors M4P2.
In another preferred embodiment of the present invention, the PCR product of this carrier 1503bp is incorporated in the pGL3-underlying carrier after handling through the propagation of using intermediary's cell, and obtains a recombinant vectors MP.
Above-mentioned two recombinant vectorss and then can be transferred in the competent host cell are to generate the recombinant cell strain of being desired.In a preferred embodiment of the present invention, this competent host cell is rat liver cancer cell Hepa-1C1C7.Come difference transfection rat liver cancer cell Hepa-1C1C7 with this recombinant vectors M4P2 and this recombinant vectors MP after, obtain rat liver cancer transformant strain Hepa1c1c7-M4P2 and rat liver cancer transformant strain Hepa1c1c7-MP.
Rat liver cancer transformant strain Hepa1c1c7-MP and Hepa1c1c7-M4P2 successively preserve and research centre (BCRC of FIRDI) (No. 331,300 Xinzhu City food roads, Taiwan) with the Biological resources of the Foodstuff Industrial and Development Inst. that was deposited at Taiwan on May 14th, 2004 May 7 2004 Christian era, deposit numbering and are respectively BCRC 960207 and BCRC 960208.This two cell strain also has the regulation according to budapest treaty, in June 18 2004 Christian era be deposited at American type culture collection (ATCC, P.O.Box 1549, Manassas, VA 20108, USA), deposit numbering and are respectively PTA-6089 and PTA-6090.
The applicant further confirms the response capacity of above-mentioned cell strain for Dioxins, wherein use 2,3,7,8-TCDD is as test compounds and change test concentrations and after the reaction times, and successfully sets up out the cognation of the expressive ability of the luciferase gene that is had on the recombinant vectors that is had in this two cell strain with respect to Dioxins concentration.So, can supply according to this recombinant cell strain of the present invention and to be used for coming qualitatively or detect quantitatively (for example: soil to be present in one from physical environment by bioanalysis (bioassay), the water source, edible meat, fish, vegetables and fruits or the like, the flue of factory or incinerator, bottom ash, bed mud or the like) the collected interior dioxin compounds of sample.
The present invention puts up with following embodiment and is described further, but will be appreciated that, this embodiment is only for illustrating usefulness, and should not be interpreted as the restriction on the invention process.
Embodiment
General experimental technique and material:
Experimental technique that is adopted among the present invention and the correlation technique that is used for dna clone (DNAcloning), be the textbook of knowing clearly and knowing: SambrookJ with reference in this area, Russell DW (2001) Molecular Cloning:a LaboratoryManual, 3rd ed.Cold Spring Harbor Laboratory Press, NewYork, for example use the DNA cleavage reaction (DNA cleavage reactionby restriction enzymes) of restriction enzyme, use the DNA gluing reaction (DNA ligation with T4 DNA ligase) of T4 DNA gluing enzyme (ligase), polymerase chain reaction (polymerase chain reaction), agarose gel electrophoresis method (agarose gelelectrophoresis), sulfuric acid ten diester sodium-polyacrylamide gel electrophoresises (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis) and plasmid conversion method (plasmid transformation) etc., and these technology all are to be familiar with this technology personage can come the implementer according to the professional attainment of itself.For example, employed plasmid conversion method is with through calcium chloride (CaCl in following embodiment 2) competent cell (calcium chloride-treated competent cell) handled carries out.
The preparation of the preparation of a small amount of plasmid (plasmid), a large amount of plasmids and the purifying of dna fragmentation reclaim be use respectively Plasmid Miniprep Purification Kit (Bertec Enterprise Co., Ltd.), Plasmid Midiprep kit (VIOGENE) and Gel Elution Kit commercialization purification kits such as (VIOGENE) carry out.
I, experiment material:
1. Dioxins [2,3,7,8-tetrachloro dibenzo-ρ-Dioxins (2,3,7,8-tetrachlorodibenzo-ρ-dioxin, 2,3,7,8-TCDD) (available from WellingtonLaboratories, Inc.) solution, original concentration are 10 μ g/ml, and it is standby to use diformazan Asia (DMSO) to be diluted to different concns before experiment;
The Dioxins standard substance [contain 17 kinds of Dioxins/furan compounds (dioxin/furancompounds): 2,3,7,8-TCDD, 2,3,7,8-TCD F, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-HpCDD, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8,9-HpCDF, OCDD, OCDF] provided by Zhengxiu Science and Technology Univ. ultramicron center;
3. following experiment material is available from Protech Technology Enterprise Co. Ltd. (Protech Technology Enterprise Co.Ltd.): tbe buffer liquid [TBEbuffer, Tris/ borate (borate)/EDTA], PBS damping fluid (PBS buffer, NaCl/KCl/Na 2HPO 4/ KH 2PO 4PH7.4), isopropyl-enzyme (isopropyl-β-D-thiogalactopyranoside, IPTG), phenol/chloroform/primary isoamyl alcohol (phenol/chloroform/isoamyl alcohol), 5-bromo-4-chloro-3-indoles-6-D-galactoside (5-bromo-4-chloro-3-indoyl-β-D-galactoside, X-Gal);
4. following experiment material be purchase Yisheng Bio tech Development Co., Ltd. (YeasternBiotech Co., Ltd.): YEA archaeal dna polymerase (YEA DNA polymerase), 10 times of damping fluids (10X buffer);
5. substratum: (Minimum essential medium is available from BioWest MEM) to minimal essential medium; Du's Bei Keshi improvement according to the Ge Shi substratum (Dulbecco ' sModified Eagles Medium, be DMEM) available from GibcoBRL; (Alpha-minimum essential medium, α-MEM) are available from BioWest to alpha minimal essential medium, and LB broth culture (LBBroth) is available from Athena EnzymeSystem Inc.; Nutrient agar (Nutrition Agar) is available from BectonDickson Inc.; Foetal calf serum Fetal Bovine serum (FBS) is available from Hyclone Inc.; And horse serum (horse serum is available from BioWest HS);
6. microbial culture microbiotic: penbritin (ampicillin) is available from Sigma (A1593); Penicillin-Streptomycin sulphate (penicillin-streptomycin) is available from Invitrogen Inc.; And GENETICIN  (G-418 vitriol) is available from GibcoBRL;
7. restriction enzyme (restriction enzymes): KpnI and BglII are available from NewEngland Biolabs, Ltd. (NEB), and HindII is available from Promega, and XhoI is available from GibcoBRL;
8.Luciferase Assay System (Cat#1500) is available from PromegaCooperation; PcDNA is available from Invitrogen Cooperation; GenePORTER TM2 Transfection Reagent are available from GST Inc.; SeaKem LE agarose (agarose) is available from FMC Bioproducts Inc.; Trypsinase-EDTA (trypsin-EDTA) is available from GibcoBRL; Methyl-sulphoxide
(DMSO) (EtBr is available from Sigma E8751) with ethidium bromide; 1Kb dna ladder (DNA Ladder) is available from Biolabs Inc. with filling dyestuff damping fluid (loading dye buffer);
9. following experiment material be available from Yisheng Bio tech Development Co., Ltd. (Yeastern Biotech Co., Ltd.): coli strain JM109 (E.colistrain JM 109), its cultivation and storing mode are the product descriptions according to the supplier; YT﹠amp; A Cloning Vector Kit includes yT﹠amp; A cloning vector, joint buffer A (ligation buffer A), joint buffer B, T 4Dna ligase (ligase), wherein yT﹠amp; The framework of A cloning vector (2728bp) as shown in Figure 1, and have ampicillin resistance gene (ampicillin resistance gene, AP), T 7Promotor (T 7Promoter), beta-galactosidase gene (β-galactosidase gene, lacZ) and multiple clone site (multiple cloning site, MCS);
10. following carrier is available from Promega Cooperation:pGL2-promoter vector (pGL2-promoter vector) framework (5790bp) as shown in Figure 2, and has ampicillin resistance gene (Amp r), SV40 promotor, luciferase gene (luc) and multiple clone site etc.; And pGL3-underlying carrier (pGL3-basicvector) framework (4818bp) as shown in Figure 3, and has ampicillin resistance gene (Amp r), luciferase gene (luc +) and multiple clone site etc.;
II, cell strain and culture condition:
The host cell that is used to carry out the transfection effect is rat liver cancer cell (mousehepatoma cell) Hepa-1C1C7 and mouse macrophage (mousemacrophage cell) J774A.1, they all are (the Food Industry Research and Development Institute of Foodstuff Industrial and Development Inst. available from Taiwan, FIRDI) Biological resources are preserved and research centre (Biosource Collection andResearch Center, BCRC) (No. 331,300 Xinzhu City food roads, Taiwan), deposit and be numbered BCRC 60104 and BCRC60140, and make succeeding transfer culture (subculture) according to following described mode separately:
1.Hepa-1C1C7 cell:
With the α-MEM that do not contain Nucleotide (nucleotide-free Alpha minimum essential mediumsupplemented with 10%FCS) of cytomixis to be added with 10% foetal calf serum (FCS), and formed cell suspending liquid is added in the culture dish, and be set to 37 ℃ and 5%CO at culture condition 2Incubator in cultivate.When the area of culture dish bottom has 80 to 90% to be covered by cultured cells, remove nutrient solution and clean cell with 3 to 5ml PBS damping fluid (pH 7.4), add then 1ml trypsinase-EDTA so that cell break away from from the bottom of culture dish.Afterwards, add in the fresh nutrient solution and dash the suction nutrient solution repeatedly to break up cell with tryptic activity and with transfer pipet (pipette), formed cell suspending liquid is dispensed in the culture dish (diameter 10cm) then, and is set to 37 ℃, 5%CO at culture condition 2Incubator in cultivate.
