CN102392073A - Analysis method for activity of mismatch repair of zebra fish embryo DNA and application thereof - Google Patents
Analysis method for activity of mismatch repair of zebra fish embryo DNA and application thereof Download PDFInfo
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Abstract
The present invention relates to an analysis method for the activity of mismatch repair of zebra fish embryo DNA, and the application of the method in toxicity evaluation of mismatch repair of environmental compound DNA. The method includes the following steps: preparing single-stranded DNA molecules with plasmids pGEM-EGFP; preparing homoduplex and heteroduplex with the single-stranded DNA molecules and linearized plasmids pGEM-EGFP and plasmids pGEM- (mt) EGFP respectively; microinjecting the homoduplex and heteroduplex to a zebra fish embryo with 1-cell stage; and quantifying the activity of mismatch repair of embryo DNA. The present invention establishes the analysis method for the activity of mismatch repair of DNA on the zebra fish embryo level for the first time, and successfully applies the method to the toxicity evaluation of mismatch repair of environmental compound DNA, which is significant for the evaluation of health risk and cancerization risk for environmental compound people. In addition, by applying the zebra fish to the evaluation of the toxicity of mismatch repair of compound DNA, the method has the advantages of simple operation, direct viewing, fastness, simple administration for tested compounds, small dosage, and the like.
Description
Technical field
The invention belongs to the biology techniques field, relate to the active analytical procedure of a kind of zebrafish embryo dna mismatch repairing effect, and the application of this analytical procedure on environmental compound dna mismatch repairing effect toxicity assessment.
Background technology
The dna mismatch reparation (mismatch repair, MMR) system extensively is present in the organism, and it is a kind of repair mode behind the dna replication dna.Its function is mispairing, insertion or the deletion mutantion that DNA plerosis is introduced in reproduction process, makes dna sequence dna recover normal.Therefore, it plays a part to strengthen dna replication dna fidelity of reproduction, DNA plerosis mispairing, reduces genovariation and keeps genome stability.In a single day the dna mismatch repairing effect weakens or lacks, and body is very easy to produce sudden change, and then brings out mutant phenotype generation canceration.Therefore, quantitative analysis DNA MMR functionally active is preventing cancer, carries out a kind of important means that the medicine risk of cancer is estimated.
At present, the active method of analyzing DNA mispairing repairing effect has a lot, and mainly containing with the phage M13mp2 and the strain of deriving thereof is material, changes through plaque and comes quantitative methods; Having with the synthetic oligonucleotide is material, changes through restriction endonuclease sites and comes quantitative methods; Have with the EGFP gene as reporter gene, change through relative green fluorescent protein and come quantitative methods.Preceding two kinds of methods are carried out the evaluation of MMR functionally active with the nucleus extract, belong to the analyzed in vitro method, have complicated operating process, and low the hanging down with accuracy of indication parameter sensitivity reaches shortcomings such as being difficult for statistics.A kind of method in back is to propose (Lei, X. first by texas,U.S university Sun Lu Zhejiang (Lu zhe-Sun) professor; Zhu, Y.; Tomkinson, A.; Sun, L., Measurement of DNA mismatch repair activity in live cells.Nucleic Acids Res 2004,32, (12), e100.), and through some optimizations and improvement (Zhou, B.; Huang, C.; Yang, J.; Lu, J.; Dong, Q.; Sun, L.Z., Preparation of heteroduplex enhanced green fluorescent protein plasmid in vivo mismatch repair activity assay.Anal Biochem 2009; 388; (1), 167-9.Zhou, B.; Dong, Q.; Ma, R.; Chen, Y.; Yang, J.; Sun, L.Z.; Huang; C., Rapid isolation of highly pure single-stranded DNA from phagemids.Anal Biochem2009,389; (2); 177-9. one Chinese patent application number: 201010586301.9, a kind of pair of luciferase plasmid, based on active analytical procedure of dna mismatch repairing effect and application thereof in its viable cell), very big progress has been arranged comparing to preceding two kinds of methods aspect operating process and the precision; But these three kinds of methods quantitatively all are only limited on cell levels DNA MMR functionally active, and the power that is difficult to MMR functionally active in the accurately real reflection organism changes.Therefore, set up and a kind ofly be used for carrying out on the body level method that DNA MMR functionally active is analyzed, and the carcinogenic risk evaluation of using it for environmental compound or medicine has very major and immediate significance.
