CN1058994C - High effect expressing carrier for animal mammary gland tissue specificity - Google Patents

Abstract

The present invention discloses a vector pBCD for the specificity expression of exogenous genes in the mammary tissues of animals. The vector contains sheep BLG genes, expression control sequences of the sheep BLG genes, mouse STAT sequences and polylinker DNA fragments, wherein the sheep BLG genes control the specificity expression of exogenous genes in the mammary tissues, the mouse STAT sequences drive the exogenous genes to highly efficiently synthesize protein, and the polylinker DNA fragments facilitating the insertion of the exogenous genes into the vector is positioned in the lower reaches of sheep BLG gene promoters and in the upper reaches of a first genetic codon ATG of lactoglobulin genes. The present invention also discloses a method for the expression of the exogenous genes (such as human growth hormones) in the mammary tissues of animals through the vector.

Description

High effect expressing carrier for animal mammary gland tissue specificity

The present invention relates to a kind of carrier that makes foreign gene specifically expressing in the animal's mammary gland tissue, specifically a kind of proteinic biological elements that can drive foreign gene at animal's mammary gland tissue rather than synthetic this genes encoding of other tissue.It comprises the regulating and controlling sequence of control foreign gene at the sheep BLG of mammary tissue specifically expressing gene, drive the mouse STAT sequence of exogenous gene high-efficient synthetic protein, be convenient to pack into the synthetic DNA fragment of carrier of foreign gene, and the P that duplicates in intestinal bacteria together with the foreign gene that inserts on it for carrier ^BluescriptThe SK sequence.

Mammary tissue is the zooblast of a group specialization, and major function is synthetic milk-protein.The ability of its synthetic protein is very strong, surpasses any other tissue of animal body.A good milk cow can produce 300 kilograms of milk-proteins in 1 year, and a sheep can produce 30 kilograms of milk-proteins in 1 year, even a family exempts from also can produce in 1 year 1.2-1.5 kilogram protein.Therefore, if adopt transgenic technology with foreign gene, particularly coding has the gene of medical value, as the human interferon gene, human insulin gene, human growth hormone gene or human erythropoietin gene etc. change in the animal body, just might create a kind of production system of producing these medical protein matter.Its advantage is the output height, cost is low and quality product near natural extract (Andrew S.et.al.1993, Biotechnology, Vol.11 1263-1269).But foreign gene changes over to after the animal, there is randomness in its insertion place and expression place, if exogenous genes products has strong physiological function, can hemopoietic cell proliferation as erythropoietin, foreign gene is arbitrarily expressed the meeting kill animals, can not extract easily, reach the purpose of utilization.

The objective of the invention is to make up a kind of carrier that has the mammary tissue specificity and the efficiently expressing exogenous gene ability is arranged, make foreign gene be contained on the carrier of the present invention and when changing animal over to, can be in animal's mammary gland the coded protein of this foreign gene of mass production.

The unique distinction of the carrier that the present invention is constructed mainly is: the first, and it has comprised whole controlling elements of sheep lactoglobulin gene (BLG), guarantees that foreign gene, can be at other any tissue expression only in the mammary tissue expression; The second, in the both sides of sheep lactoglobulin gene, assembled the dna sequence dna (STAT sequence) of mouse, the function of this sequence is to guarantee the expression of exogenous gene high-efficient rate ground; The 3rd, before first genetic code of sheep lactoglobulin gene, inserted the DNA of one section synthetic, its concrete sequence is 5 ' AAGTCGACAACCCGGGAAATCGATAA3 ', contain SalI in this section sequence, the site of SmaI and three restriction enzymes of ClaI guarantees that foreign gene inserts after among above-mentioned three restriction enzyme sites any one, can express prior to sheep lactoglobulin gene in mammary gland.The acting in conjunction of the various components of this carrier, be the foreign gene that guarantees to be contained in its top after the mammary gland cell that changes animal or isolated culture over to, can both obtain the specific and high efficiency expression of mammary tissue.No matter they are inserted into which the bar karyomit(e) of animal, no matter it is inserted into which concrete site on the karyomit(e), the tissue specificity of its expression and expression high-efficiency characteristics all can not disappear yet.The other parts of carrier are that institute is necessary when supplying carrier and contained foreign gene thereof to increase in intestinal bacteria, are not that the present invention is distinctive.