2.J774A.1 cell:
With cell be added to contain 4mM L-glutaminate (L-glutamine) and be adjusted to the yellow soda ash that contains 1.5g/L and the glucose of 4.5g/L and be added with 10% foetal calf serum Du Bei Keshi improvement according in the Ge Shi substratum (DMEM), and formed cell suspending liquid is added in the culture dish, and be set to 37 ℃ and 5%CO at culture condition 2Incubator in cultivate.When the area of culture dish bottom has 80 to 90% to be covered by cultured cells, remove nutrient solution and clean cell with 3 to 5ml PBS damping fluid (pH7.4), then with scraper streak gently culture dish the bottom so that cell break away from from the bottom of culture dish.Afterwards, add fresh nutrient solution and dash the suction nutrient solution repeatedly to break up cell, formed cell suspending liquid is dispensed in the culture dish (diameter 10cm) then, and is set to 37 ℃, 5%CO at culture condition with transfer pipet (pipette) 2Incubator in cultivate.
III, be derived from the pre-treatment (Preliminary treatments of dioxin-containing samples fromdifferent natural sources) of the sample that contains Dioxins of different natural sources:
1. the pre-treatment of environmental sample:
Environmental sample mainly is the sample that is taken from following source: soil (soil), water source, the flue of factory or incinerator, flying dust, bottom ash and bed mud etc.
(1) extraction step (extraction step):
Take by weighing an an amount of sample and put into to a cylindrical filter paper that had cleaned (Thimble Filter) (Toyo Roshi Kaisha, Ltd.) in, then cylindrical filter paper is put into soxhlet's extractor return line (soxhlet), the anhydrous sodium sulphate that is then added 2g, and the toluene of getting 150ml in addition places the Florence flask of a 250ml, and sample carried out return-flow type extraction treatment (extraction treatment with refluxing) last 24 hours.Afterwards, utilize concentrating under reduced pressure that the toluene extraction liquid is concentrated into about 1ml for subsequent disposal.
(2) acid pickling step (acid-wash step):
The resulting toluene extract of step (1) is added in the 24ml sample bottle with coffin bottle cap in the Teflon, then add the normal hexane (n-hexane) of 7ml and this sample bottle that vibrates and last about 5 seconds, the vitriol oil (98%) that adds 4ml then gives pickling and this sample bottle of thermal agitation lasts about 20 seconds.Afterwards, carry out centrifugal (2000rpm, 2 minutes) to produce layering and to collect sulfuric acid acid layer, the normal hexane layer then gives pickling with sulfuric acid again and becomes white (being no more than 3 pickling) up to sulfuric acid acid layer.The normal hexane layer is collected in the 50ml sample bottle.Each sulfuric acid acid layer is again with the normal hexane of 7ml extracting twice one by one.All normal hexane layers are collected in this 50ml sample bottle, and are evaporated to about 1ml for subsequent disposal.
(3) purifying step (purification step using amulti-layered silica gel column) of use multilayer silica gel tubing string:
Load with glass wool (glass wool) pointed bottom in a multilayer silica gel tubing string (diameter 0.5cm, long 20cm), inserts 0.5g silica gel (silica gel), 0.5g Silver Nitrate silica gel (AgNO then in order 3-silica gel), 0.5g silica gel, 0.5g sodium hydroxide silica gel (NaOH-silica gel), 0.5g silica gel, 5g sulfuric acid silica gel (sulfuric acid-silicagel), 0.5g silica gel and 0.5g anhydrous sodium sulphate (anhydrcus sodium sulfate), and give compacting with glass stick when filling (packing).With the good tubing string of 30ml normal hexane prewashing (pre-wash) filling and abandon washing lotion.
The 1ml acid-leached product of gained in the step (2) is moved in the multilayer silica gel tubing string of prewashing, after treating that acid-leached product all enters in the tubing string, clean the sample bottle that contains acid-leached product with the normal hexane of 1ml and amount to 3 times, and the normal hexane washing lotion is moved in this tubing string again.Then, come wash-out (eluting) tubing string to amount to twice, be collected in the Erlenmeyer flask of a 300ml then with the normal hexane wash-out tubing string of 120ml, and with eluate (eluate) with the normal hexane of 5ml.With nitrogen purging (nitrogen purge) collected eluate (eluate) is blown near dried.So far dioxin compound (dioxin compounds), non-planar polychlorinated biphenyl compound (non-planarpolychlorinated biphenyl compounds) and flush type polychlorinated biphenyl compound (planar polychlorinated biphenyl compounds) are contained in resulting purified product the inside.
If desire is analyzed the total concn of contained above-mentioned three compounds in this purified product the inside, this purified product can be dissolved in the DMSO, and use and to detect in the total concn that described bioanalytical method hereinafter carries out these compounds.
And if desire is analyzed the concentration out of the ordinary of this contained dioxin compound in purified product the inside, non-planar polychlorinated biphenyl compound and flush type polychlorinated biphenyl compound, below must carrying out again, this purified product uses acidic alumina tubing string and activated carbon tubing string purifying step, in order to do so that dioxin compound, non-planar polychlorinated biphenyl compound and flush type polychlorinated biphenyl compound are separated, then again carrying out concentration analysis in described bioanalytical method hereinafter.
(4) purifying step of use acidic alumina tubing string:
Glass wool is inserted in pointed bottom in an acidic alumina tubing string (acid aluminum oxide column) (diameter 0.5cm, long 20cm), inserts 1g silica gel, 6g acidic alumina, 1g silica gel and 1g anhydrous sodium sulphate afterwards in order.Come the prewashing tubing string that filling is good and abandon washing lotion with the 20ml normal hexane.
The eluate that multilayer silica gel tubing string from step (3) is gone out by wash-out by nitrogen purging after become 1ml, moved in the acidic alumina tubing string of prewashing, normal hexane with 5ml carries out wash-out twice altogether then, then be collected in the Erlenmeyer flask of a 150ml with the normal hexane wash-out tubing string of 90ml, and with eluate (eluate).So far the non-planar polychlorinated biphenyl compound is contained in resulting eluate the inside.
If analyze the concentration of the non-planar polychlorinated biphenyl compound that this eluate contained, this eluate can be done near with nitrogen purging, be dissolved in the DMSO then, and use and will carry out the compound concentrations analysis in described bioanalytical method hereinafter.
After finishing the normal hexane wash-out, the acidic alumina tubing string is again with methylene dichloride/normal hexane (methylene chloride/n-hexane) (20/80 of 20ml, v/v) carry out wash-out, and eluate is collected in the sample bottle of a 50ml, do near with nitrogen purging lentamente, the mixture of dioxin compound and flush type polychlorinated biphenyl compound is contained in so far resulting eluate the inside.This eluate is dissolved in the 1ml normal hexane and is loaded in the sample bottle, so that carry out following activated carbon tubing string purifying step.
(5) purifying step of use activated carbon tubing string:
In one activated carbon/diatomite (activated carbon/celite) tubing string (diameter 0.5cm, long 20cm) glass wool is inserted in pointed bottom, insert in order afterwards 0.5g silica gel, 0.5g activated carbon/diatomite (18/82, v/v) and 0.5g silica gel, when filling, give compacting with glass stick.Afterwards, the good tubing string of filling is in order respectively to be methyl alcohol (methanol), toluene (toluene), the methylene chloride/toluene (75/20/5 of 5ml, v/v/v), hexanaphthene/methylene dichloride (cyclohexane/methylenechloride) (50/50, v/v) and normal hexane give prewashing and washing lotion abandoned.
