CN1216986C - Method for expressing modified human alpha antitrypsin gene in sheep's mammar gland - Google Patents

Abstract

The present invention discloses a human antitrypsin factor producing method. The present invention comprises the following steps that (1) a ewe with a normal reproduction function is selected as a donor, and the donor is treated so that the donor realizes superovulation; (2) an embryo at a pronucleus stage is acquired; (3) the microinjection of corrected human alpha antitrypsin genes into the embryo at a pronucleus stage is carried out, and a fresh embryo is transferred; (4) a transgenic sheep is obtained; (5) human antitrypsin factors are extracted from mink liquid of the transgenic animal. When the method of the present invention is used, the mammary glands of sheep can be used for producing human alpha antitrypsin factors in a large scale with low cost, and medicine markets can enriched.

Description

The method that the modifier alpha antitrypsin gene is expressed in sheep mammar gland

Technical field

The present invention relates to the method that the modifier alpha antitrypsin gene is expressed in sheep mammar gland.

Background technology

Animal mammary gland bioreactor (Mammary Bioreactor) is that a kind of animal mamma as raw material that utilizes that occurs early 1990s comes scale operation medical treatment and proteinic biological high-technology for health care as biological factory.

The technology of utilizing the microinjection method that foreign gene is imported the mouse genome and producing transgenic mice is very ripe.As far back as nineteen eighty-two, people such as Palmiter have just imported the mouse genome to rat growth hormone gene (rGH) and have obtained first transgenosis " super mouse " in the world.After 1985, transgene rabbit, transgenic pig, transgenic sheep, genetically engineered fish and transgenic chicken etc. are come out one after another.1987, people such as Simons at first successfully expressed sheep lactoglobulin gene in the transgenic mice breast, and the content of sheep lactoglobulin reaches 23 grams per liters in the mouse milk, is more than 400 times of animal cell culture technological expression level.After this, cow's milk albumin gene (Vilotte etc., 1989), people organize profibr(in)olysin activating factor gene (Gordon etc., 1987), human growth hormone gene (Brem etc., 1991; Reddy etc., 1991), human body antitrypsin gene (Archibald, 1990), human urokinase gene (Meade etc., 1990) and human interferon gene (Stinnake etc., 1991) etc., all obtained expressing in the transgenic mice breast, expressed proteins has normal biological activity.Changeing has people's antitrypsin gene, human blood coagulation IX gene and people to organize transgenic sheep (Clark etc., 1989 of profibr(in)olysin incitant (tPA) gene; Wright etc., 1991; Elbert etc., 991; Carver etc., 1994), being integrated with HRBC stimulates the transgenic cattle (Hyttnen etc., 1994) that generates the factor (EP0) gene also to produce in succession.

People's antitrypsin can prevent the pneumonocyte fibrosis, in time the removing obstacles pneumonocyte is exercised the material of oxygen exchange, it is the efficient pharmaceutical protein of a kind of treatment alveolar fibrotic disease (pulmonary emphysema), respiratory distress syndrome (i.e. a series of pathologies of having difficulty in breathing and causing) and heredity lung fibrocyst there are special efficacy, are called as the street cleaner of lungs.In China, it is international the first that number of smokers and ratio all occupy, and 80% senile respiratory distress syndrome (ivrds) patient is in China in the world, and is very huge to the demand of protease inhibitor.1991, people such as Wright at first successfully expressed people's antitrypsin gene (ATT) in the breast of sheep.

Modifier alpha antitrypsin gene (mAAT) is on the basis of people's antitrypsin gene, has implemented the point mutation at some positions, and its nucleotide sequence is seen SEQ ID NO:1 (wherein comprising intron 2,3 and 4).The modifier alpha antitrypsin gene more helps expressing.

Summary of the invention

The purpose of this invention is to provide a kind of modifier alpha antitrypsin gene great expression in sheep mammar gland, produce the method for people's anti-trypsin.

