CN109321600A - A kind of method and its application of ox that cultivating production low irritability milk - Google Patents

Abstract

The invention discloses a kind of method and its application of ox for cultivating production low irritability milk.This method comprises the following steps: importing Zinc finger nuclease carrier pZFN to bovine fibroblasts, obtains the donorcells that genotype is diallele saltant type；Diallele saltant type is that frameshift mutation occurs for the nucleic acid molecule of the β-LG gene on two homologues of ox；The nucleus of donorcells is moved into the bovine oocyte for removing nucleus, development forms reconstruct embryo, then moves into cow uteri, and childbirth obtains the ox of production low irritability milk.Successful incubation of the present invention without exogenous DNA integration, can produce no beta lactoglobulin milk and mutation passed to by follow-on β-LG diallele by normal breeding and knock out ox.The present invention is to solve the problems, such as that milk allergy lays important foundation, while also will provide unlimited possibility for the preparation of humanization milk.The present invention has important application value.

Description

A kind of method and its application of ox that cultivating production low irritability milk

Technical field

The invention belongs to field of biotechnology, and in particular to it is a kind of cultivate production low irritability milk ox method and its Using.

Background technique

Milk is the healthy food that the important nutrition of worldwide edible one kind is in admirable proportion, including protein abundant, Carbohydrate, fat element and minerals.Although Nutrition Management and Quantitative Genetics improve ox by the Breeding Strategies of many years The yield of milk, still, the ingredient of milk there is no significant changes.It is especially right with the development of biotechnology and genetic engineering Animal husbandry breed improvement provides bigger chance to prepare " the design mode milk " of the suitable mankind.Up to now, various Gao Pin Matter transgenic cow, which is reported that, for example expresses restructuring lactoferrin ox, increases milk casein content ox, anti-mammitis Ox, anti-rabid ox disease ox, improve animal welfare polled cattle.But the allergic reaction problem of milk at present still cannot be fine Ground solves.

Beta lactoglobulin (β-lactoglobulin, β-LG) is the main allergen in milk, and Milk allergy is in baby children It is most commonly seen in youngster's food hypersenstivity.Foreign Epidemic disease investigation result shows that the disease incidence of infant's cow milk protein allergy is 0.3%-3.5%, and have the tendency that constantly rising, it seriously affects infant and nutriment in dairy products is absorbed and utilized.So far Until the present, researcher has attempted to multiple technologies to reduce the sensitization of β-LG in cow's milk, mainly includes heat treating process, high pressure Method, enzymatic isolation method and glycosylation modification method etc..Although these methods reduce the sensitization of β-LG, milk to a certain extent In the structure and functions of other albumen destroyed, the trophism of milk substantially reduces, and the sensitization of β-LG can not yet It completely eliminates.β-LG sensitization how is effectively reduced, while keeping the original structure and function of albumen as far as possible again, at present state Inside and outside all not effective and feasible technical method.

2001, Bibikova et al. was successfully mediated in Xenopus Oocytes using Zinc finger nuclease homologous for the first time Recombination, Zinc finger nuclease technology causes the extensive attention of biological field, be widely used in animal and plant gene deletion, Reparation, large fragment DNA are deleted and the fixed point of gene is inserted into etc..Zinc finger nuclease-mediated High-efficiency gene Knockout technology is It is combined with human disease treatment, phase clinical trial is successfully completed by the Zinc finger nuclease that Sigma Co., USA designs, As a result exciting.They formulate Zinc finger nuclease (the HIV disease of specific bond mankind's CCR5 gene by way of genetic engineering Poison utilizes CCR5 gene coded protein, infects the CD4+ cell of people) so that the gene inactivates, to resist invading for inhibition of HIV Dye.The CD4+ cell (CCR5 gene knockout) that Zinc finger nuclease modified is injected by patient HIV receives 6 HIV diseases for the treatment of In people, there is 5 immunocyte quantity to significantly rise.Urnov et al. utilizes the reparation of Zinc finger nuclease-mediated IL2R γ gene (immune deficiency disorder), can with the mutation IL2R γ gene (efficiency is up to 18%) of repair cell, to restore the gene expression, The T cell of immune performance is shown as, provides a good therapy approach for immunodefiiciency patient.Hockemeyer etc. People modifies OCT4 and PITX3 gene using Zinc finger nuclease in human stem cells, it was demonstrated that Zinc finger nuclease can be dry thin The gene of different expressions is efficiently acted in born of the same parents, has established certain basis for stem-cell therapy.Zinc finger nuclease-mediated High-efficiency gene Knockout technology is combined with somatic cell clone technique, and Whyte et al., which has been obtained, utilizes Zinc finger nuclease The clone pig of GFP gene knockout is mediated, gene knockout efficiency is up to 5%.In addition, the scientific research of Chinese scholar professor Lai Liangxue leader Team also successfully utilizes the PPAR- γ gene knockout of Zinc finger nuclease-mediated pig, obtains corresponding gene knockout clone pig.

Summary of the invention

The purpose of the present invention is the oxen of production low irritability milk or cultivation production low irritability milk.

The present invention protects the method for cultivating the ox of production low irritability milk first.

The present invention is to be protected to cultivate the method for producing the ox of low irritability milk, concretely method one, it may include such as Lower step: inhibit to set out the synthesis of β-LG albumen and/or expression quantity and/or activity in cow genome group, obtains transgenic cow；With go out Hair ox is compared, and the milk sensitization of transgenic cow production reduces.

In the above method one, described " inhibition is set out the synthesis of β-LG albumen and/or expression quantity and/or work in cow genome group Property " can be carried out by way of gene site-directed editor, RNA interference, homologous recombination or gene knockout；The gene site-directed editor can It is carried out using cow genome group editor's carrier.

In the above method one, described " inhibition is set out the synthesis of β-LG albumen and/or expression quantity and/or work in cow genome group Property " it can be that frameshift mutation is occurred into for the nucleic acid molecule of the β-LG gene on two homologues for the ox that sets out.

The present invention is to be protected to cultivate the method for producing the ox of low irritability milk, concretely method two, it may include step Suddenly (1): importing cow genome group editor's carrier to bovine fibroblasts, it is thin to obtain the donor that genotype is diallele saltant type Born of the same parents；

The diallele saltant type is that the nucleic acid molecule of the β-LG gene on two homologues of ox occurs Change, it is this to change the synthesis for inhibiting β-LG albumen and/or expression quantity and/or activity.

It is described " nucleic acid molecule of the β-LG gene on two homologues of ox changes " in the above method two Frameshift mutation can occur for the nucleic acid molecule of the β-LG gene on two homologues of ox.

Any of the above-described the method two may also include step (2): after completing step (1), by the cell of the donorcells Core moves into the bovine oocyte for removing nucleus, and development forms reconstruct embryo, then moves into cow uteri, it is low that childbirth obtains production The ox of sensitization milk.

In the above method two, the bovine fibroblasts can specifically be digested by bovine fetal fibroblast system to be obtained.

In the above method two, described " importing cow genome group editor carrier to bovine fibroblasts " is concretely to ox at fibre Tie up the mRNA that cell imports cow genome group editor's carrier.

Any of the above-described cow genome group editor carrier can be Zinc finger nuclease carrier pZFN；The Zinc finger nuclease carrier The target DNA that pZFN is identified in cow genome group is the DNA fragmentation for encoding β-LG albumen.

The recognition site of any of the above-described Zinc finger nuclease carrier pZFN is concretely in sequence table shown in sequence 4 DNA molecular.

The method of the present invention ox to be protected for cultivating production low irritability milk, concretely method three, can be selection The Niu Jinhang breeding for the production low irritability milk that any of the above-described method is cultivated.

The present invention also protect it is a kind of detection or auxiliary detection milk sensitization method, it may include following steps: detection to β-LG the gene surveyed on two homologues of ox is wild type or diallele saltant type, and genotype is double equipotential bases The sensitization of the milk produced by the ox to be measured of saltant type be lower than or it is candidate lower than genotype be wild type ox to be measured；It is described double Allelic variants are that frameshift mutation occurs for the nucleic acid molecule of the β-LG gene on two homologues of ox；It is described Wild type is that frameshift mutation does not occur for the nucleic acid molecule of the β-LG gene on two homologues of ox.

In the above method, the anaphylactogen of the sensitization can be β-LG albumen.

The present invention also protects the Zinc finger nuclease carrier pZFN of cow genome group editor a kind of；The Zinc finger nuclease carrier The target DNA that pZFN is identified in cow genome group can be the DNA fragmentation of coding β-LG albumen.

The recognition site of the Zinc finger nuclease carrier pZFN can be DNA molecular shown in sequence 4 in sequence table.