With in the above-mentioned steps (4) from moving into this activated carbon/diatomite tubing string through resulting 1ml hexane solution behind methylene dichloride/normal hexane wash-out, after treating that this hexane solution all enters in the tubing string, normal hexane with 1ml cleans sample bottle 3 times altogether, and the normal hexane washing lotion is moved in this tubing string again.
Then, hexanaphthene/methylene dichloride (50/50 with 2ml, v/v) this activated carbon of wash-out/diatomite tubing string amounts to 4 times, again with methylene chloride/toluene (75/20/5 of 1ml, v/v/v) the wash-out tubing string amounts to twice, all eluates are incorporated in the sample bottle of a 50ml, and so far resulting eluate contains the flush type polychlorinated biphenyl compound.
If desire is analyzed the concentration of this flush type polychlorinated biphenyl compound, with nitrogen purging collected eluate liquid is blown near doing, be dissolved in the DMSO then, and use will be carried out the compound concentrations analysis in described bioanalytical method hereinafter.
After finishing above-mentioned wash-out, this activated carbon/the diatomite tubing string gives wash-out with the toluene of 35ml again, and eluate is collected in the Erlenmeyer flask of a 150ml, and so far the resulting eluate of step contains dioxin compounds.
If desire the separate analysis dioxin compound, with nitrogen purging will be collected this toluene eluate is blown near doing, be dissolved in the DMSO then, and use will be carried out the concentration analysis of dioxin compounds in described bioanalytical method hereinafter.
2. the pre-treatment of plant sample:
(1) extraction of plant sample:
Take by weighing an an amount of sample and put into brown serum bottle, then add the 37.5%HCl of 50ml normal hexane, 50ml methylene dichloride, 25ml and the ultrapure water of 25ml, and rock this brown serum bottle of vibration and last 24 hours to a 250ml.Afterwards, formed mixture is poured into to a separating funnel, and amounted to twice, and washing lotion is also poured into to this separating funnel with this brown serum bottle of normal hexane cleaning of 50ml.This separating funnel is left standstill, and forms an organic upper strata (organic upper layer) and a water-based lower floor (aqueous lower layer) therefrom.Collect this organic upper strata, and clean this water-based lower floor with the normal hexane of 50ml and amount to twice, then with twice normal hexane washing lotion with should the merging of organic upper strata, and formed organic mixture concentrating under reduced pressure is become the enriched material of about 1ml.
Afterwards, the enriched material of this 1ml can use the purifying step of multilayer silica gel tubing string, purifying step and (5) that (4) use the acidic alumina tubing string to use the purifying step of activated carbon tubing string to further process according to (2) acid pickling step in above-mentioned " pre-treatment of environmental sample ", (3).
3. contain the pre-treatment of fat sample:
(1) contain the extraction of fat sample:
Contain the fat sample and mainly be meant the sample that is taken from following source: meat, feed, blood and dairy products etc.
Take by weighing an an amount of sample and put into, add the 150ml methylene dichloride and also rock this brown bottle of vibration and last 24 hours to a 250ml brown bottle.Afterwards.Dichloromethane extraction liquid is poured in the flask, and cleaned this brown bottle with the methylene dichloride of 50ml and amount to twice, and washing lotion is all poured into to this flask, then formed mixture concentrating under reduced pressure is become the enriched material of about 1ml.
(2) mensuration of oil-contg per-cent (detection of oil content%):
Resulting 1ml enriched material in the above-mentioned extraction step (1) is fed to a tubing string (diameter 0.5cm, long 20cm) that contains 0.5g silica gel, come this tubing string of wash-out, and collected eluate is concentrated into dried, promptly obtain a grease with the methylene dichloride of 50ml.The weight that this grease institute scale gets can be calculated the oil-contg per-cent that this contains the fat sample divided by this weight that contains the fat sample.
(3) acid pickling step:
Resulting grease in the above-mentioned steps (2) is added in the sample bottle with 50ml of coffin bottle cap in the Teflon, then add the normal hexane of 10ml and this sample bottle that vibrates lasts about 5 seconds, the vitriol oil (98%) that adds 7ml then gives pickling and this sample bottle of thermal agitation lasts about 20 seconds.Carry out centrifugal (2000rpm, 2 minutes) afterwards, obtain a normal hexane layer and a sulfuric acid layer by this.Collect sulfuric acid acid layer, the normal hexane layer then gives pickling with sulfuric acid again and becomes white (being no more than 3 pickling) up to sulfuric acid acid layer.The normal hexane layer is collected in the 50ml sample bottle.Each sulfuric acid acid layer gives extracting twice with the normal hexane of 7ml respectively again.All normal hexane layers are collected in this 50ml sample bottle, and give concentrating under reduced pressure and become the enriched material of about 1ml for subsequent disposal.
(4) purifying step (purification step usingan acid silica gel column) of use acidic silica gel tubing string:
Load with glass wool point bottom in an acidic silica gel tubing string (diameter 0.5cm, long 20cm), inserts 20g silica gel, 100g sulfuric acid silica gel, 20g silica gel and 10g anhydrous sodium sulphate then in order, and give compacting with glass stick when filling.Come the good tubing string of prewashing filling and abandon washing lotion with the 50ml normal hexane.
Resulting 1ml enriched material in the above-mentioned acid pickling step (3) is moved in this acidic silica gel tubing string of prewashing, after treating that this enriched material all enters in the tubing string, the sample bottle that cleans this 50ml with the normal hexane of 1ml amounts to 3 times, and the normal hexane washing lotion all is added in this acidic silica gel tubing string.Then, come the wash-out tubing string to amount to twice,, and eluate is collected in the Erlenmeyer flask of a 1000ml then with the normal hexane wash-out tubing string of 500ml with the normal hexane of 5ml.With nitrogen purging collected eluate is blown near dried.
Afterwards, resulting thus resistates can use the purifying step of multilayer silica gel tubing string, purifying step and (5) that (4) use the acidic alumina tubing string to use the purifying step of activated carbon tubing string to further process according to (3) in above-mentioned " pre-treatment of environmental sample ".
Embodiment 1. makes up recombinant vectors M4P2 and the MP (Construction of recombinant plasmids M4P2 and MPcontaining dioxin-response elements) that contains the Dioxins fragment reaction
Deposited numbering (accession number) AF210905[house mouse (Mus musculus) CYP1A1 gene according to what login in the NCBI network address, promoter region and partial sequence] when shown in nucleotide sequence and deposit numbering X01681[house mouse (Mus musculus) CYP1A1 gene] when shown in nucleotide sequence, and utilize software Primer3 Version and design following two groups of primers at the Dioxins fragment reaction (DREs) in house mouse (Mus musculus) the CYP1A1 gene order to (primer pairs), wherein the nucleotide sequence of these primers is to entrust Protech Technology Enterprise Co. Ltd. to synthesize:
Forward primer (forward primer) 1
5 '-cagagagcacctgcaaaaca-3 ' (sequence identification numbering: 1)
Reverse primer (reverse primer) 1
5 '-ggctacaaagggtgatgctt-3 ' (sequence identification numbering: 2)
Forward primer 2
5 '- CtcgagGagggcaggtgaaggtgttag-3 ' (sequence identification numbering: 3)
Reverse primer 2
5 '- AagcttAagtgaagagtgttctctagg-3 ' (sequence identification numbering: 4)
Wherein forward primer 2 and reverse primer 2 are designed to have the cleavage site (as bottom line sign person) of restriction enzyme XhoI and HindII respectively.
Therefore, when with house mouse (Mus musculus) CYP1A1 gene order as template (template), use is carried out polymerase chain reaction (PCR) by first primer that forward primer 1 and reverse primer 1 are constituted to (first primer pair), can obtain one and be the PCR product of 479bp (sequence identification numbering: 5) (referring to Fig. 4), and use second primer that is constituted by forward primer 2 and reverse primer 2 that (second primer pair) carried out PCR, can obtain one and be the PCR product of 1503bp (sequence identification numbering: 6) (referring to Fig. 5).These two PCR products have may be the peculiar nucleotide sequence of Dioxins fragment reaction (cacgc and gcgtg), and is used to make up recombinant vectors M4P2 and MP in following experiment.