Embodiment of the present invention are as follows: 1, produce the method for people's anti-trypsin, may further comprise the steps:

1) ewe of selecting to have normal reproductive capability is handled it as donor, makes it superovulation;

2) obtain pronuclear-stage embryos;

3) has SEQ ID № in the sequence table to the pronuclear-stage embryos microinjection: the modifier alpha antitrypsin gene of 1 nucleotide sequence, bright embryonic implantation;

4) obtain transgenic sheep;

5) in the milk liquid of above-mentioned transgenic sheep, extract people's anti-trypsin.

Described ewe donor can be a goat, also can be sheep.

The method that adds lutropin with follitropin makes the superovulation of described donor sheep.

Described superovulation be chosen in compare in March in 1 year or May suitable.

After the superovulation, fertilize an egg with nature or artificial method, and then obtain pronuclear-stage embryos.

Modifier alpha antitrypsin gene to the pronuclear-stage embryos microinjection, need combine with carrier, the construction step of its transgene carrier is: the 5 ' end that 5 ' control region of sheep β casein gene is connected in mAAT structure gene, insert the caseic 3 ' control region of β then, be connected on the T carrier, with Not I endonuclease digestion recombinant plasmid, reclaim the external source fragment of 14kb, obtain transgene carrier.

The acceptor of described donor or spontaneous estrus is given in described bright embryonic implantation of having injected the modifier alpha antitrypsin gene.

The method of judging the described transgenic sheep that has successfully changed the modifier alpha antitrypsin gene over to is, extract total DNA of the livings lamb of institute, with the detection of PCR-Southern method change foreign gene coding region N-end over to and 3 ' control region partial sequence whether positive.

The present invention utilizes and imports the modifier alpha antitrypsin gene in the pronuclear-stage embryos of microinjection normal direction sheep, the preparation transgenic sheep, utilize the mammary gland of sheep to produce people's alpha-1 antitrypsin factor on a large scale, at low cost, the content of people's alpha-1 antitrypsin factor is up to 30 grams per liters in the goat milk.For scale operation is that the medicine of the treatment pulmonary disorder of activeconstituents provides reliable guarantee with the alpha-1 antitrypsin factor, and then enrich the medical supplies market of China.

Can the quantity of superovulation and transplanting embryo all be the keys that successfully obtain transgenic sheep.Result of study of the present invention shows that the quantity of superovulation and transplanting embryo has confidential relation with the time that operation is carried out.Result shown in the table 1 shows, annual March and two month of May, the effect that super ovulation is handled is significantly better than December, and super row's effect is best during with May, and available embryo number is maximum.3,5, can obtain 19.50,21.70 and 16.06 pieces of average ovulations December respectively, the zygote number is respectively 4.31,6.48 and 3.57 pieces.ANOVA showed significant adopts the FSH+LH combined techniques that goat is surpassed row in March and May, does not have evident difference (P＜0.05) between super row's effect, and December is minimum, and difference is (P＜0.01) extremely significantly.Analyzing its reason, mainly is because the cold of weather this moment has caused low, the available embryo's comparatively small amt of the rate of recovery.

The superovulation effect of table 1 goat

Month	Number of elements	Ovulation count (on average)	Reclaim the ovum number	The unfertilized egg number	The rate of recovery (%)	The transplanting embryo number
							May 3 month December	17 26 13	273(16.06) 507(19.50) 282(21.70)	177 399 212	65 224 61	64.84 78.70 75.00	3.57(75/21) 4.31(125/29) 6.48(136/21)

The acceptor of donor or spontaneous estrus is given in embryo transfer after the microinjection, transplants conception rate and is respectively 18.18% and 25.00%.Show that the ewe with spontaneous estrus is embryo's a acceptor, conception rate is higher.

Below in conjunction with accompanying drawing embodiments of the invention are described further.