The present invention also protects the nucleic acid molecule of the β-LG gene on two homologues by ox that frameshit occurs and dashes forward The method or product of change or will inhibit in cow genome group the synthesis of β-LG albumen and/or expression quantity and/or active method or Product in the ox and/or reduction milk sensitization of cultivating production low irritability milk and/or produces answering in low irritability milk With.

In above-mentioned application, frameshit occurs for the nucleic acid molecule of the β-LG gene on two homologues by ox The product of mutation concretely any of the above-described Zinc finger nuclease carrier pZFN.

In above-mentioned application, frameshit occurs for the nucleic acid molecule of the β-LG gene on two homologues by ox The method of mutation concretely any of the above-described the method two.

" it is prominent that frameshit occurs for the nucleic acid molecule of the β-LG gene on two homologues of ox described in any of the above-described Become " concretely 265-281 nucleotide deletion (nucleosides of β-LG gene at this time of β-LG gene on one article of homologue Acid sequence is as shown in sequence 2 in sequence table) while β-LG 265-282 nucleotide of gene on another article of homologue It lacks (nucleotide sequence of β-LG gene is as shown in sequence 3 in sequence table at this time).

The nucleotide sequence of any of the above-described β-LG gene can be as shown in sequence 1 in sequence table.

The amino acid sequence of any of the above-described β-LG albumen is as shown in sequence 5 in sequence table.

The β-of the present inventor's first passage Zinc finger nuclease (ZFNs) technology successful incubation without exogenous DNA integration LG diallele knocks out ox, and generates the milk without beta lactoglobulin.Balb/c is carried out to the milk of no beta lactoglobulin The Allergenicity assessment of mouse model is tested, it is found that it can trigger more compared with ordinary milk (milk that non-transgenic cow produces) Low allergic reaction, the generation including allergen specificity Immunoglobulin IgE；The milk without beta lactoglobulin is in 2min simultaneously When be easy to by pepsin digestion, and the beta lactoglobulin in ordinary milk (non-transgenic cow produce milk) is after 60min Digestion completely not yet, the combination of the IgE and the milk without beta lactoglobulin of milk allergy (CMA) patient are significant to be lower than ordinary milk (milk that non-transgenic cow produces).Importantly, exogenous DNA will not be introduced receptor by the technology, base can be mitigated in this way The bio-safety problem being related to by editor animal.Finally, β-LG diallele knocks out ox can will be mutated by normal breeding Pass to the next generation.These researchs also will be " humanization milk " to solve the problems, such as that milk allergy lays important foundation Preparation offer is infinitely possible, so that milk is more suitable for human health, and improve the functional characteristic of milk.The present invention has important answer With value.

Detailed description of the invention

Fig. 1 is the flow chart cultivated β-LG diallele and knock out ox.

Fig. 2 is the recognition site position view of pZFN.

Fig. 3 is the photo that β-LG diallele knocks out ox.

Fig. 4 is the testing result of β-LG albumen in 2 milk of embodiment.

Fig. 5 is the testing result of simulate the gastric juice digestion experiment.

Fig. 6 is the testing result of people's serum test.

Fig. 7 is the testing result of animal model experiment.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

Wild holstein cow is non-transgenic cow.

The PBS buffer solution (hereinafter abbreviation PBS buffer solution) of pH7.2,0.01mM, FBS and DMEM culture medium are Gibco The product of company.A23178 is the product of Sigma company.Shock by electricity the product that liquid is Lonza company.

Embodiment 1 cultivates β-LG diallele knockout ox using Zinc finger nuclease

Ox is diploid animal, when Zinc finger nuclease plays a role, on same two intracellular homologues Two allele be likely to be edited, generate same-type or different types of mutation.β-the LG that the present embodiment obtains Diallele knocks out ox (β-LG-^/Ox) be two homologues β-LG gene be mutated and mutant form not Together.The present inventor cultivates β-LG diallele using Zinc finger nuclease and knocks out ox, considers the industrialization in later period, avoids Biosafety issues do not use riddled basins, while using ZFNs mRNA, can cultivate no exogenous DNA integration in this way β-LG diallele knock out ox, technology path is detailed in Fig. 1.

One, ZFN plasmid is synthesized

According to the sequence information of ox β-LG gene (shown in sequence 1 in sequence table), zinc finger core is synthesized by the design of sigma company Sour zymophore pZFN.PZFN is made of two plasmids, and targeting sequence is located at the First Exon of β-LG gene.Specific such as Fig. 2 Shown in (BLG, that is, β-LG gene).

The recognition site of pZFN are as follows: CCCAGGCCCTCATTGTCacccagACCATGAAGGGCCTGGA (sequence in sequence table Column 4), β-LG 257-296 nucleotide of gene shown in sequence 1 in corresponding sequence table.Wherein, two terminal sequence (CCCAGGCCCTCATTGTC and ACCATGAAGGGCCTGGA) is zinc finger binding site, and middle section sequence (acccag) is to cut Cut site.

Two, the fibroblastic acquisition of saltant type

1, the acquisition of bovine fetal fibroblast system

Take the ear skin tissue of holstein cow, it is clear with 70% (v/v) alcohol water blend after ear's lower edge back side unhairing Wash clean, then pick from ear's lower edge back side with blade that take area be 1cm²The skin of left and right, is placed in 0 DEG C of DMEM/F12 culture medium In transport laboratory back as early as possible, with PBS buffer solution and 70% (v/v) alcohol water blend cleaning shred into 1mm afterwards several times³What is controlled is small Block, DMEM culture medium plants block in batches after cleaning 2 times in the DMEM culture medium culture bottle containing 1mL containing 10% (v/v) FBS, (specification is 25cm²) in, the DMEM culture medium containing 10% (v/v) FBS is added again after tissue block adherent is secured to 6mL, 37 DEG C, 5%CO₂Training Case culture 6-7d is supported, during which every 2d is changed liquid 1 time, after cell growth converges, passed on 2-3 times with 0.25% trypsin digestion, It is frozen in batches with the DMEM culture medium containing 20% (v/v) FBS, 10% (v/v) DMSO.In this way, through originally culture, secondary culture, The operation of the in vitro cultures such as freezing, establishes holstein cow fibroblast.

2, the mRNA transfection of ZFNs

(1) 2d before transfecting obtains bovine fibroblasts with trypsin digestion bovine fetal fibroblast system to unicellular (also referred to as wild type fibroblast)；By 1 × 10⁶A bovine fibroblasts are forwarded to culture bottle (specification 100mL), are added 4mL contains the DMEM culture solution of 10% (v/v) fetal calf serum, 37 DEG C, 5%CO₂It cultivates to logarithmic growth phase.

(2) after completing step (1), the bovine fibroblasts in logarithmic growth phase are taken, with trypsin digestion, then 1000g is centrifuged 5min, collects precipitating.

(3) after completing step (2), the precipitating is taken, is washed 1 time with PBS buffer solution, is then resuspended with 100 μ L electric shock liquid, Obtain cell suspension.

(4) take cell suspension (containing 1 × 10⁶A cell), it (will be in step 1 by the 4 μ g ZFNs mRNA being transcribed in vitro The Zinc finger nuclease carrier pZFN of sigma company design synthesis is linearized and using it as template, according to the mRNA of Ambion company The operating procedure of in-vitro transcription kit obtains) it mixes well rear corotation and enters the cup that shocks by electricity, shocked by electricity (electric field strength 1.2KV/ Cm, burst length 1ms).

(5) after completing step (4), the cell after electric shock is transferred to culture bottle (specification 100mL), 4mL is added and contains 10% (v/v) the DMEM culture solution of fetal calf serum, 37 DEG C, 5%CO₂1-2d is cultivated, Transfected cells are obtained.

3, infinite dilution method prepares single cell clone and genotype identification

(1) when the Transfected cells culture for obtaining above-mentioned steps 2 to fusion rate reaches 80%-90%, with the 0.1% of 37 DEG C Cell is collected by centrifugation after the DMEM culture medium containing 10% (v/v) FBS terminates digestion in pancreatin digestion.

(2) cell is resuspended with the DMEM culture medium containing 20% (v/v) FBS, takes part cell to be counted, cell is diluted To 100/mL, cell suspending liquid is obtained.In the culture dish for having added DMEM culture medium of the 9mL containing 20% (v/v) FBS to 20 It is each that 1mL cell suspending liquid, 37 DEG C, 5%CO are added₂, cultivate under the conditions of saturated humidity.

(3) when the cell clone in culture dish grows to diameter and reaches 2mm or more, culture medium is removed, after DPBS is rinsed, is used Clone's ring covers in cloning cluster, and 0.1% pancreatin of 37 DEG C of about 100 μ L is added dropwise thereto, after digesting about 3min, is added dropwise and contains 20% (v/v) the DMEM culture medium of FBS terminates digestion, transfers them to after gently blowing and beating and expands culture in 48 orifice plates.