One. be used for BLG promoter expression vector (pBCD) that mouse and sheep mammar gland express referring to accompanying drawing 1:

Fig. 1 is the structure of pBCD mammary tissue specific efficient expression vector of the present invention, the cloning site of carrier pBluescript sk is NotI, gray area is polylinker joint (containing SalI, SmaI and CLaI site) among the figure, is inserted in the PvuII site of BLG.Can be in the required expressed exogenous gene of Insert Here.Black region is 5 ' mouse chromosome fragment (containing the STAT sequence that karyomit(e) is unwind) among the figure, 4.6kb.The left side of adjacent black region tiltedly drawn area is sheep BLG5 ' zone, 4.5kb.Right tiltedly scribe area is sheep BLG3 ' zone among the figure, 8.3kb.White portion among the figure is 3 ' mouse chromosome fragment (containing the STAT sequence that karyomit(e) is unwind), 5.0kb.

PBCD of the present invention (CGMCC NO 0312) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 18th, 1997.

The preparation of two .pBCD

1. each several part is originated

A. polylinker

Artificial synthetic oligonucleotide's 5 ' AAGTCGACAACCCGGGAAATCGATAA3 ' sequence wherein contains Sall, Smal and Clal restriction enzyme site.

B. sheep BLG5 ' and 3 ' zone (are respectively 4.5kb, 8.3kb)

Ordinary method makes up genome gene library (Lambda EMBL3 carrier), screens with the probe of one section PCR method synthetic BLG promoter region, picks out the positive colony that contains BLG5 ', 3 ' sequence respectively, connects into complete BLG gene.

Discharge the regional 4.5kb fragment of BLG5 ' with Sall (being positioned at BLG5 ' end) and Pvull (being positioned at first exon, part digestion), clone Sall and Pvull site, be built into the P5 plasmid that contains BLG5 ' sequence in the pSP72 carrier.

Discharge the regional 6.5kb fragment of BLG3 ' with Pvull (be positioned at first and show son, part digestion) and Xbal, clone Pvull and Xbal site, be built into the P3 plasmid (concrete operations are referring to accompanying drawing 2) that contains BLG3 ' sequence in the pSP72 carrier.

C. the segmental clone of mouse chromosome

The transgenic mouse that is efficiently expressed people's trypsase gene (AAT) by a mammary gland is (expression amount and integration site are irrelevant, are directly proportional and inheritance stability with the integration copy number), makes up its genome gene library (in Super-cos-1 cosmid carrier).Filter out the recon of the foreign gene that contains integration, the neighbour mouse chromosome sequence in gene both sides.Discharge the 5 ' mouse with NotI and Sall and dye right body region (4.6kb), discharge mouse chromosome 3 ' zone (5.0kb), clone called after SK4.5 and SK5.0 respectively with NotI and Hindlll.(concrete operations are referring to accompanying drawing 3)

2.pBCD structure (referring to accompanying drawing 4), △ represents that this site has lacked the Sall of SK carrier among Fig. 4, Clal, HindIII, EcoRI, Pstl and Smal site.

The restriction enzyme mapping of three .pBCD mammary gland Expression elements: (referring to accompanying drawing 5)

Polylinker joint and near dna sequence dna thereof among four .pBCD:

--TATAA---enters exons 1---AAGCGACCCCAGGTCGACAA

The Sall of BLG

The TATA box

CCCGGGAAATCGATCTGCAGCCATGAAGTGC---

—— —— ——

Smal Clal

Annotate: the translation initiation codon that uses foreign gene itself.

Five, the function of pBCD carrier each several part and using method

Accompanying drawing 1 has been described the one-piece construction of mammary gland-specific efficient expression vector, accompanying drawing 2,3,4 has illustrated the source of each funtion part and has obtained their method, accompanying drawing 5 provides the restriction map spectrum of pBCD carrier core, and expression uses cited restriction enzyme each element can be cut off in different loci.Restriction enzyme mapping is to describe the section of DNA feature, distinguishes one of means of it and other dna segment dissimilarity.Therefore, the restriction enzyme mapping in the accompanying drawing 5 is that carrier pBCD is peculiar, does not contain any section of DNA in the world and provides identical zymogram.