Description of drawings

Fig. 1 is expression vector establishment figure

Fig. 2 by the PCR of living lamb individuality detect collection of illustrative plates

Fig. 3 by the PCR-Southern detected result of living lamb individuality

Fig. 4 carries out the collection of illustrative plates that Southern is hybridized for being probe with the primer I amplified fragments to the living lamb genes of individuals group DNA of institute

Embodiment

Embodiment 1: produce people's anti-trypsin with the goat mammary gland bio-reactor

1, laboratory animal: select to grow up, 100 of healthy Wendeng City milk goats, 80 of the goats of wherein growing up, 20 of reserve goats carry out conventional feeding and management, and wherein 56 are carried out superovulation as donor and handle, and other sheep are only as acceptor.

2, test drug: follitropin (FSH) and lutropin (LH) are produced by the Ningbo aquatic products; Prostaglandin(PG) (PG) is produced by the Nanjing aquatic products; Sheep vagina bolt (CIDR) is New Zealand Pharmacia﹠amp; The product of Upjohn; Taq enzyme, Not I and BamH I restriction endonuclease are the products of U.S. Biolab company; The T carrier is the product of Chinese magnificent company.

3, the processing of animal superovulation: meat sheep used as donor heeling-in vaginal suppository 8-14 days, morning every day, 8 difference intramuscular injection in evening FSH 50IU injected 3 days continuously then.Intramuscular injection PG 1ml when FSH injects the 3rd pin takes out vaginal suppository during the 6th injection FSH, and oestrus next day and identify and breeding, and intramuscular injection LH 300IU.The ewe that every superovulation is oestrused is at least with 2 rams breedings 3-4 time.Prick ram examination feelings searching spontaneous estrus ewe with separating.

4, the structure of transgene carrier: the 5 ' end that 5 ' control region of goat β casein gene is connected in mAAT structure gene with ordinary method, insert the caseic 3 ' control region of β then, form complete transgenic structure, be connected on the T carrier, ordinary method increases, extraction and purifying.Constructed expression vector as shown in Figure 1, wherein β casein gene 5 ' control region is that 7987bp, mAAT structure gene (containing intron) are 1526bp for 4805bp, β casein gene 3 ' control region; 5 ' end of β casein gene 5 ' control region has Not I and Not I/BamH I restriction enzyme site, and BamH I restriction enzyme site is arranged in the mAAT structure gene, and 3 ' end of β casein gene 3 ' control region has BamH I restriction enzyme site.Nucleotide sequence to the transgene carrier that obtained carries out sequencing, and the checking sequence is accurate.

5, the preparation of injection dna solution: with Not I endonuclease digestion recombinant plasmid, reclaim the external source fragment of 14kb, be dissolved in the TE damping fluid (Promaga Catalog, calendar year 2001) of transgenosis special use, adjusting DNA concentration is 2g/ml, and the molecule number that makes DNA in every ml solution is 4.8 * 10 ¹¹

6, microinjection and transplanting: the employing operation is dashed in 24-28 hour in last breeding back towards the ovum method and is got the embryo, and the embryo male pronucleus that is fertilized is carried out microinjection, and the amount of each procaryotic injection foreign gene is 1-2pl.To make bright embryo adopt modus operandi to transplant acceptor rapidly then, guarantee that each acceptor transplants two pieces of quality embryo preferably at least to donor or spontaneous estrus.The embryo breeds in acceptor, and mature spontaneous labor obtains lamb.

7, in resulting lamb, filter out the individuality of transgenic positive.

8, in the milk liquid of above-mentioned transgenic sheep, extract people's anti-trypsin with conventional method.