(A) structure of recombinant vectors M4P2:
According to the method described in top " general experimental technique " joint, go out chromosomal DNA (chromosomal DNA) from mouse macrophage J774A.1 isolation and purification and be used as template, and use first primer that is constituted by forward primer 1 and reverse primer 1 to carrying out the PCR reaction, be the PCR product of 479bp and amplify one, wherein the nucleotide sequence of this PCR product is corresponding to the nucleotide residue 210-688 that deposits numbering AF210905 that is logined in the NCBI network address, and in the middle of contain 7 and may be peculiar nucleotide sequence cacga of Dioxins fragment reaction (DREs) and gcgtg (referring to the part of being got up by frame among Fig. 4).
Use yT﹠amp; A cloning vector test kit (yT﹠amp; A Cloning Vector Kit, Yeastern Biotech Co., Ltd.) and with this 479bp PCR product cloning that is amplified out to yT﹠amp; A cloning vector (2728bp, its framework is as shown in Figure 1) in, then according to the plasmid conversion method described in top " general experimental technique " joint with formed recombinant vectors transfection to e. coli jm109 cell (Yeastern Biotech Co., Ltd) in, and utilize the solid-state culture dish that contains penbritin to screen the anti--penbritin bacterium colony (ampicillin-resistant colonies) that is grown by transfected Bacillus coli cells (transformed E.coli cells).
Afterwards, use Plasmid Miniprep Purification Kit (BertecEnterprise Co., Ltd.) and be purified into plasmid with as template from the recombinant type amicillin resistance intestinal bacteria bacterium colony that filters out, and use this first primer to carrying out the PCR reaction, and perform an analysis with agarose gel electrophoresis method (agarose gel electrophoresis), thus, one to contain a tool nucleotide sequence be recombinant type yT﹠amp corresponding to the tool person's of this 479bpPCR product institute dna fragmentation substantially; The A cloning vector is screened to be gone out.
Described in top " general experimental technique " joint, use restriction enzyme KpnI/BglII to cut this screened recombinant type yT﹠amp that goes out; The A cloning vector, and obtain a KpnI-BglII dna fragmentation (0.5Kb).
Afterwards, this KpnI-BglII dna fragmentation is incorporated into the pGL2-promoter vector [5790bp that cuts to KpnI/BglII, its framework as shown in Figure 2, wherein have luciferase gene (luc)] in (Promega Cooperation), to contain a tool nucleotide sequence be recombinant vectors M4P2 corresponding to this 479bp PCR product tool person's of institute dna fragmentation substantially and obtain one, and the KpnI-BglII dna fragmentation that wherein is merged in is seated in the upstream end of the luciferase gene (luc) of pGL2-promoter vector.
According to the plasmid conversion method described in top " general experimental technique " joint, this recombinant vectors M4P2 by transfected to coli strain JM109 and duplicated in large quantities.Afterwards, extract go forward side by side performing PCR reaction of recombinant vectors M4P2 according to foregoing operating method, in order to do confirming whether contain a dna fragmentation corresponding to this 479bpPCR product among this recombinant vectors M4P2, and this recombinant vectors M4P2 also by sequencing to do further affirmation.
Recombinant vectors M4P2 through confirming to coli strain JM109 and duplicated in large quantities, gives purifying with Plasmid Midiprep Purification Kit by transfected then, then the recombinant vectors M4P2 that is purified is stored in-20 ℃ times standby.
(B) structure of recombinant vectors MP:
According to the method described in top " general experimental technique " joint, go out chromosomal DNA (chromosomal DNA) from mouse macrophage J774A.1 isolation and purification and be used as template, and use second primer that is constituted by forward primer 2 and reverse primer 2 to carrying out the PCR reaction, be the PCR product of 1503bp and amplify one, wherein the nucleotide sequence of this PCR product is to add the nucleotide residue 22-63 that deposits numbering X01681 corresponding to the nucleotide residue 108-1557 that deposits numbering AF210905 that is logined in the NCBI network address substantially, and in the middle of contain 12 and may be peculiar nucleotide sequence cacga of Dioxins fragment reaction (DREs) and gcgtg (referring to the part that is risen by frame among Fig. 5).
Use yT﹠amp; A cloning vector test kit (yT﹠amp; A Cloning Vector Kit, Yeastern Biotech Co., Ltd.) and with this 1503bp PCR product cloning that is amplified out to yT﹠amp; In the A cloning vector, then according to the plasmid conversion method described in top " general experimental technique " joint with formed recombinant vectors transfection to the e. coli jm109 cell, and utilize the solid-state culture dish that contains penbritin to screen by transfected anti--penbritin bacterium colony that Bacillus coli cells grew.
Use Plasmid Miniprep Purification Kit (BertecEnterprise Co., Ltd.) and be purified into plasmid with as template from the recombinant type amicillin resistance intestinal bacteria bacterium colony that filters out, and use this second primer to carrying out the PCR reaction, and perform an analysis with agarose gel electrophoresis method (agarose gel electrophoresis), thus, one to contain a tool nucleotide sequence be substantially corresponding to the recombinant type yT﹠amp of this 1503bp PCR product tool person's of institute dna fragmentation; The A cloning vector is screened to be gone out.
Described in top " general experimental technique " joint, use restriction enzyme HindIII/XhoI to cut this screened recombinant type yT﹠amp that goes out; The A cloning vector, and obtain a HindIII-XhoI dna fragmentation (approximately 1.5Kb).Afterwards, [4818bp, its framework wherein have luciferase gene (luc as shown in Figure 3 to the pGL3-underlying carrier that cuts with restriction enzyme HindIII/XhoI with this HindIII-XhoI dna fragmentation time cloning +)] in (Promega Cooperation), to contain a tool nucleotide sequence be substantially corresponding to the recombinant vectors MP of this 1503bp PCR product tool person's of institute dna fragmentation and obtain one, and wherein this HindIII-XhoI dna fragmentation is seated in the luciferase gene (luc of pGL3-underlying carrier +) upstream end.
According to the plasmid conversion method described in top " general experimental technique " joint, this recombinant vectors MP by transfected to coli strain JM109 and duplicated in large quantities.Afterwards, extract go forward side by side performing PCR reaction of recombinant vectors MP according to foregoing operating method, confirming whether contain a dna fragmentation corresponding to this 1503bp PCR product among this recombinant vectors MP, and this recombinant vectors MP also by sequencing to do further affirmation.
Recombinant vectors MP through confirming to coli strain JM109 and duplicated in large quantities, gives purifying with Plasmid Midiprep Purification Kit by transfected then, then the recombinant vectors MP that is purified is stored in-20 ℃ times standby.
Embodiment 2. uses recombinant vectors M4P2 and MP to come transfection rat liver cancer cell Hepa-1C1C7 (Transfection of mouse hepatoma cellsHepa-1C1C7 with recombinant vectors M4P2 and MP)
The Hepa-1C1C7 cell strain can be according to following preparation procedure A or preparation procedure B and by recombinant vectors MP or the transfection of M4P2 institute, wherein the training method of Hepa-1C1C7 cell strain is to carry out according to the schedule of operation described in top " cell strain and culture condition " joint.
Preparation procedure A:
1. with the basic DMEM of 172 μ l and the GenePORTER of 28 μ l TM2Transfection Reagent (GST Inc.) mixes to form one first solution, the DNA and the pcDNA (InvitrogenCooperation) of in addition that the New DNA diluent B (GST Inc.) of 200 μ l and the DNA[of 8 μ g is wherein selected recombinant vectors respectively are 4 μ g] mix forming one second solution, and allow this first and second solution leave standstill respectively to last 5 minutes; Afterwards, with this two solution uniform mixing, allow formed thus mixture leave standstill then and last 20 minutes;
2. with 2 * 10 7The Hepa-1C1C7 cell of cell/ml is added to a 10cm 2Culture dish in, and be set at 37 ℃, 5%CO in one 2Constant incubator in cultivate and last about 18-20 hour, and make area bottom the culture dish have 70%～80% to be covered by cell; Afterwards, remove substratum and clean cell 2 times altogether, add the fresh basic DMEM substratum of 5ml then with 1X PBS;
3. ready mixture in the step 1 is added in the culture dish, and in 37 ℃, 5%CO 2Constant incubator in cultivate and last about 4 hours; Afterwards, (DMEM+20%FBS) is added in the culture dish with an amount of substratum, and continues to cultivate and last 20 hours;
4. remove substratum, cleaning cell with 1X PBS amounts to 2 times and removes washing lotion, trypsinase-the EDTA that adds 1ml then handles cell and lasts and be no more than 5 minutes, the substratum (DMEM+10%FBS) that then adds 9ml is assigned to formed cell suspension thing (the resultant cell suspension) then and continues in the culture plate of 6-hole to cultivate;
5. to become tool one cell concn be 1 cell/100 μ l to the cell dilution of step 4 being turned out with substratum (DMEM+10%FBS), is seeded to the interior overnight incubation of 96-hole culture plate (100 μ l/ hole) then;
6. whether has only a cell in each hole in the culture plate of microscopically screening 96-hole, and this cell moved on in the culture plate of 6-hole continue to cultivate, with the indivedual pure strain (individual clones of transfected cells) of setting up transfected cell; And
7. with regard to step 4 institute's cultured cells in 6, the ability whether cell has the performance luciferase gene be can detect by the Dioxins revulsion (dioxin induction) described in the following embodiment 3, selected recombinant vectors M4P2 or MP had really in order to do the cell strain of being set up thus with affirmation.