Zebra fish is a kind of tropical fresh water fish, is used to developmental biology and genetics research at first.Because zebra fish has the special advantages characteristic: small, be prone to captive breeding, in vitro fertilization, oviparity and egg laying amount is big, the embryo is transparent, fetal development is fast, sexual maturing period is short, can directly add in the embryo medium with Human genome height homology, pollutent and medicine, zebra fish has become at present studies the bioactive a kind of important animal model of macromolecular substance.
Along with green fluorescent protein (Green Fluorescent Protein; GFP) application on zebra fish model of transgenic technology success; Use zebra fish model, especially gfp transgene via zebra fish embryo model to come the biological activity of macromolecular substance in the qualitative, quantitative research body to become a kind of possibility.Compare with other animal models, the transgenic zebrafish embryo model have economy, fast, advantage such as convenient and efficient.
Summary of the invention
In view of this; The active analytical procedure of zebrafish embryo dna mismatch repairing effect and this method of the purpose of this invention is to provide a kind of simple to operate, valid metric directly perceived are in the application of estimating on the environmental compound dna mismatch repairing effect toxicity; Only be confined in the cell to overcome traditional dna mismatch repairing effect activation analysis method, can not reflect the active deficiency of dna mismatch repairing effect on the integral level truly, exactly.
For achieving the above object, the present invention adopts following technical scheme:
The present invention provides a kind of zebrafish embryo dna mismatch repairing effect active analytical procedure, and said analytical procedure may further comprise the steps:
1) prepares single strand dna with plasmid pGEM-EGFP, said plasmid pGEM-EGFP ability normal expression green fluorescent protein;
2) plasmid pGEM-EGFP and pGEM-(mt) EGFP with the single strand dna colinearityization of obtaining in the step 1) prepares homology and heteroduplex respectively, and there is a nonsense mutation (TGG-＞TAG) in the 58th codeword triplet place in the green fluorescent protein EGFP gene of said plasmid pGEM-(mt) EGFP;
3) homology for preparing and heteroduplex are distinguished microinjection to the zebrafish embryo of 1-cell stage;
4) embryo's dna mismatch repairing effect activity is carried out quantitatively.
Further, above-mentioned steps 1) comprising: with plasmid pGEM-EGFP is material, uses helper phage M13ko7, obtains single strand dna through the method that infects.
Further, above-mentioned steps 1) comprising: with plasmid pGEM-EGFP is material, uses and incises the open loop plasmid that enzyme formation band is incised, and uses exonuclease to carry out digestion reaction then and obtains single strand dna.
Further, the above-mentioned enzyme of incising is Nb.Bsrd I or Nb.Bpu10I.
Further, above-mentioned exonuclease is exonuclease III.
Further; Above-mentioned steps 2) comprising: with single strand dna with annealing through restriction enzyme BstXI or the linearizing plasmid pGEM-EGFP of Mlu I and pGEM-(mt) EGFP; The annealing after product relies on enzyme PSAD digestion through the plasmid security, removes residual single strand dna and linearizing plasmid and can obtain high-purity homology and heteroduplex.
Further; Above-mentioned steps 2) plasmid pGEM-(mt) EGFP described in is mutagenic obtained through PCR through plasmid pGEM-EGFP; Promptly; The 58th TGG of codeword triplet place mutagenesis of EGFP gene is TAG, and plasmid pGEM-EGFP and pGEM-(mt) EGFP has only the difference (owing to introduced a nonsense mutation, thereby pGEM-(mt) EGFP can't the normal expression green fluorescent protein) of a base in based composition.