The evaluation of embodiment 2, transgenosis individuality

1、PCR

From the ear of living lamb gather tissue block ,-80 ℃ of refrigerators are preserved, and hold (primer I I) two pairs of primers to carry out pcr amplification respectively with 5 ' end (primer I) and 3 ' after extracting genomic dna, the detection positive individuals, the sequence of primer is as follows:

Primer I: forward: 5 ' ATTGGACAGAAGGAGGAGACTGG-3 '

Oppositely: 5 ' TGGCATACAGCAGGTGATGGAC-3 '

Primer I I: forward 5 ' TTGTGGAGAGTGAAAGGCTGTC-3 '

Oppositely: 5 ' CTCCGTCTCTACCAGGAATGGC-3 '

5 ' terminal expanding fragment length is 313bp (primer I); 3 ' control region expanding fragment length is 289bp (primer I I).

Pcr amplification condition: 94 ℃ 5min-94 ℃ 30sec-60 ℃ 30sec-72 ℃ 30sec (30cycles)-72 ℃ 5min; Amplified production is analyzed with 2% agarose gel electrophoresis, and detected result as shown in Figure 2.Among the figure, the meaning of each symbol is M: molecular weight standard; C: blank; H: people's genome contrast; P: the positive control that contains people mAAT gene plasmid; 3,15,90,06 is the numbering of transgenic positive lamb individuality; 6,47 is the numbering of the negative lamb individuality of transgenosis; The arrow indication is the purpose fragment.

2, PCR-Southern detects

The reliability of amplified fragments in order to guarantee, to living lamb individuality carried out the PCR-Southern detection again.Require to adopt above-mentioned two pairs of primers that hybridization probe is carried out the DIG mark according to test kit, measure labeling effciency.To carrying out Southern hybridization behind the PCR product commentaries on classics film.The result as shown in Figure 3.Among the figure, the meaning of each symbol is Arabic numerals: be the numbering of lamb individuality, wherein 3,15,90,06 for PCR detects positive individuals, and 6,47 is the transgenosis negative individuals; H: people's genomic dna; P: contain the segmental plasmid clone of external source; M: plasmid PBR322 enzyme is cut molecular weight standard; The arrow indication is the purpose fragment.

3, Southern detects

The content of hybridizing used genomic dna by spectrophotometric determination, and quantitatively arrive the 8g/ sample, utilize BamH I restriction endonuclease to digest then, the external source fragment that changes over to should be 3.1kb, again through changeing processes such as film, hybridization, colour developing, primer I is carried out the DIG mark become probe, and utilize this probe to hybridize processing.The result as shown in Figure 4.Among the figure, the meaning of each symbol is M: molecular weight standard; P: contain the segmental plasmid of external source; H: people's genomic dna; Arabic numerals: the numbering of living lamb individuality, 06,90,15,3 positive individualities wherein, 6,47,8 negative individualities; The arrow indication is the purpose fragment.

Sequence table

(1) general information:

(I) applicant: China Agricultural University

Beijing Xing Lvyuan three high-tech limited liability companys

(II) denomination of invention: the method that the modifier alpha antitrypsin gene is expressed in sheep mammar gland