When cell confluency is up to 90% in (4) 48 orifice plates, take a semicell for cell clone genotype identification after digestion, Remaining half continuation is cultivated in the orifice plate.

(5) cell for identifying genotype abandons supernatant after 1000g is centrifuged 5min, is added according to cell precipitation amount 10-20 μ L cell pyrolysis liquid.

(6) drawing 3 μ L cell pyrolysis liquids is that template carries out PCR identification, (is set according to ox β-LG gene with the primer in table 1 Count and synthesize) carry out PCR amplification.

Table 1

Reaction system is 20 μ L, (dense by 1.0 μ L DNA profilings, 0.4 μ L primer P1 (concentration is 10 μM), 0.4 μ L primer P2 Degree is 10 μM), 0.4 μ L dNTP, 0.3 μ L LA archaeal dna polymerase, 2.0 μ 10 × PCR of L Buffer and 15.5 μ L ddH2O groups At.

Response procedures: 94 DEG C of 5min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72 DEG C of 5min, 4 DEG C of preservations.

Pcr amplification product is purified, is sequenced.If producing mutation in ZFNs action site, sequencing result will appear Double-peak Phenomenon determines that the cell line is mutant.And then the PCR product of mutant is connected to pMD-19t plasmid vector, it converts After E. coli competent, the multiple single colonies of picking are sequenced, and sequencing result is compared with wild-type beta-LG gene, is obtained Obtain detailed abrupt information.

The result shows that in 128 single cell clones, obtain one be diallele be mutated and cause frameshift mutation (- Single cell clone 16bp/-17bp).Specifically, β-LG gene is the β-LG base of a homologue in the single cell clone 265-281 nucleotide deletions of cause, and the missing of the β-LG 265-282 nucleotide of gene of another article of homologue. The single cell clone is named as saltant type fibroblast.

β-LG gene in wild type fibroblast genome is wild-type beta-LG gene (1 institute of sequence in sequence table Show).β-LG gene in saltant type fibroblast genome is by β-LG gene 265-281 of one article of homologue The β-of nucleotide deletion (nucleotide sequence of β-LG gene is as shown in sequence 2 in sequence table at this time) and another homologue The missing (nucleotide sequence of β-LG gene is as shown in sequence 3 in sequence table at this time) of LG 265-282 nucleotide of gene, obtains The DNA molecular arrived.

It follows that saltant type fibroblast and wild type is fibroblastic is different only in that: being on β-LG gene It is no that there are the missings of 265-281 nucleotide and 265-282 nucleotide in sequence 1.

Three, β-LG diallele knocks out the cultivation of ox

1, the saltant type fibroblast in logarithmic growth phase is taken, with 0.25% trypsin digestion 5min, obtains list Cell.

2, in the unicellular immigration non-nucleus egg mother cell oolemma for obtaining step 1, first it is placed in Zimmerman liquid (100mL Zimmerman liquid is by sucrose 0.9854g, Magnesium acetate (Mg(OAc)2) tetrahydrate 10.7mg, Calcium diacetate monohydrate 1.8mg, phosphoric acid hydrogen two Potassium 7.4mg, reduced glutathione 3.1mg, bovine serum albumin 1.0mg and water composition) in balance 3-5min, then be placed in integration slot Interior rotation egg cell contacts donorcells with egg mother cell and vertical with electric field, while in the direct current arteries and veins that field strength is 2.5kV/cm It rushes in field, (fusion instrument is BTX public for fusion under conditions of the burst length is 10 μ s, pulse number is 2 times, the pulse spacing is 1s The product of department, model ECM-2001), rapid M199 liquid (product of Gibco company) of the immigration containing 10% (v/v) FBS, 37 DEG C, 5%CO2 cultivate 30min, obtain reconstructed embryo.

3, the CR1aa culture solution for the Calcium ionophore A23178 that concentration is 5 μM is added in the reconstructed embryo for taking step 2 to obtain (100mL CR1aa culture solution is by 0.67g sodium chloride, 0.023g potassium chloride, 0.22g sodium bicarbonate, 2mg Sodium Pyruvate, 100 μ l Phenol red and water composition) processing 5min；Liquid phase is abandoned, is added containing 5 μ g/mL cytochalasin Bs and 10 μ g/mL cycloheximides CR1aa culture solution handles 5h (purpose is activation reconstituted embryo)；Abandoning liquid phase, CR1aa culture solution of the addition containing 5% (v/v) FBS, 37 DEG C, 5%CO2 cultivate 48h, observe cleavage rates, 7-8d observe blastocyst rate.

4, the excellent transgene clone blastaea of the form that 7d is cultivated in step 3 is moved into the cornua uteri of the receptor cow of the same period Interior (co-transplantation receptor cow 3) obtains β-LG diallele and knocks out ox (i.e. No. 111027 oxen).

Using blood and tissue gene group reagent box (QIAGEN company) extract ox to be measured (No. 111027 oxen or wild lotus this Smooth milk cow) ear tissue genomic DNA, and using it as template, using the primer pair of primer P1 and primer P2 (shown in table 1) composition PCR amplification is carried out, pcr amplification product is obtained.Pcr amplification product is purified, is sequenced.

Sequencing result shows that genotype of the wild holstein cow based on β-LG gene is wild type (β-LG+^/+), Genotype of No. 111027 oxen based on β-LG gene is diallele saltant type (β-LG-^/-).Diallele saltant type is β-LG 265-281 nucleotide deletions of gene of one article of homologue, and the β-LG gene of another article of homologue The missing of 265-282 nucleotide.Due to lacking 16bp and 17bp respectively, β-LG gene frameshift mutation, and then β-LG egg are caused White afunction.

No. 111027 oxen survive normally at present, see Fig. 3.

Embodiment 2, detection β-LG diallele knock out and whether there is β-LG albumen in the milk of ox

One, SDS-PAGE, which detects β-LG diallele and knocks out, whether there is β-LG albumen in the milk of ox

1, with ox to be measured (β-LG diallele knock out ox or wild holstein cow) the 1st day, the 3rd day lactation period or the The milk of production in 5 days extracts total protein.

2, the total protein and β-LG protein standard substance (product of Sigma company) for the ox to be measured for taking step 1 to obtain respectively, into Row SDS-PAGE.

Experimental result is shown in A in Fig. 4 (M is albumen Marker, and β-LG is β-LG protein standard substance).The result shows that β-LG is double The total protein that allele knocks out ox does not detect the expression of β-LG albumen, it is seen that β-LG diallele knocks out the ox that ox produces There is no β-LG albumen in milk.

Two, Western blot, which detects β-LG diallele and knocks out, whether there is β-LG albumen in the milk of ox

1, with ox to be measured (β-LG diallele knock out ox or wild holstein cow) the 1st day, the 3rd day lactation period or the The milk of production in 5 days extracts total protein.

2, the total protein and β-LG protein standard substance for the ox to be measured for taking step 1 to obtain respectively carry out Western blot.It adopts Use Bovine beta-lactoglobulin Antibody (product of BETHYL company) as primary antibody, goat antibody (middle China fir The product of company of Golden Bridge) as secondary antibody detection β-LG albumen.

Experimental result is shown in B in Fig. 4 (β-LG is β-LG protein standard substance).The result shows that β-LG diallele knocks out ox Total protein do not detect the expression of β-LG albumen, it is seen that β-LG diallele, which knocks out in the milk that ox produces, does not have β-LG egg It is white.

After the above results show that the β-LG gene of β-LG diallele knockout ox is destroyed, do not contained in the milk of production β-LG albumen.

Embodiment 3, β-LG diallele knock out the sensitization detection of the milk of ox

Dry powder is made in the milk that ox to be measured (β-LG diallele knocks out ox or wild holstein cow) produces, is named as Milk powder to be measured.

One, simulate the gastric juice digestion experiment

A kind of native protein will keep the sensitization of itself, and the amino acid fragment at the IgE binding site of the albumen is general It can be resistant to the digestion of internal gastrointestinal tract, be possible to be absorbed and be immunoreacted after arrival mucous membrane of small intestine in this way.Therefore, in body Still remaining protein fragments after outer simulation digestion 60min, are considered extremely difficult digestion, and have the possibility of sensitization.