Preparation procedure B:
1. prepare one first mixture according to the step 1 described in the preparation procedure A, but be to use the pcDNA (Invitrogen Cooperation) of 8 μ g to prepare this second solution;
Following step 2 to 3 is same as the step 2 described in the above-mentioned preparation procedure A to 3, proceeds the following step then:
4. remove substratum and add the substratum [90% basic DMEM+10%FBS is added with G-418] that contains GENETICIN  and cultivate and lasted for 1 week;
5. prepare one second mixture according to the step 1 described in the preparation procedure A, but be to use the selected recombinant vectors DNA of 8 μ g to prepare this second solution;
6. the cell that screens for the GENETICIN  by step 4 uses the second prepared mixture of step 5 to come the step 2 described in the repetition preparation procedure A to 3, in order to do making cell by selected recombinant vectors M4P2 or the transfection of MP institute; And
7. repeat the step 4 described in the preparation procedure A to 7.
The applicant uses two the recombinant vectors M4P2 and the MP that are set up among the embodiment 1 to come transfection Hepa-1C1C7 cell, and obtains two rat liver cancer cell strains through transfection, also is Hepa1c1c7-MP and Hepa1c1c7-M4P2.This two cell strain is preserved and research centre (BCRC of FIRDI) respectively at May 7 2004 Christian era and the Biological resources of the Foodstuff Industrial and Development Inst. that was deposited at Taiwan on May 14th, 2004, deposits numbering and is respectively BCRC 960207 and BCRC 960208.This two cell strain also has the regulation according to budapest treaty, in June 18 2004 Christian era be deposited at American type culture collection (ATCC, P.O.Box 1549, Manassas, VA 20108, USA), deposit numbering and are respectively PTA-6089 and PTA-6090.
Embodiment 3. sets up the cognation (Establishment of the correlation of dioxinconcentration vs.the light intensity caused by luciferase) of Dioxins concentration with respect to the light intensity that luciferase caused
A. the luciferase gene in cells transfected is at the post-stimulatory performance of Dioxins (Expression of luciferase gene in transfected cells after thestimulation of dioxin):
Will be through cells transfected (4 * 10 5Cells/well) is placed in 24 holes-culture plate, and adds the substratum (DMEM+10%FCS) of 1ml in every hole, then culture plate is placed on 37 ℃, 5%CO 2Constant incubator in and left standstill 2 hours so that cell attaches.
With DMSO is the Dioxins (2 of 10 μ g/ml with concentration, 3,7,8-TCDD) stock solution (stock solution) is done 10 times of serial dilutions (10-fold serial dilution), each diluent is got 3.22 μ l respectively and is added in each hole of culture plate, be respectively 10nM, 1nM, 100pM, 50pM, 10pM, 1pM and 0.1pM in order to do the Dioxins ultimate density that makes each experimental group, and each experimental group is done 4 repetitions.Normal control group (normal control) is a cell of not doing any processing, and the cell that solvent control group (solve ntcontrol) is to use 3.22 μ l DMSO to be handled.
Handling with Dioxins after cells transfected is lasted a preset time (5hrs, 10hrs, 15hrs and 20hrs), remove substratum and in every hole, add 150 μ l 1X cytolysis solution (cell lysis solution) (in be contained in Luciferase AssaySystem (Cat#1500), Promega Cooperation), jiggle culture plate then and last 15-20 minute so that cell is dissolved fully.Afterwards, the solution that will be arranged in every hole moves to an Eppendorf tube (microtube), and under 4 ℃, lasting 2 minutes so that 12000rpm is next centrifugal, resulting thus supernatant liquor (supernatant) is exactly the transfectional cell lysate of handling with Dioxins (lysate of dioxin-treatedtransfected cell).
Each is organized resulting lysate respectively gets 100 μ l and is added in each hole of opaque 96 holes-culture plate (non-transparent 96-well plate), then add 50 μ l fluorescein (luciferin) (in be contained in Luciferase Assay System, (Cat#1500), Promega Cooperation), use Beckman coulterDU640B spectrophotometer[set(ting)value to be PMT:1100 at last, read length (read length): 0.5, and Gain:1～100] detect the light intensity (lightintensity) in 20 seconds automatically.Resulting experimental result is analyzed drawing by statistical analysis method, and whether has statistical significance (statistic significance) in the middle of inspecting.
B. result:
Fig. 6 and Fig. 7 show that respectively foundation rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention and Hepa1c1c7-MP are at the Dioxins (2 with 100pM, 3,7,8-TCDD) handled 5,10,15 and 20 hours measured light intensity results afterwards, wherein the detection sensitivity when detecting the light intensity of Hepa1c1c7-M4P2 is set to 100, then is set at 10 when detecting Hepa1c1c7-MP.
Show by the result, rat liver cancer transformant strain Hepa1c1c7-M4P2 is at the Dioxins (2,3,7 with 100pM, 8-TCDD) handled after 15 hours and have stable and the most best light intensity result (Fig. 6), and the background value of normal group and DMSO group is also very low.But after Dioxins was handled 20 hours, the reaction of rat liver cancer transformant strain Hepa1c1c7-M4P2 dropped significantly.
As for rat liver cancer transformant strain Hepa1c1c7-MP, it has stable and the most best light intensity result (Fig. 7) after handling 20 hours with Dioxins, and also can produce enough significantly reaction after being handled 15 hours with Dioxins, and the light intensity read value is very high and the susceptibility of fluorescence analyser need be dropped to 10 and could all read.
According to these experimental results, soon the Dioxins treatment time all is set at 15 hours in ensuing experiment.
Fig. 8 and Fig. 9 show that respectively foundation rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention and Hepa1c1c7-MP are at the Dioxins (2 with different concns (0.1pM, 1pM, 5pM, 10pM, 50pM, 100pM and 1000pM), 3,7,8-TCDD) handled 15 hours measured light intensity results afterwards, wherein the detection sensitivity when detecting the light intensity of Hepa1c1c7-M4P2 is set to 10, then is set at 1 when detecting Hepa1c1c7-MP.
Referring to Fig. 8, detect Dioxins (2 when using according to rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention, 3,7, in the time of 8-TCDD), with respect to the light intensity result of control group, only use 2 of 5pM, 3,7,8-TCDD handles the variation that this cell just can be observed tangible light intensity.
And as shown in Figure 9, detect Dioxins (2,3,7 when using according to rat liver cancer transformant strain Hepa1c1c7-MP of the present invention, in the time of 8-TCDD), also can be at 2,3 of 5pM, 7,8-TCDD handles the variation of observing tangible light intensity down, and more more obvious than Hepa1c1c7-M4P2.In addition, with respect to the light intensity result of control group, present a positive relationship that is perfectly clear between Dioxins concentration and the measured light intensity.
Sum up, the Dioxins limit of detection of foundation rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention and Hepa1c1c7-MP all is 5pM.And when the light intensity of considering control group (value as a setting), the light intensity difference that rat liver cancer transformant strain Hepa1c1c7-MP is produced is bigger, therefore, with regard to rat liver cancer cell Hepa-1C1C7, recombinant vectors MP may be a preferable conversion carrier.
The detection of the dioxins concentration of embodiment 4. flying dust samples
A. the preparation of Dioxins standard substance (Preparation of dioxinstandards):
In an appropriate containers, use DMSO that 17 kinds of Dioxins/furan compounds (dioxin/furanc ompounds) listed in the following table 1 are mixed with total concn according to the ratio of the toxic factor of each compound and be 0.5ng-TEQ and 0.1ng-TEQ.Afterwards, blow near with nitrogen and to do, then with 128.8 μ l DMSO with Hui Rong (redissolve).