Further, there is a G/T mispairing in the 58th codeword triplet place in the EGFP gene of above-mentioned heteroduplex.
Further, above-mentioned steps 3) comprising: homology and heteroduplex to the embryo injects same isoconcentration≤100ng/ μ L respectively are preferably 30ng/ μ L; The injection site is tenuigenin or yolk sac, is preferably yolk sac; Every piece of embryo is 500pL, and the indicator phenol red concentration is controlled at 0.025-0.050%, the embryo of each strain homology group and allos group injection equivalent amount:>=100 pieces.
Further; Above-mentioned steps 4) comprising: observe behind the embryonal vaccination 12-36h; Be preferably and observe behind the 24h and take pictures; With NIS-BR2.3 software analysis embryo green fluorescence expression intensity, and statistics institute embryonal vaccination green fluorescence expression rate, embryo's dna mismatch repairing effect activity is represented with the relative egfp expression rate of this strain; The egfp expression rate is calculated by following formula and is obtained relatively:
Relative egfp expression rate=allos group luciferase expression rate * allos group average fluorescent strength/homology group luciferase expression rate * homology group average fluorescent strength.
Further; Above-mentioned plasmid pGEM-EGFP is through the CMV-EGFP-PolyA fragment is scaled off from plasmid pEGFP-N1 (U55762.1GI:1377911 the gene pool) enzyme; Utilize the T4 ligase enzyme to be connected among the carrier pGEM5Zf (+) (X65308.2GI:5701825 in the gene pool) then and obtain (Nucleic Acids Res 2004; 32, (12), e100).
Simultaneously, the present invention also provides the application of a kind of above-mentioned analytical procedure in environmental compound dna mismatch repairing effect toxicity assessment.
We utilize green fluorescence protein gene as reporter gene; The direct acting tape error of preparation mismatch repair system is joined the double-strandednucleic acid substrate of base; This substrate base mismatch is under the situation of being repaired by mismatch repair system, and the embryo can green-emitting fluorescence, is not repaired then not green-emitting fluorescence.Utilize microinjection technique that this substrate is injected in the zebra fish 1-cell stage embryo; The strong and weak directly indication of the green fluorescence signal that 24hpf (after fertilization 24 hours) zebrafish embryo sends endogenous mispairing repairing effect is active strong and weak; We have just successfully set up and a kind ofly have been used for the active analytical procedure of zebrafish embryo inner analysis dna mismatch repairing effect like this, and successfully method are applied to the toxic evaluation of environmental compound dna mismatch repairing effect.
The beneficial effect that the present invention produces is following:
Analytical procedure provided by the invention has overcome traditional DNA MMR functionally active and has been only limited to the deficiency on the cell levels; First on the zebra fish integral level and in vivo realize the analysis of DNA MMR functionally active; It can reflect truly, accurately that organism DNA MMR functionally active changes than traditional method, and this risk of cancer evaluation for environmental compound or poisonous substance is of great immediate significance; In addition, zebra fish than other animal models have volume little, be prone to raise, the embryo is transparent, growths is fast, sexual maturing period is short, dosage is few and advantage such as easy, thereby this method more has concurrently and expends low, directly perceived, the convenient, fast plurality of advantages such as efficiently of cost; And its experimental period is short, simple to operate, directly perceived fast, the test-compound administration is simple, consumption is few and a large amount of analyzing samples (the body physiological environment of living animal) can be provided on research environment compound or medicine daughter DNA MMR function toxicity.