Simulation peptic digest liquid is pH value, salt ionic concentration and the digestion of pepsin concn setting simulated in human gastric juice Liquid detects simulation gastro-intestinal Fluid exogenous proteins with reference to Ministry of Agriculture's genetically modified organism and products thereof edible safety and digests stability test Method carries out, and pepsin (pepsin) (product of the Sigma company) vigor used in this standard cannot be below 2000U/mg, Additive amount A=5 × 10 of pepsin in 100mL simulate the gastric juice⁶(A-pepsin additive amount, unit are milligram to/19B (mg)；B-peptic activity of stomach, unit are every milligram of unit vigor (U/mg)).Preparation system: 0.2g sodium chloride is weighed (NaCl) and Amg pepsin 70mL double distilled water, is added, is added 730 μ L hydrochloric acid, then with hydrochloric acid tune pH to 1.2, adds water constant volume To 100mL.Matching while using.

Protein sample sample-loading buffer is the product of green skies company.

Specific step is as follows:

1, centrifuge tube is taken, 1.9mL is added and simulates peptic digest liquid, 37 DEG C of water bath with thermostatic control 5min.

2, after completing step 1,100 μ L milk amidin (concentration 5g/L) to be measured or double distilled water is added (as right According to), vortex oscillation, then 37 DEG C of water-baths 0s, 15s, 2min, 15min, 30min or 60min sample 200 μ L.

3, a new centrifuge tube is separately taken, the sodium bicarbonate that the sampling and 70 μ L concentration that step 2 is added are 0.2mol/L is water-soluble Liquid, ice bath 1min.

4, after completing step 3, the centrifuge tube is taken, 70 μ L protein sample sample-loading buffers are added, boiling water bath 5min takes out It is cooled to room temperature after centrifuge tube.

5, by the liquid and pepsin (as control) progress SDS-PAGE in the centrifuge tube for completing step 4.

Experimental result is shown in Fig. 5, (left figure is wild holstein cow, and right figure is that β-LG diallele knocks out ox, and M is albumen Marker, Pepsin are pepsin).The result shows that after simulate the gastric juice digests 60min, in non-transgenic cow milk powder β-LG albumen does not digest yet, and the β-LG albumen that β-LG diallele knocks out in milk powder is easy to digest, has low irritability.

Two, human serum is tested

The test serum that this experiment is chosen is that (anaphylactogen of patient may be more in milk for the serum of milk allergy patient Kind of allergic protein) and non-milk allergy healthy volunteer serum.

Allergen specificity antibody IgE expression in detection test serum is tested using Elisa.Suffer from if it is milk allergy Person, serum and milk powder interaction, allergen specificity antibody IgE expression can be high, and the milk that β-LG gene knockout ox generates, If the anaphylactogen of autopath is β-LG albumen, the expression that sensitization reduces IgE in then serum can be reduced. But for healthy volunteer, allergen-specific antibody in serum IgE expression does not have king-sized influence.Specific steps are such as Under: 1) non-transgenic cow milk powder with β-LG diallele is knocked out into milk powder bicarbonate buffer (pH9.6) and dissolved, and It is separately added into 96 orifice plates in (2 hole μ g/), 4 DEG C of overnight incubations；2) by 96 orifice plates with PBST solution (0.05%Tween 20/PBS) Washing is three times, each that 200 μ LBSA solution (1%BSA/PBS), 37 DEG C of incubation 1h are added；It 3), will be to be measured after PBST solution washs 3 times Serum is added separately in 96 orifice plates in (100 hole μ L/) with 1:10 dilution, and 37 DEG C of incubation 1h are simultaneously washed 3 times with PBST solution；4) with Biotin labeling is anti-human-and IgE antibody (product of Abcam company) is used as primary antibody, and rabbit source biotin antibody is as secondary antibody (Abcam The product of company), after 37 DEG C of incubation 1h, microplate reader measures IgE expression.

Experimental result is shown in A in Fig. 6.The results show that β-LG gene knockout milk powder makes ox compared with non-transgenic cow milk powder Milk autopath organizes IgE antibody expression in No. 9 and No. 10 patients serums and is remarkably decreased (P < 0.001), illustrates that β-LG very may be used It can be the main allergen of No. 9 and No. 10 patients；IgE antibody expression in 7,8, No. 11 patients serums of milk allergy patient group Decline (P < 0.01), IgE antibody expression does not have significant changes in No. 6 patients serums, thus it is speculated that β-LG be not its mainly or most Main anaphylactogen.The above result shows that β-LG protein knockout, can reduce milk sensitization.,

The total protein for extracting milk powder to be measured carries out Western blot.Specific step is as follows: 1) milk powder total protein to be measured Loading carries out SDS-PAGE respectively；2) in 350mA constant current transferring film to nitrocellulose filter；3) nitrocellulose filter is put into self-styled In bag, 1h is closed with the diluted BSA closing room temperature of 5%TBST；4) using the serum of 9, No. 10 patient more sensitive to β-LG Serum pond is blended together as primary antibody, and 4 DEG C are incubated overnight；5) with the anti-human-IgEdiluted 1:1000 (production of Abcam company of source of mouse Product) it is secondary antibody, development.

Experimental result is shown in B in Fig. 6.The result shows that with non-transgenic cow milk powder (the i.e. milk of wild type milk cow milk preparation Powder) it compares, β-LG diallele, which knocks out milk powder (i.e. the milk powder of β-LG gene knockout ox milk preparation), to be had significantly Low irritability.

Three, animal model experiment

Cleaning grade female Balb/c mouse is the product of Beijing Vital River Experimental Animals Technology Co., Ltd., quality certification number SCXK (capital) 2012-0001.

Cleaning grade normal diet is to assort the level of main nutrient composition, is processed into pellet；Then it passes through⁶0Co spoke Sterilizing is penetrated, feed is made to reach cleaning grade.The experiment that the level of cleaning grade normal diet reaches listed in GB14924-2010 is dynamic Object trophic level.

Rearing conditions: this experiment carries out in SPF grades of animal houses of China Agricultural University's food and nutrition engineering college, qualified Card number: SYXK (capital) 2010-0036.22~25 DEG C of environment temperature, relative humidity 40%~60%, 12h artificial light/dark is followed Ring, 15 times/h of rate of ventilation.With every 4, the cage nursings of group animal, ad lib and drinking-water.

Take 96, the cleaning grade female Balb/c mouse that weight is 16-20g, cleaning grade normal diet adaptable fed 5 days Afterwards, positive controls, negative control group, non-transgenic cow milk powder group 1, non-transgenic cow milk powder group 2, not are randomly divided by weight Transgenic cow milk powder group 3, β-LG diallele knock out milk powder group 1, β-LG diallele knocks out milk powder group 2 and β-LG Diallele knocks out totally 8 groups of milk powder group 3, and every group 12, being then handled as follows (needs to weigh mouse before each stomach-filling Average weight, for adjusting the concentration of stomach-filling liquid):

Positive controls: respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th day, every mouse oral is filled Stomach 0.2mL OVA solution；It tests the 42nd day, every mouse oral stomach-filling 2mL OVA solution；The concentration of OVA solution is 5mg/mL, Solvent is PBS buffer solution；

Negative control group: respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th day, every mouse oral is filled Stomach 0.2mL physiological saline；It tests the 42nd day, every mouse oral stomach-filling 2mL physiological saline；

Non- transgenic cow milk powder group 1: every small respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th day The non-transgenic cow milk power solution of the oral stomach-filling 0.2mL of mouse (using the total protein content in non-transgenic cow milk powder as tested material, dosage For 0.5mg total protein/gBW)；It tests the 42nd day, every non-transgenic cow milk power solution of mouse oral stomach-filling 0.2mL is not (to turn Total protein content in gene milk powder is tested material, and dosage is 5mg total protein/gBW)；

Non- transgenic cow milk powder group 2: every small respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th day The non-transgenic cow milk power solution of the oral stomach-filling 0.2mL of mouse (using the total protein content in non-transgenic cow milk powder as tested material, dosage For 1mg total protein/gBW)；It tests the 42nd day, every non-transgenic cow milk power solution of mouse oral stomach-filling 0.2mL is not (to turn base Because the total protein content in milk powder is tested material, dosage is 10mg total protein/gBW)；

Non- transgenic cow milk powder group 3: every small respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th day The non-transgenic cow milk power solution of the oral stomach-filling 0.2mL of mouse (using the total protein content in non-transgenic cow milk powder as tested material, dosage For 2mg total protein/gBW)；It tests the 42nd day, every non-transgenic cow milk power solution of mouse oral stomach-filling 0.2mL is not (to turn base Because the total protein content in milk powder is tested material, dosage is 20mg total protein/gBW)；

β-LG diallele knocks out milk powder group 1: respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th It, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution and (knocks out milk with β-LG diallele Total protein content in powder is tested material, and dosage is 0.5mg total protein/gBW)；It tests the 42nd day, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution, and (knocking out the total protein content in milk powder with β-LG diallele is Tested material, dosage are 5mg total protein/gBW)；

β-LG diallele knocks out milk powder group 2: respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th It, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution and (knocks out milk with β-LG diallele Total protein content in powder is tested material, and dosage is 1mg total protein/gBW)；It tests the 42nd day, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution, and (knocking out the total protein content in milk powder with β-LG diallele is Tested material, dosage are 10mg total protein/gBW)；

β-LG diallele knocks out milk powder group 3: respectively in experiment the 0th day, the 7th day, the 14th day, the 21st day and the 28th It, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution and (knocks out milk with β-LG diallele Total protein content in powder is tested material, and dosage is 2mg total protein/gBW)；It tests the 42nd day, every mouse oral stomach-filling 0.2mL β-LG diallele knocks out milk powder solution, and (knocking out the total protein content in milk powder with β-LG diallele is Tested material, dosage are 20mg total protein/gBW).