(2) information of SEQ ID NO:1

(i) sequence signature

(A) length: 6237 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

(xi) sequence description: SEQ ID NO:1

1 GGATCCCCAG GGAGATGCTG CCCAGAAGAC AGATACATCC CACCATGATC AGGATCACCC

61 AACCTTCAAC AAGATCACCC CCAACCTGGC TGAGTTCGCC TTCAGCCTAT ACCGCCAGCT

121 GGCACACCAG TCCAACAGCA CCAATATCTT CTTCTCCCCA GTGAGCATCG CTACAGCCTT

181 TGCAATGCTC TCCCTGGGGA CCAAGGCTGA CACTCACGAT GAAATCCTGG AGGGCCTGAA

241 TTTCAACCTC ACGGAGATTC CGGAGGCTCA GATCCATGAA GGCTTCCAGG AACTCCTCCG

301 TACCCTCAAC CAGCCAGACA GCCAGCTCCA GCTGACCACC GGCAATGGCC TGTTCCTCAG

361 CGAGGGCCTG AAGCTAGTGG ATAAGTTTTT GGAGGATGTT AAAAAGTTGT ACCACTCAGA

421 AGCCTTCACT GTCAACTTCG GGGACACCGA AGAGGCCAAG AAACAGATCA ACGATTACGT

481 GGAGAAGGGT ACTCAAGGGA AAATTGTGGA TTTGGTCAAG GAGCTTGACA GAGACACAGT

541 TTTTGCTCTG GTGAATTACA TCTTCTTTAA AGGTAAGGTT GCTCAACCAG CCTGAGCTGT

601 TTCCCATAGA AACAAGCAAA AATATTTCTC AAACCATCAG TTCTTGAACT CTCCTTGGCA

661 ATGCATTATG GGCCATAGCA ATGCTTTTCA GCGTGGATTC TTCAGTTTTC TACACACAAA

721 CACTAAAATG TTTTCCATCA TTGAGTAATT TGAGGAAATA ATAGATTAAA CTGTCAAAAC

781 TACTGACGCT CTGCAGAACT TTTCAGAGCC TTTAATGTCC TTGTGTATAC TGTATATGTA

841 GAATATATAA TGCTTAGAAC TATAGAACAA ATTGTAATAC ACTGCATAAA GGGATAGTTT

901 CATGGAACAT ACTTTACACG ACTCTAGTGT CCCAGAATCA GTATCAGTTT TGCAATCTGA

961 AAGACCTGGG TTCAAATCCT GCCTCTAACA CAATTAGCTT TTGACAAAAA CAATGCATTC

1021 TACCTCTTTG AGGTGCTAAT TTCTCATCTT AGCATGGACA AAATACCATT CTTGCTGTCA

1081 GGTTTTTTTA GGATTAAACA AATGACAAAG ACTGTGGGGA TGGTGTGTGG CATACAGCAG

1141 GTGATGGACT CTTCTGTATC TCAGGCTGCC TTCCTGCCCC TGAGGGGTTA AAATGCCAGG

1201 GTCCTGGGGG CCCCAGGGCA TTCTAAGCCA GCTCCCACTG TCCCAGGAAA ACAGCATAGG

1261 GGAGGGGAGG TGGGAGGCAA GGCCAGGGGC TGCTTCCTCC ACTCTGAGGC TCCCTTGCTC

1321 TTGAGGCAAA GGAGGGCAGT GGAGGCAAGC CAGGCTGCAG TCAGCACAGC TAAAGTCCTG

1381 GCTCTGCTGT GGCCTTAGTG GGGGCCCAGG TCCCTCTCCA GCCCCAGTCT CCTCCTTCTG

1441 TCCAATGAGA AAGCTGGGAT CAGGGGTCCC TGAGGCCCCT GTCCACTCTG CATGCCTCGA

1501 TGGTGAAGCT CTGTTGGTAT GGCAGAGGGG AGGCTGCTCA GGCATCTGCA TTTCCCCTGC

1561 CAATCTAGAG GATGAGGAAA GCTCTCAGGA ATAGTAAGCA GAATGTTTGC CCTGGATGAA

1621 TAACTGAGCT GCCAATTAAC AAGGGGCAGG GAGCCTTAGA CAGAAGGTAC CAAATATGCC

1681 TGATGCTCCA ACATTTTATT TGTAATATCC AAGACACCCT CAAATAAACA TATGATTCCA