Description of drawings
Fig. 1 shows the technological line figure that the present invention is provided for zebrafish embryo DNA MMR activation analysis method;
Fig. 2 shows single strand dna preparation flow synoptic diagram provided by the invention;
Fig. 3 shows same, heteroduplex preparation flow synoptic diagram provided by the invention;
Fig. 4 shows the present invention and is provided for the used agarose injection groove of zebrafish embryo microinjection;
Fig. 5 shows the mensuration of injection droplet dia in the microinjection step of the present invention;
Fig. 6 is the luciferase expression situation of injecting zebrafish embryo AB system and MLH1 defective system with, heteroduplex respectively and green fluorescence expression rate relatively;
Fig. 7 is the luciferase expression situation and the relative green fluorescence expression rate that are injected to the sudden and violent poison product filial generation embryo of institute of zebra fish parental generation B [a] p with, heteroduplex respectively;
Fig. 8 is the luciferase expression situation and the relative green fluorescence expression rate that are injected to the sudden and violent poison product filial generation embryo of institute of zebra fish parental generation PFOS with, heteroduplex respectively.
Embodiment
Be noted that following specifying all is exemplary, being intended to provides further explanation to the present invention.Only if statement is arranged in addition, all Science and Technology terms that this paper uses have with the present invention under the identical meanings of person skilled common sense.
Below in conjunction with specific embodiment the present invention is further described.
Be described further below in conjunction with accompanying drawing:
Shown in Figure 1 is the active analytical procedure of zebrafish embryo DNA MMR, and the practical implementation step is following.
One, the obtaining of single strand dna (schema is seen Fig. 2)
A, utilize helper phage M13ko7 to infect UltraMax DH5 α/p111 engineering bacteria legal system to be equipped with ssDNA (M13KO7 available from Beijing NEB company), wherein pGEM-EGFP is p111, and pGEM-(mt) EGFP is p189:
1) carry previous day with ultraDH5 α/p111 bacterium liquid drawing board on AMP (ammonia benzyl mycin) plate of preserving, and in bacteriological incubator 37 ℃ of incubated overnight;
2) get the flat board that grows single bacterium colony; The several single bacterium colonies of picking (can connect 5-6 when bacterium colony is big; Meet 10-20 when bacterium colony is little) be inoculated in the LB substratum (making the AMP final concentration is 100 μ g/mL) of 100mL, place 37 ℃ in constant-temperature shaking culture case, 250rpm/min cultivates;
3) (note observation at any time) after 2-4 hour and (make OD with its OD value of spectrophotometric determination
600＜0.05), add helper phage M13ko7 (100 μ L) shaking culture 1-1.5 then and as a child added card and receive mycin in the back, making its final concentration is 70 μ g/mL;
4) incubated overnight 14-18 hour, 4 ℃, the centrifugal 10min of 8000rpm changed supernatant in another new 50mL centrifuge tube over to, continue centrifugal 10min, behind centrifugal the finishing, supernatant were transferred in the clean 250mL tip bottle;
5) phage precipitation buffering liquid (phage precipitation buffering fluid component: PEG8000 (polyoxyethylene glycol 8000) 30% (V/V) (the green skies) and the 2.5M NaCl (sodium-chlor of (being 20mL) of 0.2 times of volume of adding in supernatant; Homemade analytical pure); Put upside down mixing; Place 1h or-20 ℃ for 4 ℃ and place 15-30min, during mixing several times;
6) in 4 ℃, the centrifugal 15min of 10000rpm outwells 90% supernatant; With a residue bacterium liquid piping and druming bottle wall, deposition is fully suspended, be transferred to 10000rpm, 10min collecting precipitation in the centrifuge tube of a 50mL; Be inverted centrifuge tube 2-3min; Supernatant is flow to end, residual supernatant on sucking-off bottle wall and the neck, with drier (at the bottom of not running into bottle with the bottle wall on the phage particle deposition of thin layer white);
7) the TE dissolution precipitation (with Vortex vortex 30s) of adding 1.6mL in the centrifuge tube of 50mL, equal-volume branch install in the EP pipe of 2 1.5mL;
8) centrifugal 1min transfers to supernatant in two new centrifuge tubes;
9) every pipe adds after 200 μ L phage precipitation buffering liquid liquid (phage precipitation buffer) place room temperature 5min, the centrifugal 10min of 13000rpm;
10) abandon supernatant,, add the resuspended deposition of 400 μ L TE with the drier deposition;
11) add isopyknic saturated phenol, leave standstill 5min behind the mixing, the centrifugal 5min of 12000rpm;
12) get supernatant in another new centrifuge tube, and then add the phenol of 200 μ L, the chloroform of 200 μ L/primary isoamyl alcohol extracting twice, vibration 20-30s leaves standstill 5min, centrifugal 12000rpm 5min;
13) in centrifuge tube, add chloroform 400 μ L extractings once, draw in the new EP pipe of supernatant to;
14) the 3M sodium-acetate of adding 1/10 volume, the absolute ethyl alcohol of the precooling of 2.5 times of volumes (cooling 30min better effects if in placing-20 ℃), the centrifugal 15min of 13000rpm;
15) washing with alcohol of adding 1mL 70%, the centrifugal 15min of 13000rpm adds 200 μ L TE dissolution precipitations after the drier.