During test, upgrowth situation (figure and features, feed amount and spiritual shape including mouse of mouse are observed weekly State etc.).

When testing the 42nd -day-old of stimulation (i.e. dosage increases to 1- times and stimulated), appropriate vaseline is smeared using probe Electronic thermometer measures Temperature changing of the mouse before and after stimulation.As a result see that (wild type milk is non-transgenic cow milk powder to Fig. 7 Group 1, non-transgenic cow milk powder group 2 and non-transgenic cow milk powder group 3 are as repeating to be averaged three times as a result, β-LG clpp gene Except milk is that β-LG diallele knocks out milk powder group 1, β-LG diallele knocks out milk powder group 2 and the double equipotential bases of β-LG Because knocking out milk powder group 3 as the result for repeating to be averaged three times).The result shows that β-LG diallele knocks out milk powder The anal intestine temperature change of administering transgenic mouse is significantly lower than non-transgenic cow milk powder, further illustrates that β-LG diallele knocks out The sensitization of milk powder reduces.

The above results show that β-LG diallele knocks out milk powder and milk sensitization is greatly lowered.

<110>China Agricultural University

<120>a kind of method and its application for the ox for cultivating production low irritability milk

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 4894

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 1

tcctgcttgg cctcgaatgg aagaaggcct cctattgtcc tcgtagagga agcaacccca 60

gggcccaagg ataggccagg ggggattcgg ggaaccgcgt ggctgggggc ccggcccggg 120

ctggctggct ggccctcctc ctgtataagg ccccgagccc actgtctcag ccctccactc 180

cctgcagagc tcagaagcgt gaccccagct gcagccatga agtgcctcct gcttgccctg 240

gccctcactt gtggcgccca ggccctcatt gtcacccaga ccatgaaggg cctggatatc 300

cagaaggttc gagggtgccc gggtgggtgg tgagttgcag ggcaggcagg ggagctgggc 360

ctcagagacc aagggaggct gtgacgtctg ggattcccat cagtcagcta gagccgcctg 420

acaaatcgcc cgccagggca gcttcaacca ggcgtttagt gtcttgcatt ctggaggctg 480

gaagcctgca atccgggcat cggcccagct ggcttctcct gcggccactc tccggggagc 540

agacagccat cttctccctg tgtcctttgc gtgccctggt ttcctcttcc tgtgaggtca 600

ccaggcctgc tggatccacg cccgcccaca cagcctcacg taacctttgt catctcttta 660

aaggccgtgt ctccagtcct gtgttgaggt tctgggggtt aatgggacac agttcagccc 720

ctaaaagagt cactctgccc ctcaaatttt ccccacctcc agctatgtct ccccaagatc 780

caaatgttgc cacgtgtgcg ggggctcatc tgggtccctc tttgggctca gagtgagtct 840

ggggagagca ttcctcaggg tgccgagttg gggggagcat ctcagggctg cccaggccag 900

ggtgggacag agagcccact gtggggctgg gggccccttc ccgcccctgg agtgcagctc 960

aaggtccctc cccaggtggc ggggacttgg tactccttgg ccatggcggc cagcgacatc 1020

tccctgctgg acgcccagag tgcccccctg agagtgtatg tggaggagct gaagcccacc 1080

cctgagggcg acctggagat cctgctgcag aaatggtggg cgtccccccc aaaaaaagca 1140

tggaaccccc actccccagg gatatggacc cccccggggt ggggtgcagg agggaccagg 1200

gccccagggc tggggaacgg ggcttggagt ttcctggtac ccctggaggt ccacccaagg 1260

ctgcttatcc agggctttct ctttcttttt ttcccccaac ttttattaat ttgatgcttc 1320

agaacatcat caaacaaatg aacacaaaac atcattttcg ttaacttgga aggggagata 1380

aaatccactg aagtggaaat gcataggaaa gatacataca gtaaggcagg tattctgaat 1440

tcgctgttag tttgaggatt acaaatgcac ttgagcaaca gagagacgtt ttcattattt 1500

ctggtctgaa cagctcagta tctaaaatga acaagatgtc atggagacaa agccggcggg 1560

ggagaggccc gtgtgaaggc cgctgggcgg ctgcagacct gggtcctcgg ggcccaggca 1620

gttcccacta ccagccctgt ccaccctcag acgggggtca gagtgcagga gagagctggg 1680

tgggtgtggg ggcagagatg gggacctgaa ccccaggact gccttttggg gtgcctgtgg 1740

tcaaggctct ccccaacctt ttctccctgg ctccatctga cttctcctgg cccatccacc 1800

cggtcacctg tggccccaga ggtgacagtg agtgcagcca aggccggttg gccagccggc 1860

cccctatgcc cacgccaccc gcctccagcc cctcctgggg ccgccttctg cccctggccc 1920

tcagttcatc ctgatgaaaa tggtccatgc ccgtggctca gaaagcagct gtctttcagg 1980

gagaacggtg agtgtgctca gaagaagatc attgcagaaa aaaccaagat ccctgcggtg 2040

ttcaagatcg atggtgagtg ctgggtcccc aggggacgcc caccaccccc cagggactgt 2100

gggcaggtgc agggggctgg cgtcaggccc cgagatgcta aggggctggt ggtgatgaag 2160

acactgccgt gccacctgct tccctggcct ccctgccacc tgcccggggc cttggggccg 2220

gtggccgtgg gcaggtcccg gctgggcagg tctgacaccc cagggtgaca cccgagctct 2280

ctttgctgag ggtggggtgg tgctcggggc cctcaggctg agctcaggag gtccctgtgc 2340

ccacccaggg gtaaccgaga gccgctgccc gctccagggg tccaggtgcc ccacgatccc 2400

agcccacccc acggctcctt catctcctga agacgaactc tgtccgccct cgctcattca 2460

cttgtttgtc ctaaatccaa gatgagaaag cttcgaggtg gggttggggt tccatcaggg 2520

cctgcccttc cgccgggcag cctgggccac atctgccctt ggcctcttca ggactcactc 2580

tgactggagg ccctgcactg actgatgcca gggtgcccag cccagggtct cctgtgccat 2640

ccggctgcac ggggtttgga tgctggtcct gcccccaagc tgcccagaca ctgcagggca 2700

gctggggcca cccgcaggcc tcggtcaggg agagccccag ctgcccccgc tcagcgctgc 2760

cccccaacaa ttccccagtc ctcaggacgc atccctcttc ccttgctggg cagtgttcag 2820

ccccacccga gatcggggga agccctattt cttgaccact ccggtccctg ggggagggcg 2880

gcctcagact gagtggtgag tgttcccaag tccaggaggt ggtggagggt ccctggcgga 2940

tccagagttg ggcttccaga gtgagggctt cctgggcccc atgtgcctgg cagtggcagc 3000

agggaagggg ccacaccatt ttggggctgg gggatgccag agggcgctcc ccaccccgtc 3060

ctcaccaagt ggtgaccccg ggggagcccc gctggttgtg gggggcgctg ggggctgacc 3120

agaaaccccc ctcctgctgg aactcacttt cctcctgtct tgatctctac cagccttgaa 3180

cgagaacaaa gtccttgtgc tggacaccga ctacaaaaag tacctgctct tctgcatgga 3240

gaacagtgct gagcccgagc aaagcctggt ctgccagtgc ctgggtgggt gccaaccctg 3300

gctgcccagg gagaccagct gtgtggtcct cgctgcaacg gggccggggg ggacggtggg 3360

agcagggagc ttgattccca ggaggaggag ggatgggggg tccccgagtc ccgccaggag 3420

agggtggtca tataccggga gccggtgtcc tgggggtctg tgggtgactg gggacggggg 3480

ccagacacac aggctgggag acggggggct gcagcgctct ggtgtgacca tcacgatgga 3540

gccggcggtc actatgaatc taacagcctt tgttaccggg gagtttcaat tatttcatca 3600

aataagaact caggcacaaa gctgtctttc aactgtcacg tcctgaaaac aaatggcagg 3660

tgacattttc catgccatag cagtgccact gggcattttc agggcccatg tgccaggagg 3720

gcgtgggcat cggcgagtgg aggctcctgg ccgtgtcagc tggcccaggg ggaggagggg 3780

acccagacag ccagaggtgg ggagcaggct ttccccctgt gacgctgcag acccaccgca 3840