The preparation of table 1. Dioxins standard substance
The compound title I-TEF Stock solution (pg/ μ l) S 1(20μl) S 2(100 μl)
2，3，7，8-TCDF 0.1 0.5 1 5
1，2，3，7，8-PeCDF 0.05 2.5 2.5 12.5
2，3，4，7，8-PeCDF 0.5 2.5 25 125
1，2，3，4，7，8-HxCDF 0.1 2.5 5 25
1，2，3，6，7，8-HxCDF 0.1 2.5 5 25
2，3，4，6，7，8-HxCDF 0.1 2.5 5 25
1，2，3，7，8，9-HxCDF 0.1 2.5 5 25
1，2，3，4，6，7，8-HpCDF 0.01 2.5 0.5 2.5
1，2，3，4，7，8，9-HpCDF 0.01 2.5 0.5 2.5
OCDF 0.001 5 0.1 0.5
2，3，7，8-TCDD 1 0.5 10 50
1，2，3，7，8-PeCDD 0.5 2.5 25 125
1，2，3，4，7，8-HxCDD 0.1 2.5 5 25
1，2，3，6，7，8-HxCDD 0.1 2.5 5 25
1，2，3，7，8，9-HxCDD 0.1 2.5 5 25
1，2，3，4，6，7，8-HpCDD 0.01 2.5 0.5 2.5
OCDD 0.001 5 0.1 0.5
Total amount 0.1ng-TE Q 0.5ng- TEQ
B. Dioxins detects:
The flying dust sample (being provided by Zhengxiu Science and Technology Univ. ultramicron center) of 2g is placed a beaker, and carry out pre-treatment, returned flying dust sample 29 solution that are dissolved in the DMSO and obtain one according to the step described in front " pre-treatment of environmental sample ".
Described in embodiment 3, getting respectively is the Dioxins through 10 times of serial dilutions (2,3,7 of 3.22 μ l, 8-TCDD) handle cell, and make the ultimate density of Dioxins in cell culture fluid be respectively 10nM, 1nM, 100pM, 50pM, 10pM, 1pM, 0.1pM.Each experimental group is done 3 repetitions.The normal control group is not make the cell of any processing, and the cell that the solvent control group is to use 3.22 μ l DMSO to be handled.
The standard model S of above-mentioned resulting flying dust sample solution 29 and two kinds of different concns in step (A), being disposed 1With S 2Also respectively get 3.22 μ l and handle cell, by this, this flying dust sample solution and this two standard model S 1With S 2Diluted 40 times of concentration.
After the Dioxins processing lasts 15 hours, detect light intensity according to mode described in the embodiment 3 at cell, wherein the light intensity detection sensitivity all is set at 1.
C. result:
Table 2 show the reference standards that will use as analytic sample Dioxins (2,3,7, the 8-TCDD) relation table of concentration and quantity.Therefore, at this flying dust sample solution of estimation and this two standard model S 1With S 2During the content of the dioxin compounds that is contained, it is right promptly to come with the light strength ratios of standard substance according to the measured light intensity of each sample, and converses flying dust sample solution and this two standard model S according to the relation table of Dioxins concentration shown in the table 2 and quantity 1With S 2The Dioxins quantity that is contained.
The relation table of table 2. Dioxins concentration and quantity
The volume that is added in the 1ml substratum Concentration Quality Toxic equivalent ppt
3.22μL 10nM 3220pg 3220pg-TEQ 3220ppt
3.22μL 5nM 1610pg 1610pg-TEQ 1610ppt
3.22μL 1nM 322pg 322pg-TEQ 322ppt
3.22μL 500pM 161pg 161pg-TEQ 161ppt
3.22μL 100M 32.2pg 32.2pg-TEQ 32.2ppt
3.22μL 50pM 16.1pg 16.1pg-TEQ 16.1ppt
3.22μL 10pM 3.22pg 3.22pg-TEQ 3.22ppt
3.22μL 5pM 1.61pg 1.61pg-TEQ 1.61ppt
3.22μL 1pM 322fg 322fg-TEQ 0.322ppt
Figure 10 and Figure 11 show that respectively foundation rat liver cancer transformant strain Hepa1c1c7-M4P2 of the present invention and Hepa1c1c7-MP are at the Dioxins (2 with different concns (0.1pM, 1pM, 10pM, 50pM, 100pM, 1nM and 10nM), 3,7,8-TCDD) and this flying dust sample solution and this two standard model S 1With S 2Handled 15 hours measured light intensity results afterwards.
As shown in figure 10, when doing the dioxins concentration detection with rat liver cancer transformant strain Hepa1c1c7-M4P2, Dioxins concentration and light intensity present a tangible positive relationship.According to standard model S shown in Figure 10, diluted 40 times 1Light intensity be 6980, and calculate, as can be known standard model S according to the Dioxins concentration standard shown in the table 2 1The Dioxins quality should drop on 3.22～16.1pg-2,3,7, between the 8-TCDD.As for diluted 40 times standard model S 2, its light intensity is 13184, therefore calculates that its contained Dioxins quality should drop on 32.2～322pg-2,3,7, and between the 8-TCDD.The fluorescence value of reading of diluting 40 times flying dust sample 29 is 4718, and its contained Dioxins quantity should drop on 0.322～3.22pg-2,3,7, and between the 8-TCDD.
Because Figure 10 shows that Dioxins concentration and light intensity present a tangible positive relationship, when during as the longitudinal axis, can obtaining a linear relationship chart as transverse axis and measured light intensity with the Dioxins quality, and standard model S 1With S 2Can be estimated more accurately according to this linear relationship chart with flying dust sample 29 contained Dioxins quantity, so obtain following result:
Standard model S 1: 7.44pg-2,3,7,8-TCDD;
Standard model S 2: 104.83pg-2,3,7,8-TCDD; And
Flying dust sample 29:2.15pg-2,3,7,8-TCDD.
Afterwards, above-mentioned institute value be multiply by 40 respectively, then standard model S 1Contained Dioxins quantity 297.65pg-2,3,7,8-TCDD, standard model S 2Contained Dioxins quantity is 4193.35pg-2,3,7, and 8-TCDD, and these flying dust sample 29 contained Dioxins quantity are 85.89pg-2, and 3,7,8-TCDD.
Similarly, when Figure 11 demonstration was done the dioxins concentration detection with rat liver cancer transformant strain Hepa1c1c7-MP, Dioxins concentration and light intensity also presented a tangible positive relationship.Diluted 40 times this two standard model S 1With S 2And this flying dust sample 29 measured light intensity be respectively 18650,33998 and 20523, calculate this two standard model S according to the Dioxins concentration of table 2 and the relation table of quantity 1With S 2And these flying dust sample 29 contained Dioxins quality should drop on 3.22～16.1pg-2 respectively, 3,7, and 8-TCDD, 16.1～32.2pg-2,3,7,8-TCDD and 3.22～16.1pg-2,3,7, in the scope of 8-TCDD.
Because Figure 11 shows that Dioxins concentration and light intensity present a tangible positive relationship, when during as the longitudinal axis, can obtaining a linear relationship chart as transverse axis and measured light intensity with the Dioxins quality, and standard model S 1With S 2Can be estimated more accurately according to this linear relationship chart with flying dust sample 29 contained Dioxins quantity, so obtain following result:
Standard model S 1: 4.81pg-2,3,7,8-TCDD;
Standard model S 2: 17.65pg-2,3,7,8-TCDD; And
Flying dust sample 29:6.24pg-2,3,7,8-TCDD.
Afterwards, above-mentioned institute value be multiply by 40 respectively, then standard model S 1Contained Dioxins quantity 192.36pg-2,3,7,8-TCDD, standard model S 2Contained Dioxins quantity is 705.97pg-2,3,7, and 8-TCDD, and these flying dust sample 29 contained Dioxins quantity are 249.536pg-2, and 3,7,8-TCDD.
Sum up, when detecting with rat liver cancer transformant strain Hepa1c1c7-M4P2, (2,3,7, detected result 8-TCDD), chemical analysis concentration are defined as the standard model S of 0.1ng-TEQ as the Dioxins of standard substance in contrast 1Biological detection result be equivalent to 297.65pg-2,3,7,8-TCDD, and chemical analysis concentration is defined as the standard model S of 0.5ng-TEQ 2Biological detection result then be equivalent to 4193.35pg-2,3,7,8-TCDD.And when detecting with rat liver cancer transformant strain Hepa1c1c7-MP, chemical analysis concentration is defined as the standard model S of 0.1ng-TEQ 1The biological test result be equivalent to 192.363pg-2,3,7,8-TCDD, and chemical analysis concentration is defined as the standard model S of 0.5ng-TEQ 2The biological test result then be 705.97pg-2,3,7,8-TCDD.