B, incise enzyme process and prepare ssDNA:
Get 50 μ g plasmid p111; Add and incise enzyme Nb.Bsrd I (available from New England Biolabs) 50U; 65 ℃ of effects are 3 hours in the metal bath, carry out alcohol precipitation after the extracting of phenol chloroform, and its product digests with ExonucleaseIII (available from the precious biotech firm in Dalian); Hatch 10min for 37 ℃; PCR purification kit purifying (available from the green skies, Jiangsu company) can obtain the single strand dna (ssp111) (incising enzyme here can also replace with Nb.Bpu10I or Nb.Bts I, and other operation is identical) of about 20-25 μ g.
Two, homology and heteroduplex preparation (schema is seen Fig. 3)
1) a large amount of preparations of plasmid p111 and p189: with plasmid p111 and p189 difference transformed competence colibacillus cell DH5 α (sky, Beijing root biochemical technology ltd); Make DH5 α/p111 and DH5 α/p189; DH5 α/p111 and DH5 α/p189 carry the previous day of drawing board on the AMP flat board; Preparatory overnight cultures in the fresh single bacterium colony of picking next day to the LB/AMP liquid nutrient medium; Get next day in bacterium liquid to the liquid nutrient medium and cultivate, the step that the no intracellular toxin that plasmid p111 extraction is produced in strict accordance with sky, Beijing root biochemical technology ltd extracts on the test kit specification sheets is in a large number carried out;
2) linearizing of plasmid and purifying: plasmid p111 and p189 handle with restriction enzyme Mlu I (the green skies), make its linearizing, and the order that adds enzyme according to component after many earlier less, at last adds successively; Mixing, 37 ℃ are spent the night, and hatch 10 minutes termination reactions for 65 ℃; The extracting of phenol chloroform; Alcohol precipitation is used 60 μ LTE wash-outs at last, and the purifying after product is got 0.5 μ L and carried out electrophoresis with 1.0% Agarose gel and identify;
3) annealing of homology and heterologous nucleic acids duplex molecule hybridization: get linearization plasmid p1116 μ g in the PCR of 200 μ L pipe, 90 ℃ were heated 4 minutes in water-bath; Taking-up is to the ss p111 that wherein adds 40 μ L (9 μ g) mixing, and the continuation effect is 2 minutes in water-bath; Taking-up places on ice immediately, to wherein adding 20 * SSC solution, 2.5 μ L mixings (20 * SSC solution component: 17.52g sodium-chlor, 8.82g Sodium Citrate, usp, Dihydrate Powder; Be dissolved in the 80mL deionized water; Regulate pH with sodium hydroxide, make its pH=7.0, be settled to 100mL; Autoclaving is subsequent use) placed 60 ℃ of incubations 10 minutes, taking-up immediately places ice to treat its cooling; After annealing hybridization finishes, get 3 μ L and identify with 1.0% Agarose electrophoresis;
4) PSAD (Plasmid-Safe ATP-Dependent DNase) digestion: the product that above-mentioned annealing hybridization obtains is transferred in the aseptic 1.5mL centrifuge tube, digested to remove residual single chain molecule and linearization plasmid with PSAD.Centrifugal mixing is put in the metal bath 37 ℃ and is spent the night.Behind the reaction terminating, get 3 μ L and carry out electrophoresis with 1.0% Agarose gel and identify;
5) homology and heteroduplex is quantitative: get 1 μ L stoste and measure concentration with Nanodrop2000 (micro-spectrophotometer).