1741 ATAAAAATGC ACAGCCACGA TGGCATCTCT TAGCCTGACA TCGCCACGAT GTAGAAATTC

1801 TGCATCTTCC TCTAGTTTTG AATTATCCCC ACACAATCTT TTTCGGCAGC TTGGATGGTC

1861 AGTTTCAGCA CCTTTTACAG ATGATGAAGC TGAGCCTCGA GGGATGTGTG TCGTCAAGGG

1921 GGCTCAGGGC TTCTCAGGGA GGGGACTCAT GGTTTCTTAT TCTGCTACAC TCTTCCAAAC

1981 CTTCACTCAC CCCTGGTGAT GCCCACCTTC CCCTCTCTCC AGGCAAATGG GAGAGACCCT

2041 TTGAAGTCAA GGACACCGAG GAAGAGGACT TCCACGTGGA CCAGGTGACC ACCGTGAAGG

2101 TGCCTATGAT GAAGCGTTTA GGCATGTTTA ACATCCAGCA CTGTAAGAAG CTGTCCAGCT

2161 GGGTGCTGCT GATGAAATAC CTGGGCAATG CCACCGCCAT CTTCTTCCTG CCTGATGAGG

2221 GGAAACTACA GCACCTGGAA AATGAACTCA CCCACGATAT CATCACCAAG TTCCTGGAAA

2281 ATGAAGACAG AAGGTGATTC CCCAACCTGA GGGTGACCAA GAAGCTGCCC ACACCTCTTA

2341 GCCATGTTGG GACTGAGGCC CATCAGGACT GGCCAGAGGG CTGAGGAGGG TGAACCCCAC

2401 ATCCCTGGGT CACTGCTACT CTGTATAAAC TTGGCTTCCA GAATGAGGCC ACCACTGAGT

2461 TCAGGCAGCG CCGTCCATGC TCCATGAGGA GAACAGTACC CAGGGTGAGG AGGTAAAGGT

2521 CTCGTCCCTG GGAACTTCCC ACTCCAGTGT GGACACTGTC CCTTCCCAAT ATCCAGTGCC

2581 CAAGGCAGGG ACAGCAGCAC CACCACACGT TCTGGCAGAA CCAAAAAGGA ACAGATGGGC

2641 TTCCTGGCAA AGGCAGCAGT GGAGTGTGGA GTTCAAGGGT AGAATGTCCC TGGGGGGACG

2701 GGGGAAGAGC CTGTGTGGCA AGGCCCAGAA AAGCAAGGTT CGGAATTGGA ACAGCCAGGC

2761 CATGTTCGCA GAAGGCTTGC GTTTCTCTGT CACTTTATCG GTGCTGTTAG ATTGGGTGTC

2821 CTGTAGTAAG TGATACTTAA ACATGAGCCA CACATTAGTG TATGTGTGTG CATTCGTGAT

2881 TATGCCCATG CCCTGCTGAT CTAGTTCGTT TTGTACACTG TAAAACCAAG ATGAAAATAC

2941 AAAAGGTGTC GGGTTCATAA TAGGAATCGA GGCTGGAATT TCTCTGTTCC ATGCCAGCAC

3001 CTCCTGAGGT CTCTGCTCCA GGGGTTGAGA AAGAACAAAG AGGCTGAGAG GGTAACGGAT

3061 CAGAGAGCCC AGAGCCAGCT GCCGCTCACA CCAGACCCTG CTCAGGGTGG CATTGTCTCC

3121 CCATGGAAAA CCAGAGAGGA GCACTCAGCC TGGTGTGGTC ACTCTTCTCT TATCCACTAA

3181 ACGGTTGTCA CTGGGCACTG CCACCAGCCC CGTGTTTCTC TGGGTGTAGG GCCCTGGGGA

3241 TGTTACAGGC TGGGGGCCAG GTGACCCAAC ACTACAGGGC AAGATGAGAC AGGCTTCCAG

3301 GACACCTAGA ATATCAGAGG AGGTGGCATT TCAAGCTTTT GTGATTCATT CGATGTTAAC

3361 ATTCTTTGAC TCAATGTAGA AGAGCTAAAA GTAGAACAAA CCAAAGCCGA GTTCCCATCT

3421 TAGTGTGGGT GGAGGACACA GGAGTAAGTG GCAGAAATAA TCAGAAAAGA AAACACTTGC

3481 ACTGTGGTGG GTCCCAGAAG AACAAGAGGA ATGCTGTGCC ATGCCTTGAA TTTCTTTTCT

3541 GCACGACAGG TCTGCCAGCT TACATTTACC CAAACTGTCC ATTACTGGAA CCTATGATCT