ctgccctggg aggaagggga gggaactggg ccaaggggga agggcaggtg tgctggaggc 3900

caaggcagac ctgcacacca ccctggagag caggggttga ccccgtcccg gccccacagt 3960

caggaccccg gaggtggacg acgaggccct ggagaaattc gacaaagccc tcaaggccct 4020

gcccatgcac atccggctgt ccttcaaccc aacccagctg gagggtgagc acccaggccc 4080

caccctgctc ctggggcagg aagccacccg gcccaggacc acctcctccc atggtgaccc 4140

ccagctcccc aggcctcccg ggaggatgga gacggggtgc agggccccga ggtggccccc 4200

tccccacccc ctccccagct ccctctgtcc tggggtgtcc agtcccatcc tgacgctccc 4260

ccgccacggc tctccctccc ccacagagca gtgccacatc taggtgagcc cctgccggcg 4320

cctctggggt aagctgcctg ccctgcccca cgtcctgggc acacacatgg ggtaggggtt 4380

cttggttggg cccgggagcc cccattaggc cctggggtcc ccccgtagga atggctggaa 4440

gctggggtcc ttcctggaga ctacagagcc ggctggccac atgctcgctc ttgtggggtg 4500

acctgtgtcc tggcctcact cacacgctga tctcctccac ctccttcctg gcagacctaa 4560

gggccaaggt ggaggctcag gaagtggaca cctaaggggg aggctagggg ggtccttctc 4620

ccaaggaggg gccgtcctga atccccagcc acggacaggc tggcaagggt ctggcaggta 4680

ccccaggaat cacaggggag ccccatgtcc atttcagagc ccgggagcct tggcccctct 4740

ggggacagac gatgtcatcc ccgcctgccc catcagggga ccaggaggaa ccgggaccac 4800

attcacccct cctgggaccc aggcccctcc aggcccctcc tggggcctcc tgcttggggc 4860

cgctcctcct tcagcaataa aggcataaac ctgt 4894

<210> 2

<211> 4878

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 2

tcctgcttgg cctcgaatgg aagaaggcct cctattgtcc tcgtagagga agcaacccca 60

gggcccaagg ataggccagg ggggattcgg ggaaccgcgt ggctgggggc ccggcccggg 120

ctggctggct ggccctcctc ctgtataagg ccccgagccc actgtctcag ccctccactc 180

cctgcagagc tcagaagcgt gaccccagct gcagccatga agtgcctcct gcttgccctg 240

gccctcactt gtggcgccca ggccccatga agggcctgga tatccagaag gttcgagggt 300

gcccgggtgg gtggtgagtt gcagggcagg caggggagct gggcctcaga gaccaaggga 360

ggctgtgacg tctgggattc ccatcagtca gctagagccg cctgacaaat cgcccgccag 420

ggcagcttca accaggcgtt tagtgtcttg cattctggag gctggaagcc tgcaatccgg 480

gcatcggccc agctggcttc tcctgcggcc actctccggg gagcagacag ccatcttctc 540

cctgtgtcct ttgcgtgccc tggtttcctc ttcctgtgag gtcaccaggc ctgctggatc 600

cacgcccgcc cacacagcct cacgtaacct ttgtcatctc tttaaaggcc gtgtctccag 660

tcctgtgttg aggttctggg ggttaatggg acacagttca gcccctaaaa gagtcactct 720

gcccctcaaa ttttccccac ctccagctat gtctccccaa gatccaaatg ttgccacgtg 780

tgcgggggct catctgggtc cctctttggg ctcagagtga gtctggggag agcattcctc 840

agggtgccga gttgggggga gcatctcagg gctgcccagg ccagggtggg acagagagcc 900

cactgtgggg ctgggggccc cttcccgccc ctggagtgca gctcaaggtc cctccccagg 960

tggcggggac ttggtactcc ttggccatgg cggccagcga catctccctg ctggacgccc 1020

agagtgcccc cctgagagtg tatgtggagg agctgaagcc cacccctgag ggcgacctgg 1080

agatcctgct gcagaaatgg tgggcgtccc ccccaaaaaa agcatggaac ccccactccc 1140

cagggatatg gacccccccg gggtggggtg caggagggac cagggcccca gggctgggga 1200

acggggcttg gagtttcctg gtacccctgg aggtccaccc aaggctgctt atccagggct 1260

ttctctttct ttttttcccc caacttttat taatttgatg cttcagaaca tcatcaaaca 1320

aatgaacaca aaacatcatt ttcgttaact tggaagggga gataaaatcc actgaagtgg 1380

aaatgcatag gaaagataca tacagtaagg caggtattct gaattcgctg ttagtttgag 1440

gattacaaat gcacttgagc aacagagaga cgttttcatt atttctggtc tgaacagctc 1500

agtatctaaa atgaacaaga tgtcatggag acaaagccgg cgggggagag gcccgtgtga 1560

aggccgctgg gcggctgcag acctgggtcc tcggggccca ggcagttccc actaccagcc 1620

ctgtccaccc tcagacgggg gtcagagtgc aggagagagc tgggtgggtg tgggggcaga 1680

gatggggacc tgaaccccag gactgccttt tggggtgcct gtggtcaagg ctctccccaa 1740

ccttttctcc ctggctccat ctgacttctc ctggcccatc cacccggtca cctgtggccc 1800

cagaggtgac agtgagtgca gccaaggccg gttggccagc cggcccccta tgcccacgcc 1860

acccgcctcc agcccctcct ggggccgcct tctgcccctg gccctcagtt catcctgatg 1920

aaaatggtcc atgcccgtgg ctcagaaagc agctgtcttt cagggagaac ggtgagtgtg 1980

ctcagaagaa gatcattgca gaaaaaacca agatccctgc ggtgttcaag atcgatggtg 2040

agtgctgggt ccccagggga cgcccaccac cccccaggga ctgtgggcag gtgcaggggg 2100

ctggcgtcag gccccgagat gctaaggggc tggtggtgat gaagacactg ccgtgccacc 2160

tgcttccctg gcctccctgc cacctgcccg gggccttggg gccggtggcc gtgggcaggt 2220

cccggctggg caggtctgac accccagggt gacacccgag ctctctttgc tgagggtggg 2280

gtggtgctcg gggccctcag gctgagctca ggaggtccct gtgcccaccc aggggtaacc 2340

gagagccgct gcccgctcca ggggtccagg tgccccacga tcccagccca ccccacggct 2400

ccttcatctc ctgaagacga actctgtccg ccctcgctca ttcacttgtt tgtcctaaat 2460

ccaagatgag aaagcttcga ggtggggttg gggttccatc agggcctgcc cttccgccgg 2520

gcagcctggg ccacatctgc ccttggcctc ttcaggactc actctgactg gaggccctgc 2580

actgactgat gccagggtgc ccagcccagg gtctcctgtg ccatccggct gcacggggtt 2640

tggatgctgg tcctgccccc aagctgccca gacactgcag ggcagctggg gccacccgca 2700

ggcctcggtc agggagagcc ccagctgccc ccgctcagcg ctgcccccca acaattcccc 2760

agtcctcagg acgcatccct cttcccttgc tgggcagtgt tcagccccac ccgagatcgg 2820

gggaagccct atttcttgac cactccggtc cctgggggag ggcggcctca gactgagtgg 2880

tgagtgttcc caagtccagg aggtggtgga gggtccctgg cggatccaga gttgggcttc 2940

cagagtgagg gcttcctggg ccccatgtgc ctggcagtgg cagcagggaa ggggccacac 3000

cattttgggg ctgggggatg ccagagggcg ctccccaccc cgtcctcacc aagtggtgac 3060

cccgggggag ccccgctggt tgtggggggc gctgggggct gaccagaaac ccccctcctg 3120

ctggaactca ctttcctcct gtcttgatct ctaccagcct tgaacgagaa caaagtcctt 3180

gtgctggaca ccgactacaa aaagtacctg ctcttctgca tggagaacag tgctgagccc 3240

gagcaaagcc tggtctgcca gtgcctgggt gggtgccaac cctggctgcc cagggagacc 3300

agctgtgtgg tcctcgctgc aacggggccg ggggggacgg tgggagcagg gagcttgatt 3360

cccaggagga ggagggatgg ggggtccccg agtcccgcca ggagagggtg gtcatatacc 3420

gggagccggt gtcctggggg tctgtgggtg actggggacg ggggccagac acacaggctg 3480

ggagacgggg ggctgcagcg ctctggtgtg accatcacga tggagccggc ggtcactatg 3540

aatctaacag cctttgttac cggggagttt caattatttc atcaaataag aactcaggca 3600

caaagctgtc tttcaactgt cacgtcctga aaacaaatgg caggtgacat tttccatgcc 3660

atagcagtgc cactgggcat tttcagggcc catgtgccag gagggcgtgg gcatcggcga 3720

gtggaggctc ctggccgtgt cagctggccc agggggagga ggggacccag acagccagag 3780

gtggggagca ggctttcccc ctgtgacgct gcagacccac cgcactgccc tgggaggaag 3840

gggagggaac tgggccaagg gggaagggca ggtgtgctgg aggccaaggc agacctgcac 3900

accaccctgg agagcagggg ttgaccccgt cccggcccca cagtcaggac cccggaggtg 3960