Three, embryo collection and selecting
(Holland introduces to collect AB system (U.S. does not have the pathogeny strain) and MLH1 defective system; Mispairing repairing effect defective) zebrafish embryo; Collection procedure is following: microinjection evening before that day in zebra fish is cultivated frame, put the brooder of collecting the embryo well, and with baffle plate the male and female zebra fish is separated; After treating the bright lamp of system in second day; Take away baffle plate; Begin to collect embryo's (the male and female fish is knocked into the back and begins to lay eggs behind the 10-20min generally speaking) behind the 30min; With zebra fish cultivating system water rinse embryo several, removal dysplasia and odd-shaped embryo can be arranged in the agarose injection groove and be used for microinjection under the Stereo microscope.
Four, microinjection
With collecting good embryo, be listed as by the position one of agarose mould and set (see figure 4), under stereoscopic microscope (nikon), the embryo is injected one by one; The embryo of two strains of injection (AB system and MLH1 defective strain); The embryo of each strain injects respectively with the homology of isoconcentration and heteroduplex (≤100ng/ μ L; Be preferably 30ng/ μ L); I.e. two groups (homology group and allos group); Two groups of embryonal vaccination numbers identical (>=100 pieces), every piece of embryo's ID is about 500pL (volume injected confirms that (see figure 5) is to measure the diameter of injection drop with objective micrometer, calculates according to formula V=π d3/6 then), injects the back that finishes with embryo medium (component: 0.04g KCl, 0.00358g Na
2HPO
4, 0.006g KH
2PO
4, 0.144gCaCl
2, 0.246g MgSO
47H
2O, 0.35g NaHCO
3, 0.1g kantlex (Kanamycin), 0.1g penbritin (Ampicillin) be dissolved in the 1L sterilization deionized water and form) embryo is eluted in the petridish, place illumination box to cultivate.
Five, DNA MMR functionally active is quantitative
After the microinjection 12,24,36h observes the embryo who is injected, be preferably 24h after.Under fluorescence Stereo microscope and fluorescence inverted microscope, embryonal vaccination is taken pictures; Add up every group of embryo's green fluorescence of each strain expression rate; Use every piece of embryo's of NIS-BR 2.3 software analysis green fluorescence intensity in addition; Calculate every group of embryo's average green fluorescence fluorescence intensity, calculate each strain embryo's relative green fluorescence expression rate (being DNA MMR efficient) (seeing table 1) then with the relative egfp expression rate of formula (Relative EGFP expression)=allos group luciferase expression rate * allos group average fluorescent strength/homology group luciferase expression rate * homology group average fluorescent strength.American AB does not have the pathogeny strain, and to produce embryo MMR function normal, and its mispairing remediation efficiency should be 100% in theory, and the MLH1 defective be the embryo because important gene mlh1 dysfunction is repaired in mispairing, thereby its DNA MMR function is also unusual.These strain characteristics are the male sterile of mlh1 defective homozygous individual, female generation triploid offspring, thereby its strain keeps keeping through hybridizing with wild system, and said generally speaking MLH1 defective system is a heterozygosis.) because MLH1 defective system is a heterozygosis, thereby its DNA MMR function is not complete defective, but with respect to AB system, its function is remarkable reduction.Utilize these two strains that method of the present invention is tested, the experimental result (see figure 6) confirms to conform to theory, thereby explains that method of the present invention is feasible.
Relative egfp expression rate behind each strain zebrafish embryo microinjection 24h of table 1.