The pBCD carrier has three functional element.The part of drawing oblique line among Fig. 5 is sheep lactoglobulin gene and expression regulation sequence thereof, comprise whole exons of this gene and intron, detailed Nucleotide composition is seen accompanying drawing 6, capitalization English letter is represented the amino acid coding (exon) of sheep lactoglobulin gene in the accompanying drawing 6, the small letter English alphabet is represented the part beyond noncoding intron and the transcriptional domain, the lactoglobulin gene DNA fragment is longer than sequence given among Fig. 6 in the pBCD carrier, mainly be when this gene of clone, to be inconjunction with one section sequence of having cloned lactoglobulin upstream region of gene and downstream, this a part of function is to guarantee that the foreign gene of inserting on the pBCD carrier is specific expressed in mammary tissue, because foreign gene is the downstream that is inserted into the lactoglobulin gene promoter, and the lactoglobulin gene is a gene that mammary tissue is specific expressed.Respectively adorn the DNA (STAT sequence) of the preceding paragraph at lactoglobulin gene DNA sequence both wings, its 5 ' dististyle segment length 4.5kb, 3 ' dististyle segment length 5.0kb from mouse.These two segmental sources are transgenic mices of mammary gland Table A AT gene.This mouse can efficiently express the AAT gene, the AAT gene that extracts from its tissue is transferred in many mouse together with these two fragments, all efficiently expressed, not inserted the site influences, according to the genetic expression principle, this fragment belongs to the adherent dna fragmentation of unwinding protein, as long as this fragment has been arranged, dna unwinding enzyme just makes karyomit(e) change to working order by off working state, and gene in its vicinity just can efficiently express.The incision enzyme map of this part dna sequence dna is referring to accompanying drawing 5.Being between two dna fragmentations of lactoglobulin gene, is an artificial synthetic polylinker dna fragmentation, and its nucleotide sequence is seen shown in Figure 2.The function of this section of DNA mainly is to contain three restriction enzyme site SalI, SmaI and ClaI, being convenient to any foreign gene inserts herein, the definite position of this synthetic fragment on carrier, be to insert herein at the lactoglobulin gene, the definite position of this synthetic fragment on carrier, it is the upstream (Fig. 7) of swimming over to first genetic code of lactoglobulin gene ATG under the TATA box in the lactoglobulin gene promoter, accompanying drawing 7 is PAGE electrophorograms of transgenic mice milk, and the arrow indication is the protein of exogenous gene expression among the figure.Any complete foreign gene (remove itself promotor after gene), as long as after inserting herein, can both be under the lactoglobulin gene promoter drives, prior to lactoglobulin genetic expression.Restriction enzyme site SalI and ClaI will be contained in enzyme and cut back generation sticky end, and SmaI uses one or two of these three sites with the blunt end in next life, any foreign gene can be inserted herein after end modified.Can both be under the lactoglobulin gene promoter drives, prior to lactoglobulin genetic expression.Restriction enzyme site SalI and ClaI will be contained in enzyme and cut back generation sticky end, and SmaI uses one or two of these three sites with the blunt end in next life, any foreign gene can be inserted herein after end modified.About the schedule of operation that foreign gene inserts, can carry out with reference to method contained in the work on hand handbook fully (the molecular cloning handbook, Manny An Disi etc. writes translations such as Jin Dongyan, Science Press published in 1996).

The invention will be further described with embodiment below, and spirit of the present invention and protection domain are in appending claims.

Embodiment 1

The pBCD carrier is expressed sheep lactoglobulin gene in transgenic mice

The purpose of this experiment is to detect pBCD mammary tissue specific efficient expression vector to have or not the specific expressed function of mammary tissue, and carrier has no adverse effects after changing animal over to.

Expression vector and gene constructed:

In other words the expression structure that experiment is used is exactly pBCD carrier itself as shown in Figure 1.The core of pBCD is a sheep lactoglobulin gene, the sheep lactoglobulin of itself just encoding complete.

The production of transgenic mice:

One experiment material

1. the hybridization mouse of laboratory animal Kunming small white mouse and kunming mice and C57 BL is available from animal portion of birth control institute

2. instrument inverted phase contrast microscope (Japanese Nikon)

Micrurgy instrument (German Leitz)

Draw pin instrument (Japanese Narishige PD-5 type)

Dissecting microscope (eastern branch office)

CO2gas incubator (pharmacia)

3. medicine tribromoethyl alcohol, 60% Sodium.alpha.-hydroxypropionate, calcium chloride, Unidasa, Sodium.alpha.-ketopropionate are all available from U.S. Sigma company, and bovine serum albumin (component V) is available from magnificent company, and PMSG, HCG are available from the institute of lab animals, Tianjin, and all the other medicines are homemade.

4. digoxin dna marker and detection kit, Boehringer company.SephaglasesBandprep kit Phamacia company.The human growth hormone radioimmunological kit is available from 301 Hospital internal secretion chamber.

5. nylon membrane is available from Boehrnger company.

6. plasmid pBCD.

Two, method

(1) preparation of main agents:

1,20 * SSC, dissolving 175.3 gram NaCl and 88.2 gram Trisodium Citrates in 800 ml waters, NaOH transfers PH to 7.0, adds water and is settled to 1 liter, autoclaving.2, sex change liquid 1.5M NaCl, 0.5M NaOH.3, neutralizer 1M Tris, Cl (pH7.4), 1.5M NaCl.4, mouse tail DNA extraction buffer 50mM Tris, Cl (pH8.0), 100mM EDTA (pH8.0), 100mM NaCl 1%SDS.5, microinjection DNA diluent:

7mM Tris.Cl (pH7.4) 0.16mM EDTA, tri-distilled water preparation, filtration sterilization.(2) M ₂With M ₁₆The preparation preparation M of preparation 1. stock solutions of nutrient solution ₂With M ₁₆The stock solution stock solution of nutrient solution becomes partial volume gram number and concentration (ml) A 10 * 100

NaCl 5.534

KCl 0.356

KH ₂PO ₄ 0.162

MgSO ₄7H ₂O 0.293

Sodium.alpha.-hydroxypropionate 2.610

Or the slurry of 4.349 grams 60%

Glucose 1.000

Penicillin 0.060

Streptomycin sulphate 0.050B 10 * 100

NaHCO ₃ 2.101

Phenol red 0.010C 100 * 10

Sodium.alpha.-ketopropionate 0.036D 100 * 10

CaCl ₂.2H ₂O 0.252E 10× 100

HEPES 5.958

The equal filtration sterilization of phenol red 0.010 above-mentioned stock solution in A liquid-20 ℃ two weeks of preservation, is preserved weeks for all the other 4 ℃.2.M ₂With M ₁₆Preparation.By stock solution preparation M ₂

Stock solution M ₂Working fluid (unit/ml)

10 50 100

A(10×) 10 5.0 10.0

B(10×) 0.16 0.8 1.6

C(100×) 0.10 0.5 1.0

D(100×) 0.10 0.5 1.0

E(10×) 0.84 4.2 8.4

BSA 40.0mg 200mg 400mg

Distilled water 7.80 39.0 78.0

By stock solution preparation M ₁₆

Stock solution M ₁₆Working fluid (unit/ml)

10 50 100

A(10×) 1.0 5.0 10

B(10×) 1.0 5.0 10

C(100×) 0.1 0.5 1

D(100×) 0.1 0.5 1

BSA 40.0mg 200mg 400mg

Distilled water 7.80 39.0 78.0

M ₂And M ₁₆Equal matching while using

(3) the transgenosis preparation of DNA

Behind the plasmid pBCD transformed into escherichia coli DH52, cultivate the bacillus coli DH 52 that contains the pBCD plasmid with liquid culture method.The cultivation amount can be 100ml-1000ml as required.After finishing, cultivation collected bacterium in centrifugal 10 minutes with centrifuging (300 rev/mins), with quick extraction process or extensive extraction process prepare pBCDDNA (detail operations is with reference to Manny An Disi chief editor's molecular cloning handbook, or any one about the handbook of molecular cloning all can).The pBCD DNA that extracts cuts with restriction endonuclease Notl enzyme, and it is the big fragment of 22.4kb and the small segment of a 3.0kb that the TAE agarose gel electrophoresis will obtain a molecular weight.22.4kb fragment be the pBCD complete sequence, after it is downcut, be equipped with injection with Sephaglass Babndprep test kit purifying again.

2. be diluted to 2 μ g/ml with microinjection with the DNA of DNA diluent after with purifying, centrifugal (12,000rpm, 30 minutes) get 1/2 volume, and packing promptly can be used as microinjection.

(4) transgenic experiments method

1. the preparation of mouse:

The mouse that transgenic experiments is used is divided into four types, i.e. the female mouse of donor, the female mouse of acceptor, the public mouse of kind and the public mouse of ligation.

1. the female mouse of donor, the female Kunming white mouse and the hybridization mouse in 8-10 age in week.The artificial induction oestrus the back with plant public mouse mating, as the female mouse of super ovulation donor.

2. the female mouse of acceptor, the female Kunming white mouse and the hybridization mouse in 8-10 age in week.Artificial induction oestrus back and the public mouse mating of ligation obtain the female mouse of false pregnancy, can be used as the acceptor of zygote after the microinjection.

3. plant public mouse, 8-10 male Kunming white mouse in age in week is bought the single cage in back back and raises, and label.1-2 after week with the female mouse mating of donor.

1. the male Kunming white mouse in the public mouse 8-10 of ligation age in week is tested first three week and makes bilateral vas ligation.

2. the vasoligation of public mouse: anesthesia: 2.5% tribromoethyl alcohol, (0.017 milliliter/gram body weight), abdominal injection.Ligation: mouse is lain on the back on the operation plate, and the abdominal cavity is opened in abdomen cropping sterilization, finds out respectively and ligation left and right sides vas deferens, and closure procedure path after also receiving closes place, abdomen fore edge and goes up a little sulfa powder in case infect.

3. super ovulation

The female mouse of donor is raised a week in light and shade round-robin Animal House, can do super ovulation and use.Is 100iu/ml with physiological saline with PMSG and hCG dilution, every mouse abdominal injection 0.1ml, and be 14:00PMSG inject time, hCG after 48 hours puts into a kind of public mouse cage with the female mouse of every donor respectively behind the injection hCG.Ovulation period be generally the injection hCG after 10-13 hour.