gacgacgagg ccctggagaa attcgacaaa gccctcaagg ccctgcccat gcacatccgg 4020

ctgtccttca acccaaccca gctggagggt gagcacccag gccccaccct gctcctgggg 4080

caggaagcca cccggcccag gaccacctcc tcccatggtg acccccagct ccccaggcct 4140

cccgggagga tggagacggg gtgcagggcc ccgaggtggc cccctcccca ccccctcccc 4200

agctccctct gtcctggggt gtccagtccc atcctgacgc tcccccgcca cggctctccc 4260

tcccccacag agcagtgcca catctaggtg agcccctgcc ggcgcctctg gggtaagctg 4320

cctgccctgc cccacgtcct gggcacacac atggggtagg ggttcttggt tgggcccggg 4380

agcccccatt aggccctggg gtccccccgt aggaatggct ggaagctggg gtccttcctg 4440

gagactacag agccggctgg ccacatgctc gctcttgtgg ggtgacctgt gtcctggcct 4500

cactcacacg ctgatctcct ccacctcctt cctggcagac ctaagggcca aggtggaggc 4560

tcaggaagtg gacacctaag ggggaggcta ggggggtcct tctcccaagg aggggccgtc 4620

ctgaatcccc agccacggac aggctggcaa gggtctggca ggtaccccag gaatcacagg 4680

ggagccccat gtccatttca gagcccggga gccttggccc ctctggggac agacgatgtc 4740

atccccgcct gccccatcag gggaccagga ggaaccggga ccacattcac ccctcctggg 4800

acccaggccc ctccaggccc ctcctggggc ctcctgcttg gggccgctcc tccttcagca 4860

ataaaggcat aaacctgt 4878

<210> 3

<211> 4877

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 3

tcctgcttgg cctcgaatgg aagaaggcct cctattgtcc tcgtagagga agcaacccca 60

gggcccaagg ataggccagg ggggattcgg ggaaccgcgt ggctgggggc ccggcccggg 120

ctggctggct ggccctcctc ctgtataagg ccccgagccc actgtctcag ccctccactc 180

cctgcagagc tcagaagcgt gaccccagct gcagccatga agtgcctcct gcttgccctg 240

gccctcactt gtggcgccca ggcccatgaa gggcctggat atccagaagg ttcgagggtg 300

cccgggtggg tggtgagttg cagggcaggc aggggagctg ggcctcagag accaagggag 360

gctgtgacgt ctgggattcc catcagtcag ctagagccgc ctgacaaatc gcccgccagg 420

gcagcttcaa ccaggcgttt agtgtcttgc attctggagg ctggaagcct gcaatccggg 480

catcggccca gctggcttct cctgcggcca ctctccgggg agcagacagc catcttctcc 540

ctgtgtcctt tgcgtgccct ggtttcctct tcctgtgagg tcaccaggcc tgctggatcc 600

acgcccgccc acacagcctc acgtaacctt tgtcatctct ttaaaggccg tgtctccagt 660

cctgtgttga ggttctgggg gttaatggga cacagttcag cccctaaaag agtcactctg 720

cccctcaaat tttccccacc tccagctatg tctccccaag atccaaatgt tgccacgtgt 780

gcgggggctc atctgggtcc ctctttgggc tcagagtgag tctggggaga gcattcctca 840

gggtgccgag ttggggggag catctcaggg ctgcccaggc cagggtggga cagagagccc 900

actgtggggc tgggggcccc ttcccgcccc tggagtgcag ctcaaggtcc ctccccaggt 960

ggcggggact tggtactcct tggccatggc ggccagcgac atctccctgc tggacgccca 1020

gagtgccccc ctgagagtgt atgtggagga gctgaagccc acccctgagg gcgacctgga 1080

gatcctgctg cagaaatggt gggcgtcccc cccaaaaaaa gcatggaacc cccactcccc 1140

agggatatgg acccccccgg ggtggggtgc aggagggacc agggccccag ggctggggaa 1200

cggggcttgg agtttcctgg tacccctgga ggtccaccca aggctgctta tccagggctt 1260

tctctttctt tttttccccc aacttttatt aatttgatgc ttcagaacat catcaaacaa 1320

atgaacacaa aacatcattt tcgttaactt ggaaggggag ataaaatcca ctgaagtgga 1380

aatgcatagg aaagatacat acagtaaggc aggtattctg aattcgctgt tagtttgagg 1440

attacaaatg cacttgagca acagagagac gttttcatta tttctggtct gaacagctca 1500

gtatctaaaa tgaacaagat gtcatggaga caaagccggc gggggagagg cccgtgtgaa 1560

ggccgctggg cggctgcaga cctgggtcct cggggcccag gcagttccca ctaccagccc 1620

tgtccaccct cagacggggg tcagagtgca ggagagagct gggtgggtgt gggggcagag 1680

atggggacct gaaccccagg actgcctttt ggggtgcctg tggtcaaggc tctccccaac 1740

cttttctccc tggctccatc tgacttctcc tggcccatcc acccggtcac ctgtggcccc 1800

agaggtgaca gtgagtgcag ccaaggccgg ttggccagcc ggccccctat gcccacgcca 1860

cccgcctcca gcccctcctg gggccgcctt ctgcccctgg ccctcagttc atcctgatga 1920

aaatggtcca tgcccgtggc tcagaaagca gctgtctttc agggagaacg gtgagtgtgc 1980

tcagaagaag atcattgcag aaaaaaccaa gatccctgcg gtgttcaaga tcgatggtga 2040

gtgctgggtc cccaggggac gcccaccacc ccccagggac tgtgggcagg tgcagggggc 2100

tggcgtcagg ccccgagatg ctaaggggct ggtggtgatg aagacactgc cgtgccacct 2160

gcttccctgg cctccctgcc acctgcccgg ggccttgggg ccggtggccg tgggcaggtc 2220

ccggctgggc aggtctgaca ccccagggtg acacccgagc tctctttgct gagggtgggg 2280

tggtgctcgg ggccctcagg ctgagctcag gaggtccctg tgcccaccca ggggtaaccg 2340

agagccgctg cccgctccag gggtccaggt gccccacgat cccagcccac cccacggctc 2400

cttcatctcc tgaagacgaa ctctgtccgc cctcgctcat tcacttgttt gtcctaaatc 2460

caagatgaga aagcttcgag gtggggttgg ggttccatca gggcctgccc ttccgccggg 2520

cagcctgggc cacatctgcc cttggcctct tcaggactca ctctgactgg aggccctgca 2580

ctgactgatg ccagggtgcc cagcccaggg tctcctgtgc catccggctg cacggggttt 2640

ggatgctggt cctgccccca agctgcccag acactgcagg gcagctgggg ccacccgcag 2700

gcctcggtca gggagagccc cagctgcccc cgctcagcgc tgccccccaa caattcccca 2760

gtcctcagga cgcatccctc ttcccttgct gggcagtgtt cagccccacc cgagatcggg 2820

ggaagcccta tttcttgacc actccggtcc ctgggggagg gcggcctcag actgagtggt 2880

gagtgttccc aagtccagga ggtggtggag ggtccctggc ggatccagag ttgggcttcc 2940

agagtgaggg cttcctgggc cccatgtgcc tggcagtggc agcagggaag gggccacacc 3000

attttggggc tgggggatgc cagagggcgc tccccacccc gtcctcacca agtggtgacc 3060

ccgggggagc cccgctggtt gtggggggcg ctgggggctg accagaaacc cccctcctgc 3120

tggaactcac tttcctcctg tcttgatctc taccagcctt gaacgagaac aaagtccttg 3180

tgctggacac cgactacaaa aagtacctgc tcttctgcat ggagaacagt gctgagcccg 3240

agcaaagcct ggtctgccag tgcctgggtg ggtgccaacc ctggctgccc agggagacca 3300

gctgtgtggt cctcgctgca acggggccgg gggggacggt gggagcaggg agcttgattc 3360

ccaggaggag gagggatggg gggtccccga gtcccgccag gagagggtgg tcatataccg 3420

ggagccggtg tcctgggggt ctgtgggtga ctggggacgg gggccagaca cacaggctgg 3480

gagacggggg gctgcagcgc tctggtgtga ccatcacgat ggagccggcg gtcactatga 3540

atctaacagc ctttgttacc ggggagtttc aattatttca tcaaataaga actcaggcac 3600

aaagctgtct ttcaactgtc acgtcctgaa aacaaatggc aggtgacatt ttccatgcca 3660

tagcagtgcc actgggcatt ttcagggccc atgtgccagg agggcgtggg catcggcgag 3720

tggaggctcc tggccgtgtc agctggccca gggggaggag gggacccaga cagccagagg 3780

tggggagcag gctttccccc tgtgacgctg cagacccacc gcactgccct gggaggaagg 3840

ggagggaact gggccaaggg ggaagggcag gtgtgctgga ggccaaggca gacctgcaca 3900

ccaccctgga gagcaggggt tgaccccgtc ccggccccac agtcaggacc ccggaggtgg 3960

acgacgaggc cctggagaaa ttcgacaaag ccctcaaggc cctgcccatg cacatccggc 4020

tgtccttcaa cccaacccag ctggagggtg agcacccagg ccccaccctg ctcctggggc 4080

aggaagccac ccggcccagg accacctcct cccatggtga cccccagctc cccaggcctc 4140