3601 GAAGAGCGTC CTGGGTCAAC TGGGCATCAC TAAGGTCTTC AGCAATGGGG CTGACCTCTC

3661 CGGGGTCACA GAGGAGGCAC CCCTGAAGCT CTCCAAGGTG AGATCACCCT GACGACCTTG

3721 TTGCACCATG GTATCTGTAG GGAAGAATGT GTGGGGGCTG CAGCACTGTC CTGAGGCTGA

3781 GGAAGGGGCC GAGGGAAACA AATGAAGACC CAGGCTGAGC TCCTGAAGAT GCCCGTGATT

3841 CACTGACACG GGACGGTGGG CAAACAGCAA AGCCAGGCAG GGGCTGCTGT GCAGCTGGCA

3901 CTTTCGGGGC CTCCCTTGAG GTTGTGTCAC TGACCCTGAA TTTCAACTTT GCCCAAGACC

3961 TTCTAGACAT TGGGCCTTGA TTTATCCATA CTGACACAGA AAGGTTTGGG CTAAGTTGTT

4021 TCAAAGGAAT TTCTGACTCC TTCGATCTGT GAGATTTGGT GTCTGAATTA ATGAATGATT

4081 TCAGCTAAAG TGACACTTAT TTTGGAAAAC TAAAGGCGAC CAATGAACAA CCTGCAGTTC

4141 CATGAATGGC TGCATTATCT TGGGGTCTGG GCACTGTGAA GGTCACTGCC AGGGTCCGTG

4201 TCCTCAAGGA GCTTCAAGCC GTGTACTAGA AAGGAGAGAG CCCTGGAGGC AGACGTGGAG

4261 TGACGATGCT CTTCCCTGTT CTGAGTTGTG GGTGCACCTG AGCAGGGGGA GAGGCGCTTG

4321 TCAGGAAGAT GGACAGAGGG GAGCCAGCCC CATCAGCCAA AGCCTTGAGG AGGAGCAAGG

4381 CCTATGTGAC AGGGAGGGAG AGGATGTGCA GGGCCAGGGC CGTCCAGGGG GAGTGAGCGC

4441 TTCCTGGGAG GTGTCCACGT GAGCCTTGCT CGAGGCCTGG GATCAGCCTT ACAACGTGTC

4501 TCTGCTTCTC TCCCCTCCAG GCCGTGCATA AGGCTGTGCT GACCATCGAC GAGAAAGGGA

4561 CTGAAGCTGC TGGGGCCATG TTTTTAGAGG CCATACCCAT GTCTATCCCC CCCGAGGTCA

4621 AGTTCAACAA ACCCTTTGTC TTCTTAATGA TTGAACAAAA TACCAAGTCT CCCCTCTTCA

4681 TGGGAAAAGT GGTGAATCCC ACCCAAAAAT AACTGCCTCT CGCTCCTCAA CCCCTCCCCT

4741 CCATCCCTGG CCCCCTCCCT GGATGACATT AAAGAAGGGT TGAGCTGGTC CCTGCCTGCA

4801 TGTGATCTGT AAATCCCTGG GATGTTTTCT CTGAGTCTCC CTTATAGTCC TAGCTGAGGC

4861 TGTATGTGGG CTCCAGGTAA CAGTGCTGTC TTCGGGCCCC CTGAACTGTG TTCATGGAGC

4921 ATCTGGCTGG GTAGGAATGC TGGGCTTGAA TCCAGGGGAC TGAATCCTCA GCTTACGGAC

4981 CTGGGCCCAT CTGTTTCTGG AGGGCTCCAG TCTTCCTTGT CCTGTCTTGG AGTCCCCAAG

5041 AAGGAATCAC AGGGGAGGAA CCAGATACCA GCCATGACTC CAGGCTCCAC CAAGCATCTT

5101 CATGTCCCCC TGCTCATCCC CCACTCCCCC CCACCCAGAG TTACTCATCC TGCCAGGGCT

5161 GGCTGTGCCC ACCCCAAGGC TGCCCTCCTG GGGGCCCCAG AACTGCCTGA TCGTGCCGTG

5221 GCCCAGTTTT GTGGCATCTG CAGCAACACA AGAGAGAGGA CAATGTCCTC CTCTTGACCC

5281 GCTGTCACCT AACCAGACTC GGGCCCTGCA CCTCTCAGGC ACTTCTGGAA AATGACTGAG

5341 GCAGATTCTT CCTGAAGCCC ATTCTCCATG GGGCAACAAG GACACCTATT CTGTCCTTGT

5401 CCTTCCATCG CTGCCCCAGA AAGCCTCACA TATCTCCGTT TAGAATCAGG TCCCTTCTCC