The data presentation of table 1 and Fig. 6; Embryo's relative green fluorescence expression rate is 0.95 (near 1) behind the zebrafish embryo injection homology of AB system and the heteroduplex substrate 24h; Embryo's relative green fluorescence expression rate is merely 0.26, significant difference (p＜0.01) behind the zebrafish embryo injection homology of Mlh1 genetic flaw system and the heteroduplex substrate 24h.The zebrafish embryo DAN MMR efficient that this reflects just in time conforms to theoretical value, explain that the present invention sets up to be used for zebrafish embryo DNA MMR functionally active analytical procedure be feasible.
Environmental compound DNAMMR function toxicity assay may further comprise the steps:
1) zebra fish is malicious cruelly:
Choose typical carcinogens benzopyrene (B [a] p) and general toxicity novel environmental pollutent PFOS (PFOS) zebra fish is carried out sudden and violent poison.3 months big adult fishes of the sudden and violent poison of B [a] p hydrostatic continue one month, and concentration is set at (0,10,50,100,200,250 μ g/L), change one time venom in per five days.PFOS is malicious cruelly, and the embryo from 6hpf (after fertilization) begins sudden and violent poison, and the sudden and violent poison of hydrostatic continues 5 months, and concentration is set at (0.01%DMSO, 0.01 μ M, 0.1 μ M, 0.5 μ M), venom of replacing in per 5 days;
2) single strand dna obtains:
Method is the same;
3) preparation of homology and heteroduplex molecule
Method is the same;
4) embryo collection:
The sub-F1 that collects each sudden and violent poison group is for the embryo, and collection method is with the above;
5) DNA MMR function toxicity assessment:
Homology group and allos group embryo's green fluorescence expression rate under each concentration of Statistics Division is handled under fluorescent microscope; Utilize NIS-BR2.3 software quantitatively to go out the average green fluorescence intensity that each concentration is handled following homology group and allos group embryo respectively then; Said formula calculates different concns and handles DNAMMR efficient down above the basis then; Relatively different concns exposes zebrafish embryo DAN MMR efficiency change down, thus the DNA MMR function toxicity of this kind of objective evaluation compound.The sudden and violent poison back filial generation of B [a] p embryo DNA MMR function toxicity result sees table 2 and Fig. 7, and the sudden and violent poison back filial generation of PFOS embryo DNA MMR function toxicity result sees table 3 and Fig. 8.Relative green fluorescence expression rate difference adopts one-way analysis of variance between the different concns group.
Table 2 B [a] p handles the relative egfp expression rate of embryo that zebra fish produces
Table 3 PFOS handles the relative egfp expression rate of embryo that zebra fish produces
Table 2 and Fig. 7 data presentation, its mispairing repairing effect of embryo that zebra fish produces of the long-term sudden and violent poison of B [a] P progressively weakens, and has significance difference (p＜0.01) during 100-250 μ g/L.
Table 3 and Fig. 8 data presentation, zebra fish PFOS long-term exposure, its filial generation mispairing repairing activity reduces gradually, but does not have significant difference (p＞0.05) between each concentration group along with sudden and violent malicious concentration increases.
Used medicine benzopyrene (B [a] p) is available from sigma company in the present embodiment, and PFOS (PFOS) is then available from the lark prestige company in Shanghai.
The above is merely the preferred embodiments of the present invention, should be understood that; For the those of ordinary skill in the present technique; Under the prerequisite that does not break away from core technology characteristic of the present invention, can also make some improvement and retouching, these retouchings and improvement also should belong to scope of patent protection of the present invention.