4. get ovum

To there be the female mouse of donor of cloudy bolt to choose, executions of craning one, the 0.2% new knot that immersion whole body that goes out is opened the abdominal cavity at abdomen, exposes two lateral oviducts and ovary, takes out two lateral oviducts respectively, it is transferred to fill M ₂Culture dish in, culture dish is put under the anatomical lens, with sharp tweezers ampulla of uterine tube is torn, ovum is transferred to gently, add a little Unidasa (0.3mg/ml).With ovum shifting tube piping and druming gently, treat that ovum disperses after, change ovum over to M ₁₆In the drop, M ₁₆After washing 4 times, put into the carbonic acid gas incubator.

5. the microinjection of zygote

1. hold the preparation of egg apparatus, the hard glass pipe of cut-off footpath 1mm, roasting soft on low baking temperature, it is pulled into the tubule of 80-150 μ m diameter, fracture at distance neck 2cm place, with burning the roasting even fracture of pin instrument.

2. the preparation of entry needle uses the Glass tubing that draws pin instrument diameter 1mm to pull into the entry needle of the about 1 μ m diameter of needle point.

3. microinjection is at M on each on the depression slide ₂Nutrient solution covers on the rearmounted microscopical Stage microscope with the aseptic Valelinum Liquidum of DNA injection liquid (both different mixing).Under the low power lens entry needle sucked an amount of DNA injection liquid.Get about 10 zygotes and inject M ₂In the drop.Go under the high power lens after egg apparatus holds one piece of ovum with holding.Entry needle is transferred to suitable angle, and needle point thrusts in the zygote male pronucleus, and the DNA injection liquid is injected about 1p1, and visible male pronucleus is expanded, and extracts entry needle rapidly out, treat ovum all on the depression slide inject finish after, immediately ovum is transferred to M ₁₆In the substratum, CO ₂Incubator is cultivated after 30 minutes and is moved ovum at once.

6. the common oviduct transplantation of zygote

1. transplant preparation: thin glass tube is burnt soft, pull into about 150 μ m diameters, put down the broken ends of fractured bone is roasting after fractureing on alcohol blast burner with ovum shifting tube.

2. the preparation of zygote: with zygote from M ₁₆Nutrient solution moves to M ₂In the nutrient solution, standby in the resorb ovum shifting tube.

3. the common oviduct transplantation of zygote

Selection has the female mouse of the false pregnancy of cloudy bolt, anesthesia, and method is with the vasectomy of public mouse.With mouse dorsal part alcohol disinfecting, about 1cm place behind the rib in the end makes the lengthwise otch of a long 1.5cm along backbone, opens the abdominal cavity, clamps fat pad on the ovary with tweezers, and ovary and uterine tube are pulled out gently.Under anatomical lens, find the fimbriae tubae opening, cut off outer tunicle, ovum is blown into after ovum shifting tube is inserted umbrella portion with iris scissors.The same method of transplanting of both sides input ovum portion is clamped otch with Small clamp after operation finishes, and removes clip after the week.

(5) extraction of mouse tail DNA:

2 ages in week, above mouse promptly can be used for extracting mouse tail DNA

1, cuts the long mouse tail of 2cm, clean with 70% ethanol earlier.Put into the Ependorff pipe after drying.

2, add mouse tail DNA extract 0.7ml, the mouse tail is shredded, adding 70 μ l concentration again is the Proteinase K of 10mg/ml, mixing.

3,55 ℃ water-soluble 18 hours, put upside down mixing frequently.

4, add RNA after being cooled to room temperature _AseMaking final concentration is 20 μ g/ml, and 37 ℃ water-soluble 1 hour.

5, add equal-volume phenol, put upside down gently and mixed 20 minutes, centrifugal.(4 ℃, 5000rpm, 10 minutes) get supernatant, repeat phenol extracting twice, phenol/chloroform extracting one time, 24: 1 chloroform: primary isoamyl alcohol extracting one time.

6, shift in the new pipe of water to, add the cold dehydrated alcohol of 2 times of volumes, mixing, precipitation occurs immediately.

7, will precipitate sucking-off, 70% ethanol washes twice, dries.

8, add 500 μ l TE, room temperature dissolving 48 hours treats that DNA dissolves back 4 ℃ of preservations.

(6) PCR method screening transgenic positive mouse

Template DNA is 1 μ g mouse tail DNA, and primer I I is redesign, and all the other conditions are with the experiment second section.

(7) the transgenic positive mouse is identified in Southern hybridization.