By top numerical value, biological detection result can be high than chemical analysis results, with regard to rat liver cancer transformant strain Hepa1c1c7-M4P2, and the standard model S of 0.1ng-TEQ 1Biological detection concentration and the ratio of chemical analysis concentration be 3.0, and the standard model S of 0.5ng-TEQ 2Ratio then be 8.4.We infer, meeting loss to some extent in the purification step of dioxins concentration under connecing when this may be chemical analysis, and cause the TEQ value on the low side.In addition, also might be because biological detecting method is different with traditional TEF value for the TEF value of various dioxin compounds.Therefore, next can use rat liver cancer transformant strain Hepa1c1c7-M4P2 to come to carry out the uciferase activity analysis at 17 kinds of different dioxin compounds respectively, to set up out the bioanalysis detection system (bioassay detection system) that complete being used to of a cover detects dioxin compounds.
And with regard to rat liver cancer transformant strain Hepa1c1c7-MP, the standard model S of 0.1ng-TEQ 1Biological detection concentration and the ratio of chemical analysis concentration be 1.9, and the ratio of the standard model S2 of 0.5ng-TEQ is 1.4.Therefore, rat liver cancer transformant strain Hepa1c1c7-MP seems to have better detection precision than rat liver cancer transformant strain Hepa1c1c7-M4P2.
On the other hand, when flying dust sample 29 was checked with rat liver cancer transformant strain Hepa1c1c7-M4P2 and Hepa1c1c7-MP respectively, resulting biological detection result was equivalent to 85.89pg-2 respectively, 3,7,8-TCDD and 249.536pg-2,3,7,8-TCDD.And this flying dust sample resulting result when performing an analysis with the traditional chemical mass spectrograph is 76pg-TEQ.If flying dust sample 29 resulting biological detection results and chemical analysis results are done one relatively, the ratio that uses the strain of rat liver cancer transformant Hepa1c1c7-M4P2 and Hepa1c1c7-MP to be produced is respectively 1.1 and 3.3.In view of this, biological detection numerical value can be greater than the chemical analysis value, and this very likely is because the TEF value of biological detecting method is different with traditional TEF value, need define one in addition and overlaps the TEF valve system that is applicable to the bioanalysis detection and have.In addition, the Dioxins amount of institute's loss also can cause measuring result TEQ value on the low side in the chemical analysis step.
As if the detection precision based on rat liver cancer transformant strain Hepa1c1c7-MP is excellent than mouse liver cancer transformant strain Hepa1c1c7-M4P2, and the following examples use rat liver cancer transformant strain Hepa1c1c7-MP to analyze the biological sample that is taken from the physical environment.
Embodiment 5. detects the dioxins concentration of flesh of fish sample
Get the 3g flesh of fish, carry out pre-treatment according to described step described in above-mentioned " containing the pre-treatment of fat sample ", wherein obtain the grease of 1.97g in the determination step of oil-contg per-cent, the oil-contg per-cent of therefore extrapolating the 3g flesh of fish is 65.7%.The grease of this 1.97g connects and is carried out the acid pickling step described in " containing the pre-treatment of fat sample ", uses the purifying step of the purifying step of acidic silica gel tubing string and the use multilayer silica gel tubing string described in " pre-treatment of environmental sample " and obtain an eluate, and with nitrogen purging collected eluate is blown near and to do, returned at last in the DMSO that is dissolved in 644 μ l, and obtained a flesh of fish sample solution.
According to the mode described in the embodiment 3, take out this flesh of fish sample solution of 3.22 μ l and handle cell, and detect the processed intensity variation that cell produced.According to Dioxins shown in experiment method described in the embodiment 4 and the table 2 (2,3,7, the 8-TCDD) relation table of concentration and quantity, this flesh of fish sample solution is estimated, and to go out to have a Dioxins concentration be 7.5pM.And again after converting, this flesh of fish sample solution is estimated as has 0.96pg-2,3,7, and 8-TCDD.
The dioxins concentration that can be obtained oppressing in the sample by The above results is the 0.32pg/g-flesh of fish (perhaps being the 0.49pg/g-grease).
In addition, because detection is to utilize 2,3,7,8-TCDD is as standard substance, and 2,3,7, the toxic equivalent value (TEQ) of 8-TCDD is set to 1.00, and the dioxins concentration of this flesh of fish sample also can be represented as the 0.32pg-TEQ/g-flesh of fish (or being the 0.49pg-TEQ/g-grease).
Embodiment 6. detects the dioxins concentration of fish meal sample
With reference to embodiment 5 described same way as, get the 100g fish meal and carry out the detection of dioxins concentration, wherein the 100g fish meal is recorded and is contained the 7.84g grease, and therefore, the oil-contg per-cent of this fish meal sample is 7.84%.Afterwards, the grease of getting 1g carries out the purifying step of the acid pickling step described in " containing the pre-treatment of fat sample ", the purifying step of using the acidic silica gel tubing string and the use multilayer silica gel tubing string described in " pre-treatment of environmental sample " and obtains an eluate, and with nitrogen purging collected eluate is blown near and to do, returned at last in the DMSO that is dissolved in 644 μ l, and obtained a fish meal sample solution.
According to the mode described in the embodiment 3, take out this fish meal sample solution of 3.22 μ l and handle cell, and detect the processed intensity variation that cell produced.According to Dioxins shown in experiment method described in the embodiment 4 and the table 2 (2,3,7, the 8-TCDD) relation table of concentration and quantity, this fish meal sample solution is estimated, and to go out to have a Dioxins concentration be 25 pM.And again after converting, this fish meal sample solution is estimated as that to have a dioxins concentration be 3.22pg-2,3,7, and 8-TCDD.
The dioxins concentration that can be obtained in the 100g fish meal by The above results is 0.25pg/g-fish meal (perhaps being the 3.22pg/g-grease).
In addition, because detection is to utilize 2,3,7,8-TCDD is as standard substance, and 2,3,7, the toxic equivalent value (TEQ) of 8-TCDD is set to 1.00, and the dioxins concentration of this fish meal sample also can be represented as 0.25pg-TEQ/g-fish meal (or being the 3.22pg-TEQ/g-grease).
All data in literature and the patent case quoted from this specification sheets are merged in this case as the reference data with its integral body.If to some extent when conflicting, the detailed description of this case (comprise be defined in) will be got the upper hand.
Though the present invention is described with reference to above-mentioned specific concrete example, make a lot of modifications and variations down what do not deviate from scope and spirit of the present invention significantly.What therefore be intended to is that the present invention only is subjected to as examine the restriction of person as shown in attached claims with literary composition.
Sequence table
＜110〉Zhengxiu Science and Technology Univ.
＜120〉a kind of performance according to luciferase gene detects the recombinant cell strain of dioxin compounds
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gctgtctcgc gtggatcctt cctccaccct ttcctccacc atacttagat agctctgcac 660
ccgccgcccg acttctctgt gttgcctgct tcagtatgta tgcacaacac tagcaagccc 720
cgggagccta cagggagctg caagcgggga ctcccgggct tgcagcttgc ctaaggtgac 780
ccttagaggt gagatctgca agcctgccat ccatccccac cctctagatg aagcagcgcg 840
aacttcggcc gatacccaat ttgtggggca cagagtcagt ccaatggcgc cactggcctt 900
cctgtcctgt gacctctggg ctggggtcgt tgcgcttctc acgcgagctt ggactcagta 960
atccagggaa gcaaagtcac cacccagctg ttctccctct accagccttt cccgggcacc 1020
cattggcttg tagtaggcaa gaggatctta cacactgaat gtttcagggg tgcaaaaata 1080
gtgaaaagga atccctatga cccggaatgg aggccccagt acttactttt taggttaccc 1140
cagaattttt cctcaaaccc ctccctcagt gggattatgc actgtccatg gagcaccttg 1200
aaagtgtggg gggtggtgac cccaaccttt attctttttc ttttcttatg tttttttgtg 1260
cctgtaccta catcaggtat ccggtatggc ttcttgccta tctcccccct cccccctgcc 1320
tagagcactc cctaaggctg tccctccctc ggtcccacca ctgggctcag ataaggaggc 1380
gtggccaaca gacacagagt cctataaagg tggtggtgcc ttcaccctaa ccctgaaggt 1440
ggtagttctt ggagcttccc cgatcctccc tagggtccta gagaacactc ttcacttaag 1500
ctt 1503

Claims (20)

1, a kind of primer of cloning for the reactive dna fragmentation of Dioxins tool of being used to is right, it is characterized in that this primer comprises a forward primer and a reverse primer, this forward primer has just like the sequence identification numbers: 1 or sequence identification numbering: the nucleotide sequence shown in 3, and this reverse primer has just like the sequence identification and number: 2 or the sequence identification number: the nucleotide sequence shown in 4.