Claims (10)
1. active analytical procedure of zebrafish embryo dna mismatch repairing effect is characterized in that said analytical procedure may further comprise the steps:
1) prepares single strand dna with plasmid pGEM-EGFP, said plasmid pGEM-EGFP ability normal expression green fluorescent protein;
2) plasmid pGEM-EGFP and pGEM-(mt) EGFP with the single strand dna colinearityization of obtaining in the step 1) prepares homology and heteroduplex respectively; There is a nonsense mutation in the 58th codeword triplet place in the green fluorescence protein gene of wherein said plasmid pGEM-(mt) EGFP, can not the normal expression green fluorescent protein;
3) homology for preparing and heteroduplex are distinguished microinjection to the zebrafish embryo of 1-cell stage;
4) embryo's dna mismatch repairing effect activity is carried out quantitatively.
2. analytical procedure according to claim 1 is characterized in that step 1) comprises: the intestinal bacteria to transform plasmid pGEM-EGFP are material, use helper phage M13KO7, obtain single strand dna through the method that infects.
3. analytical procedure according to claim 1; It is characterized in that; Said step 1) comprises: with plasmid pGEM-EGFP is material; Enzyme Nb.Bsrd I is incised in use or Nb.Bpu10I forms the open loop plasmid that band is incised, and uses exonuclease III to carry out digestion reaction then and obtains single strand dna.
4. analytical procedure according to claim 1; It is characterized in that; Step 2) comprising: with single strand dna with annealing respectively through restriction enzyme BstX I or the linearizing plasmid pGEM-EGFP of Mlu I and pGEM-(mt) EGFP; The annealing after product is removed residual single strand dna and linearizing plasmid and can be obtained high-purity homology and heteroduplex through plasmid security enzyme PSAD digestion.
5. analytical procedure according to claim 1 is characterized in that step 2) described in plasmid pGEM-EGFP and pGEM-(mt) EGFP in based composition, have only the difference of a base.
6. analytical procedure according to claim 1 is characterized in that step 2) described in the heteroduplex in the EGFP gene the 58th codeword triplet place have a G/T mispairing.
7. analytical procedure according to claim 1; It is characterized in that step 3) comprises: the embryo is injected same, the heteroduplex of 30～100ng/ μ L respectively, and equate with, heteroduplex injection volume; The injection site is tenuigenin or yolk sac; Every piece of embryo is 500pL, and the indicator phenol red concentration is controlled at 0.025-0.050%, the embryo of each strain homology group and allos group injection equivalent amount: >=100 pieces.
8. analytical procedure according to claim 1; It is characterized in that; Step 4) comprises: observe behind the embryonal vaccination 12-36h and take pictures; With software analysis embryo green fluorescence expression intensity, and statistics institute embryonal vaccination green fluorescence expression rate, embryo's dna mismatch repairing effect activity is represented with the relative egfp expression rate of this strain; Said relative egfp expression rate is calculated by following formula and is obtained:
Relative egfp expression rate=allos group luciferase expression rate * allos group average fluorescent strength/homology group luciferase expression rate * homology group average fluorescent strength.
9. according to arbitrary described analytical procedure among the claim 1-8, it is characterized in that said plasmid pGEM-EGFP is through the CMV-EGFP-PolyA fragment is scaled off from plasmid pEGFP-N1 enzyme, utilize the T4 ligase enzyme to be connected among the carrier pGEM5Zf (+) then and obtain.
10. the application of the described analytical procedure of claim 1 in environmental compound dna mismatch repairing effect toxicity assessment.
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| CN109402170A (en) * | 2018-11-01 | 2019-03-01 | 湖南文理学院 | A kind of method for building up of fish male sterility model |
| CN112889755A (en) * | 2021-01-21 | 2021-06-04 | 周娟 | Construction method of zebra fish animal model for screening cardiovascular disease drugs |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109402170A (en) * | 2018-11-01 | 2019-03-01 | 湖南文理学院 | A kind of method for building up of fish male sterility model |
| CN109402170B (en) * | 2018-11-01 | 2023-10-27 | 湖南文理学院 | Method for establishing fish male sterility model |
| CN112889755A (en) * | 2021-01-21 | 2021-06-04 | 周娟 | Construction method of zebra fish animal model for screening cardiovascular disease drugs |
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Application publication date: 20120328 |