1, the enzyme of mouse tail DNA is cut:

Get mouse tail DNA 20 μ g, the BamH1 enzyme is cut, and the enzyme system of cutting is

Mouse tail DNA20 μ g

10×bufer?E?20μl

BamH1 enzyme 50ul

Add distilled water to 200 μ l

37 ℃ of enzymes are cut and are spent the night

2, electrophoresis: the DNA after enzyme is cut gets electrophoretic examinations in a small amount, and enzyme with sample phenol/chloroform extracting, is dissolved among the 20 μ l TE 0.8% agarose gel electrophoresis, 3-4v/cm, 8 hours after cutting entirely behind the ethanol sedimentation.

3, change film

1. transfer groove is cleaned with clear water, distilled water flushing is spread the plastic film of being with window.

2. cut one than the big nylon membrane of window, redistilled water soaked after 2 minutes, was placed on the window.

3. the gel device on nylon membrane, no longer mobile after putting well.

4. install vacuum unit, start vacuum pump.Transferring vacuum pressure is that 40mbar kept 2 minutes.

5. inhale the last hydrochloric acid that removes photoresist and add sex change liquid, make it the rubber cover face, kept 30-60 minute.

6. inhaling removes photoresist goes up sex change liquid, adds neutralizer, makes it the rubber cover face, keeps 20 minutes.

7. inhale and remove neutralizer, add 20 * SSC, pressure regulation kept 60 minutes to 90mbar.

8. change film and finish, close vacuum pump, inhale and remove SSC solution, remove gel, take off nylon membrane, put vacuum transfer device and plastic film in order.

9. with nylon membrane with drying on the rearmounted filter paper of 6 * SSC rinsing, did roasting 2 hours for 80 ℃, DNA promptly is fixed on the nylon membrane.

4, the mark of probe, hybridization is all undertaken by Dig DNA absolute altitude and detection kit specification sheets.

(8) detection of expression product in the mouse milk

1. the preparation of mouse milk sample:

Female mouse in the 8th day postpartum isolates more than 3 hours with newborn mouse earlier, the pitocin of abdominal injection 0.3IU, 10 minutes pneumoretroperitoneum injection tribromoethyl alcohols.After the anesthesia, massage mammary gland gently, milk gently with the tweezers that twine adhesive plaster, with micro sample adding appliance with the milk sucking-off.Milk adds 5 times of dilutions of aseptic double-distilled water, the centrifugal butterfat that goes.Per 200 μ l add 4-5 μ l 1N HCl, and are centrifugal behind the mixing.Supernatant liquor is whey, can use for detecting.

2. the SDCpAGE electrophoresis of whey-protein: 5% trichloroacetic acid precipitation degrease milk-protein, centrifugal, remove supernatant, acetone is washed precipitation once, is dissolved in the electrophoresis sample-loading buffer.6% concentrated glue, 1.5% separation gel.15v/cm electrophoresis 3 hours.Coomassie brilliant blue staining.

Electrophoresis result sees that Fig. 7 represents: among the figure the 1, the 2nd, and the milk that part is purified, wherein the bands of a spectrum of Chu Xianing are caseins.No. 3 samples are the full milk of ox, and 4-6 number is the milk of transgenic mice, and No. 7 is cow's milk sphaeroprotein standard specimen product, and No. 8 is the milk of non-transgenic mice.As can be seen from the figure, occurred sheep lactoglobulin bands of a spectrum in No. 4 samples, illustrated that sheep lactoglobulin gene expresses in mouse.And no sheep milk-globule egg bands of a spectrum in No. 5 and No. 6 samples are identical with No. 8 samples (non-transgenic mouse), prove that foreign gene does not give expression to albumen in milk.

Embodiment 2: with the growth hormone gene of pBCD expressing human in the mammary gland culturing cell

Human growth hormone gene is a kind of gene of tissue specific expression, and people and other Mammals, growth hormone gene is only expressed in the Anterior pituitary cell, does not express in other any tissue.In order to confirm the mammary tissue specificity of pBCD carrier, people's growth hormone gene is assembled on the pBCD carrier, in order to transform the goat mammary gland cell of cultivating.Experiment showed, that the growth hormone gene that the pBCD carrier can drive the people efficiently expresses in mammary gland cell.Concrete experimental arrangement is as follows:

1, the structure and the sequence that are used for human growth hormone (hGH) gene that sheep mammar gland expresses:

----TATAA---- ( BLG1 )----AAGCACGACCCCAGGTCGACAACCChGH：GATCCCAAGGCCCAACTCCCCGAACCACTCAGGGTCCTGTGGACGCTCACCTAGCTGCA ATG GCT ACA G GTAAGCGCCCCTAAAATCCCTTTGGGCACAATGTGTCCTGAGGGGAGAGGCAGCGACCTGTAGATGGGACGGGGGCACTAACCCTCAGGTTTGGGGCTTCTGAATGAGTATCGCCATGTAAGCCCAGTATGGCCAATCTCAGAAAGCTCCTGGTCCCTGGAGGGATGGAGAGAGAAAAACAAACAGCTCCTGGAGCAGGGAGAGTGCTGGCCTCTTGCTCTCCGGCTCCCTCTGTTGCCCTCTGGTTTCTCCCCAG GC TCC CGG ACG TCC CTG CTCCTG GCT TTT GGC CTG CTC TGC CTG CCC TGG CTT CAA GAG GGC AGTGCC TTC CCA ACC ATT CCC TTA TCC AGG CTT TTT GAC AAC GCT AGTCTC CGC GCC CAT CGT CTG CAC CAG CTG GCC TTT GAC ACC TAC CAGGAG TTTGTAAGCTCTTGGGGAATGGGTGCGCATCAGGGGTGGCAGGAAGGGGTGACTTTCCCCCGCTGGGAAATAAGAGGAGGAGACTAAGGAGCTCAGGGTTTTTCCCGAAGCGAAAATGCAGGCAGATGAGCACACGCTGAGTGAGGTTCCCAGAAAAGTAACAATGGGAGCTGGTCTCCAGCGTAGACCTTGGTGGGCGGTCCTTCTCCTAG GAA GAA GCC TAT ATC CCA AAG GAA CAG AAG TAT TCATTC CTG CAG AAC CCC CAG ACC TCC CTC TGT TTC TCA GAG TCT ATTCCG ACA CCC TCC AAC AGG GAG GAA ACA CAA CAG AAA TCCGTGAGTGGATGCCTTGACCCCAGGCGGGGATGGGGGAGACCTGTAGTCAGAGCCCCCGGGCAGCACAGGCCAATGCCCGTCCTTCCCCTGCAG AAC CTAGAG CTG CTC CGC ATC TCC CTG CTG CTC ATC CAG TCG TGG CTG GAGCCC GTG CAG TTC CTC AGG AGT GTC TTC GCC AAC AGC CTG GTG TACGGC GCC TCT GAC AGC AAC GTC TAT GAC CTC CTA AAG GAC CTA GAGGAA GGC ATC CAA ACG CTG ATG GGG GTGGGGGTGGCGCTAGGGGTCCCCAATCTTGGAGCCCCACTGACTTTGAGAGCTGTGTTAGAGAAACACTGCTGCCCTCTTTTTAGCAGTCCAGGCCCTGACCCAAGAGAACTCACCTTATTCTTCATTTCCCCTCGTGAATCCTCTAGCCTTTCTCTACACCCTGAAGGGGAGGGAGGAAAATGAATGAATGAGAAAGGGAGGGAGCAGTACCCAAGCGCTTGGCCTCTCCTTCTCTTCCTTCACTTTGCAG AGG CTG GAA GATGGC AGC CCC CGG ACT GGG CAG ATC TTC AAG CAG ACC TAC AGC AAGTTC GAC ACA AAC TCA CAC AAC GAT GAC GCA CTA CTC AAG AAC TACGGG CTG CTC TAC TGC TTC AGG AAG GAC ATG GAC AAG GTC GAG ACATTC CTG CGC ATC GTG CAG TGC CGC TCT GTG GAG GGC AGC TGT GGCTTCTAG CTGCCC ( pBCD ) GGGAAATCGATCTGCAGCCATGAAGTGC------：。 2, the conception of expression structure:make up human chromosome group gene library (in Lambda FIX II carrier), screening contains the recon of human growth hormone gene.Discharge the hGH gene fragment with BamHI and Smal (part digestion), mend flat end, it is connected with the Smal site of pBCD.Obtain containing the fusion structure (pBCD-hGH) of hGH encoding gene, referring to accompanying drawing 8.

3, the pBCD-hGH fusion gene is expressed in the goat mammary gland cell:

(1) the experiment material laboratory animal is used 24 days she-goat lactation period of lambing.

1. plasmid pBCD-hGH

2.Lipofectin ^TMReagent, substratum MEM, 199, foetal calf serum is available from BRL company.

3. collagenase II type, Sigma I8405, Cortisol, prolactin is available from Sigma company.

4. Tissue Culture Flask is available from Corning company.

(2) preparation of main agents

1.PBS damping fluid (rises, PH7.4)

Na ₂HPO ₄H ₂The O2.9 gram, KH ₂PO ₄0.2 gram

NaCl 18.0 grams, KCL 0.2 gram

2.MEM, 199 nutrient solutions.The redistilled water preparation, filtration sterilization.

3. two anti-stock solutions (100 *)

Crystalline penicillin sodium salt 1,000,000 units

Streptomycin sulphate 1,000,000 units

100 milliliters of aqua sterilisas

The bottle packing, every bottle of 3-4ml ,-20 ℃ of preservations.

4. trypsinase stock solution (10 *)

NaCl 2.0 grams, KCL 0.4 gram, glucose 1.0 grams

NaHCO ₃0.58dpb, EDTA0.2dqb, phenol red 0.02 gram.