ccgggaggat ggagacgggg tgcagggccc cgaggtggcc ccctccccac cccctcccca 4200

gctccctctg tcctggggtg tccagtccca tcctgacgct cccccgccac ggctctccct 4260

cccccacaga gcagtgccac atctaggtga gcccctgccg gcgcctctgg ggtaagctgc 4320

ctgccctgcc ccacgtcctg ggcacacaca tggggtaggg gttcttggtt gggcccggga 4380

gcccccatta ggccctgggg tccccccgta ggaatggctg gaagctgggg tccttcctgg 4440

agactacaga gccggctggc cacatgctcg ctcttgtggg gtgacctgtg tcctggcctc 4500

actcacacgc tgatctcctc cacctccttc ctggcagacc taagggccaa ggtggaggct 4560

caggaagtgg acacctaagg gggaggctag gggggtcctt ctcccaagga ggggccgtcc 4620

tgaatcccca gccacggaca ggctggcaag ggtctggcag gtaccccagg aatcacaggg 4680

gagccccatg tccatttcag agcccgggag ccttggcccc tctggggaca gacgatgtca 4740

tccccgcctg ccccatcagg ggaccaggag gaaccgggac cacattcacc cctcctggga 4800

cccaggcccc tccaggcccc tcctggggcc tcctgcttgg ggccgctcct ccttcagcaa 4860

taaaggcata aacctgt 4877

<210> 4

<211> 4877

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 4

cccaggccct cattgtcacc cagaccatga agggcctgga 40

<210> 5

<211> 252

<212> PRT

<213>artificial sequence

<220>

<223>

<400> 5

Met Lys Cys Leu Leu Leu Ala Leu Ala Leu Thr Cys Gly Ala Gln Ala

1 5 10 15

Leu Ile Val Thr Gln Thr Met Lys Gly Leu Asp Ile Gln Lys Val Ala

20 25 30

Gly Thr Trp Tyr Ser Leu Ala Met Ala Ala Ser Asp Ile Ser Leu Leu

35 40 45

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

50 55 60

Thr Pro Glu Gly Asp Leu Glu Ile Leu Leu Gln Lys Trp Glu Asn Gly

65 70 75 80

Glu Cys Ala Gln Lys Lys Ile Ile Ala Glu Lys Thr Lys Ile Pro Ala

85 90 95

Val Phe Lys Ile Asp Ala Leu Asn Glu Asn Lys Val Leu Val Leu Asp

100 105 110

Thr Asp Tyr Lys Lys Tyr Leu Leu Phe Cys Met Glu Asn Ser Ala Glu

115 120 125

Pro Glu Gln Ser Leu Ala Cys Gln Cys Leu Val Arg Thr Pro Glu Val

130 135 140

Asp Asp Glu Ala Leu Glu Lys Phe Asp Lys Ala Leu Lys Ala Leu Pro

145 150 155 160

Met His Ile Arg Leu Ser Phe Asn Pro Thr Gln Leu Glu Gly Glu His

165 170 175

Pro Gly Pro Thr Leu Leu Leu Gly Gln Glu Ala Thr Arg Pro Arg Thr

180 185 190

Thr Ser Ser His Gly Asp Pro Gln Leu Pro Arg Pro Pro Gly Arg Met

195 200 205

Glu Thr Gly Cys Arg Ala Pro Arg Trp Pro Pro Pro His Pro Leu Pro

210 215 220

Ser Ser Leu Cys Pro Gly Val Ser Ser Pro Ile Leu Thr Leu Pro Arg

225 230 235 240

His Gly Ser Pro Ser Pro Thr Glu Gln Cys His Ile

245 250

Claims (10)

1. the method for cultivating the ox of production low irritability milk includes the following steps: to inhibit the β-LG albumen in cow genome group that sets out Synthesis and/or expression quantity and/or activity, obtain transgenic cow；Compared with the ox that sets out, the milk sensitization of transgenic cow production It reduces.

2. the method as described in claim 1, it is characterised in that: described " inhibition is set out the synthesis of β-LG albumen in cow genome group And/or expression quantity and/or activity " carried out by way of gene site-directed editor, RNA interference, homologous recombination or gene knockout；Institute Gene site-directed editor is stated to carry out using cow genome group editor's carrier.

3. method according to claim 1 or 2, it is characterised in that: described " inhibition is set out the conjunction of β-LG albumen in cow genome group At and/or expression quantity and/or activity " be to send out the nucleic acid molecule of the β-LG gene on two homologues for the ox that sets out Raw frameshift mutation.

4. the method for cultivating the ox of production low irritability milk, including step (1): importing cow genome group to bovine fibroblasts and compile Carrier is collected, the donorcells that genotype is diallele saltant type are obtained；

The diallele saltant type is that the nucleic acid molecule of the β-LG gene on two homologues of ox changes, It is this to change the synthesis for inhibiting β-LG albumen and/or expression quantity and/or activity.

5. method as claimed in claim 4, it is characterised in that: it is described " β-LG gene on two homologues of ox Nucleic acid molecule changes " be ox two homologues on β-LG gene nucleic acid molecule occurs frameshit dash forward Become.

6. method as described in claim 4 or 5, it is characterised in that: the method also includes step (2): completing step (1) Afterwards, the nucleus of the donorcells is moved into the bovine oocyte for removing nucleus, development forms reconstruct embryo, then moves into Cow uteri, childbirth obtain the ox of production low irritability milk.

7. the method as described in claim 2,4,5 or 6, it is characterised in that: cow genome group editor's carrier is zinc finger nucleic acid Zymophore pZFN；The target DNA that the Zinc finger nuclease carrier pZFN is identified in cow genome group is the DNA for encoding β-LG albumen Segment.

8. a kind of method of detection or auxiliary detection milk sensitization, includes the following steps: two homologous dyes for detecting ox to be measured β-LG gene on colour solid is wild type or diallele saltant type, and genotype is the ox to be measured of diallele saltant type The sensitization of the milk of production is lower than or the candidate ox to be measured lower than genotype for wild type；

The diallele saltant type is that the nucleic acid molecule of the β-LG gene on two homologues of ox moves Code mutation；The wild type does not occur frameshit for the nucleic acid molecule of the β-LG gene on two homologues of ox and dashes forward Become.

9. the Zinc finger nuclease carrier pZFN of cow genome group editor a kind of, it is characterised in that: the Zinc finger nuclease carrier pZFN The target DNA identified in cow genome group is the DNA fragmentation for encoding β-LG albumen.

10. the nucleic acid molecule of the β-LG gene on two homologues of ox to that the method or production of frameshift mutation occur Product will inhibit the synthesis of β-LG albumen and/or expression quantity and/or active method or product in cow genome group, give birth to cultivating It produces the ox of low irritability milk and/or reduces milk sensitization and/or produce the application in low irritability milk.