2, right according to the primer of claim 1, it is characterized in that this forward primer has just like the sequence identification to number: the nucleotide sequence shown in 1, and this reverse primer has just like the sequence identification and numbers: the nucleotide sequence shown in 2.
3, right according to the primer of claim 1, it is characterized in that this forward primer has just like the sequence identification to number: the nucleotide sequence shown in 3, and this reverse primer has just like the sequence identification and numbers: the nucleotide sequence shown in 4.
4, a dna fragmentation, it has a nucleotide sequence, it is characterized in that this nucleotide sequence is one of following:
(1) in fact corresponding to one by using the CYP1A1 gene order as template and use primer according to claim 1 to carrying out the nucleotide sequence that dna fragmentation had that the PCR reaction is amplified out;
(2) in fact corresponding to numbering: the nucleotide sequence shown in 5 just like the sequence identification;
(3) in fact corresponding to numbering: the nucleotide sequence shown in 6 just like the sequence identification;
(4) in fact corresponding to one with the complementary nucleotide sequence mutually of the nucleotide sequence described in the nucleotide sequence of the dna fragmentation described in (1) or (2) or (3).
5,, it is characterized in that it is in fact corresponding to numbering just like the sequence identification that this dna fragmentation has a nucleotide sequence: the nucleotide sequence shown in 5 according to the dna fragmentation of claim 4.
6,, it is characterized in that it is in fact corresponding to numbering just like the sequence identification that this dna fragmentation has a nucleotide sequence: the nucleotide sequence shown in 6 according to the dna fragmentation of claim 4.
7, a kind of recombinant vectors it is characterized in that this recombinant vectors has a reporter gene, and a dna fragmentation as claimed in claim 4 is positioned at the upstream end of this report gene.
8,, it is characterized in that this report gene is a luciferase gene according to the recombinant vectors of claim 7.
9,, it is characterized in that it is in fact corresponding to numbering just like the sequence identification that this dna fragmentation has a nucleotide sequence: the nucleotide sequence shown in 5 according to the recombinant vectors of claim 7.
10,, it is characterized in that this recombinant vectors is recombinant vectors M4P2 according to the recombinant vectors of claim 9.
11,, it is characterized in that it is in fact corresponding to numbering just like the sequence identification that this dna fragmentation has a nucleotide sequence: the nucleotide sequence shown in 6 according to the recombinant vectors of claim 7.
12,, it is characterized in that this recombinant vectors is recombinant vectors MP according to the recombinant vectors of claim 11.
13, a kind of recombinant cell strain is characterized in that this recombinant cell strain is by to come transfection one host cell to be formed just like each recombinant vectors in the claim 7 to 12.
14,, it is characterized in that this host cell that is used to transfection is rat liver cancer cell Hepa-1C1C7 according to the recombinant cell strain of claim 13.
15,, it is characterized in that this recombinant cell strain is to be deposited at the rat liver cancer transformant strain Hepa1C1C7-MP of American type culture collection or secondly to cultivate the offspring to deposit numbering PTA-6089 according to the recombinant cell strain of claim 14.
16,, it is characterized in that this recombinant cell strain is to be deposited at the rat liver cancer transformant strain Hepa1c1c7-M4P2 of American type culture collection or secondly to cultivate the offspring to deposit numbering PTA-6090 according to the recombinant cell strain of claim 14.
17, a kind of bioanalysis that is used for detecting the dioxin compounds of an environmental sample, it is characterized in that this bioanalysis is to utilize the sample that contains dioxin compounds just like each recombinant cell strain in the claim 13 to 16, and cause the reporter gene that this recombinant vectors had of position in this recombinant cell strain to be showed, the gene product that is generated thus can produce a detectable phenomenon, and this detectable phenomenon is used as the index of the existence of qualitative and quantitative dioxin compounds.
18,, it is characterized in that the reporter gene that is had on this recombinant vectors is a luciferase gene, and this detectable phenomenon is the light intensity that is produced by after luciferase and the luciferin reaction according to the bioanalysis of claim 17.
19,, it is characterized in that this recombinant cell strain is to be deposited at the rat liver cancer transformant strain Hepa1c1c7-MP of American type culture collection or secondly to cultivate the offspring with PTA-6089 according to the bioanalysis of claim 17.
20,, it is characterized in that this recombinant cell strain is to be deposited at the rat liver cancer transformant strain Hepa1c1c7-M4P2 of American type culture collection or secondly to cultivate the offspring to deposit numbering PTA-6090 according to the bioanalysis of claim 17.
CN 200410074417 2004-09-15 2004-09-15 Recombinant cell strain for detecting dioxin compounds according to behaviour of leuciferinase gene Pending CN1749403A (en)

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CN102851312A (en) * 2012-03-07 2013-01-02 深圳华大基因研究院 Carrier suitable for cell transformation, recombinant cell and application thereof
CN103197062A (en) * 2013-04-03 2013-07-10 涿州市恒通达科贸有限公司 Method for detecting dioxin and special enzyme linked immunoassay kit for same
CN103820496A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103820497A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103820495A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103834688A (en) * 2014-03-18 2014-06-04 中国科学院生态环境研究中心 Recombinant vector and cell used for biologically detecting dioxins
CN103834689A (en) * 2014-03-18 2014-06-04 中国科学院生态环境研究中心 Recombinant vector and cell used for biologically detecting dioxins
CN103849651A (en) * 2014-03-18 2014-06-11 中国科学院生态环境研究中心 Recombinant vector and cell for biological detection of dioxin substances
CN104034708A (en) * 2014-05-23 2014-09-10 孟令启 Device for detecting content of dioxin in food
CN105755106A (en) * 2016-03-23 2016-07-13 厦门大学 Method for detecting dioxin persistent organic pollutants
CN105886532A (en) * 2015-05-21 2016-08-24 中国科学院生态环境研究中心 Humanized recombinant vector and cell for dioxin-type substance biological detection
CN111208270A (en) * 2020-03-11 2020-05-29 天津市生态环境监测中心 Application of trichiurus haumela CYP1 family gene in preparation of water pollution detection biomarker and detection method thereof

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* Cited by examiner, † Cited by third party
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CN102851312A (en) * 2012-03-07 2013-01-02 深圳华大基因研究院 Carrier suitable for cell transformation, recombinant cell and application thereof
CN103197062A (en) * 2013-04-03 2013-07-10 涿州市恒通达科贸有限公司 Method for detecting dioxin and special enzyme linked immunoassay kit for same
CN103834689A (en) * 2014-03-18 2014-06-04 中国科学院生态环境研究中心 Recombinant vector and cell used for biologically detecting dioxins
CN103820497A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103820495A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103834688A (en) * 2014-03-18 2014-06-04 中国科学院生态环境研究中心 Recombinant vector and cell used for biologically detecting dioxins
CN103820496A (en) * 2014-03-18 2014-05-28 中国科学院生态环境研究中心 Recombinant vector used for biologically detecting dioxin substances
CN103849651A (en) * 2014-03-18 2014-06-11 中国科学院生态环境研究中心 Recombinant vector and cell for biological detection of dioxin substances
CN103820497B (en) * 2014-03-18 2016-06-29 中国科学院生态环境研究中心 Recombinant vector for dioxin-like chemical biological detection
CN104034708A (en) * 2014-05-23 2014-09-10 孟令启 Device for detecting content of dioxin in food
CN104034708B (en) * 2014-05-23 2017-05-17 安徽宸瑞节能环保科技工程有限公司 Device for detecting content of dioxin in food
CN105886532A (en) * 2015-05-21 2016-08-24 中国科学院生态环境研究中心 Humanized recombinant vector and cell for dioxin-type substance biological detection
CN105755106A (en) * 2016-03-23 2016-07-13 厦门大学 Method for detecting dioxin persistent organic pollutants
CN111208270A (en) * 2020-03-11 2020-05-29 天津市生态环境监测中心 Application of trichiurus haumela CYP1 family gene in preparation of water pollution detection biomarker and detection method thereof

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