Trypsinase 0.5-0.6 gram, 100 milliliters of redistilled waters, filtration sterilization.

The bottle packing ,-20 ℃ of preservations.

5. 8%NaHCO ₃Solution.The redistilled water preparation, filtration sterilization.

6. collagenase liquid.10mg/ml, PBS preparation, filtration sterilization.

7. Sigma I8405 is made into 1mg/ml with 199 nutrient solutions, filtration sterilization.

8. prolactin is made into 1mg/ml with 199 nutrient solutions, filtration sterilization.

9. hydrocortisone is made into 2mg/ml with ethanol, filtration sterilization.

10. cell growth medium

MEM?45ml?199?45ml

Two anti-1ml glutamine 1ml

NaHCO ₃(8%) transfers to pink (PH7.1-7.3)

Foetal calf serum 10ml

(3) cultivation of goat mammary epithelial cell of former generation

1. kill sheep, clean mammary gland with 75% alcohol earlier, bind up with gauze with double-layer sterile behind the cutting-out mammary gland, put operation in the Bechtop immediately.

2. cut off fatty tissue with the aseptic operation apparatus, with the mammary tissue (1cm that is cut into small pieces ³/ piece).

3.MEM substratum rinsing 3 times.

4. tissue block is shredded (about 1mm ³/ piece), add the rinsing of MEM substratum.

5. centrifugal (room temperature, 1000rpm7 minute) removes supernatant.

6. it is inferior to repeat to give a baby a bath on the third day after its birth.

7. add MEM and collagenase solution in the tissue precipitation, the collagenase final concentration is 1mg/ml.

8. 37 ℃ of shaking baths are 2 hours.

9. centrifugal (room temperature 1000rpm 7 minutes) removes supernatant.

10.MEM wash five times.

11. refinement intracellular growth liquid 20ml.

12. the mixture branch that obtains is filled to culturing bottle, inoculation 2-3ml (to exceed at the bottom of the bedding bottle) in each culturing bottle.

13. 37 ℃, 5%CO ₂Cultivated about 1 hour in the incubator.

14. refinement intracellular growth liquid is to 5ml.

15. 37 ℃, 5%CO ₂Cultivate in the incubator.

(4) transfection of plasmid

1. each nutrient solution of cultivating bottleneck is inclined to, 199 wash twice, add the MEM/199 substratum that the 3ml serum-free adds glutamine.

2. get 200ul lipofectin and mix with 200ul pBCD-hGH plasmid, room temperature left standstill 15 minutes.

3. every culturing bottle adds lipofectin-DNA mixture 200ul, dropwise adds, and jog mixes.

4. 37 ℃, 5%CO ₂Incubator is cultivated sampling detection after 18 hours.

(5) radioimmunoassay of human growth hormone

The human growth hormone Determination on content adopts radioimmunology in the culturing cell, measures used kit and is buied by Clonetech (Shanghai) company, and the working method of employing is undertaken by the method described in the specification sheets of producer, shown in expression of results sees the following form.

Table 2-1 goat mammary gland epithelial cell of former generation expressing human tethelin ria-determination result (ug/10 ⁶Cell)

Hours mammary gland cell nutrient solution behind the hormone induction

0 ND

12 48.4

24 37.56

36 41.6

48 29.56

60 36.64

72 171.8

84 666.0

96 579.2

108 496.4

ND: the hGH content of not measuring the mammary gland cell lysate is 1.5ng/ml, does not measure human growth hormone without the nutrient solution of the mammary gland cell of plasmid transfection.

Under the situation of no hormone induction, 18 hours pBCD-hGH genes are not expressed (result is unlisted) after the former generation goat mammary gland epithelial cell pBCD-hGH transfection.By table 2-1 as can be seen, hormone induction is after 12 hours, and the BLG/hGH gene begins to express, and expression amount raise suddenly in 72 hours, to the highest reduction was arranged slightly thereafter in 84 hours.And, illustrate that this is to be secreted into extracellular human growth hormone after producing because what measure is nutrient solution.From the detected result of mammary gland cell lysate as can be seen, human growth hormone content is extremely low in the mammary gland cell, illustrate after expression product synthesizes and secrete to nutrient solution immediately, prove that mammary gland cell can itself guide the human growth hormone of peptide secrete the extracellular effectively with having.

In former generation,, the goat cell transformed the back growth normally through pBCD-hGH, showed that the foreign gene pair cell do not poison.Growing state is as shown in Figure 9 under cultivation conditions for the goat mammary gland cell.

Claims (1)

1. high effect expressing carrier for animal mammary gland tissue specificity pBCD is characterized in that being included among the intestinal bacteria CGMCC No.0312.

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