5461 CCAGATGAAG AGGAGGGTCT CTGCTTTGTT TTCTCTATCT CCTCCTCAGA CTTGACCAGG

5521 CCCAGCAGGC CCAGAAGACC ATTACCCTAT ATCCCTTCTC CTCCCCAGTC ACATGGCCAT

5581 AGGCTGCTGA TGGCTCAGGA AGGCCATTGC AAGGACTCCT CAGCTATGGG AGAGGAAGCA

5641 CATCACCCAT TGACCCCCGC AACCCCTCCC TTTCCTCCCC TGAGTCCCGA CTGGGGCCAC

5701 ATGCAGCCTG ACTTCTTTGT GCCTGTTGCT GTCCCTGCAG TCTTAGAGGG CCACCGCAGC

5761 TCCAGTGCCA CGGCAGGAGG CTGTTCCTGA ATAGCCCCTG TGGTAAGGGC CAGGAGAGTC

5821 CTTCCATCCT CCAAGGCCCT GCTAAAGGAC ACAGCAGCCA GGAAGTCCCC TGGGCCCCAT

5881 GCTGAAGGAC AGCCTGCTCC CTCCGTCTCT ACCAGGAATG GCCTTGTCCT ATGGAAGGCA

5941 CTGCCCATCC CAAACTAATC TAGGAATCAC TGTCTAACCA CTCACTGTCA TGAATGTGTA

6001 CTTAAAGGAT GAGGTTGAGT CATACCAAAT AGTGATTTCG ATAGTTCAAA ATGGTGAAAT

6061 TAGCAATTCT ACATGATTCA GTCTAATCAA TGGATACCGA CTGTTTCCCA CACAAGTCTC

6121 CTGTTCTCTT AAGCTTACTA CTGACAGCCT TTCACTCTCC ACAAATACAT TAAAGATATG

6181 GCCATCACCA AGCCCCCTAG GATGACACCA GACCTGAGAG TCTGAAGACC TGGATCC

Claims (8)

1, produce the method for people's anti-trypsin, may further comprise the steps:

1) ewe of selecting to have normal reproductive capability is handled it as donor, makes it superovulation;

2) obtain pronuclear-stage embryos;

3) has SEQ ID № in the sequence table to the pronuclear-stage embryos microinjection: the modifier alpha antitrypsin gene of 1 nucleotide sequence, bright embryonic implantation;

4) obtain transgenic sheep;

5) in the milk liquid of above-mentioned transgenic sheep, extract people's anti-trypsin.

2, the method for production people anti-trypsin according to claim 1, it is characterized in that: described ewe donor is a goat.

3, the method for production people anti-trypsin according to claim 1, it is characterized in that: described ewe donor is a sheep.

4, the method for production people anti-trypsin according to claim 1, it is characterized in that: the method that adds lutropin with follitropin makes the superovulation of described donor sheep.

5, the method for production people anti-trypsin according to claim 1 is characterized in that: after the described superovulation, fertilize an egg with nature or artificial method, and then obtain pronuclear-stage embryos.

6, the method for production people anti-trypsin according to claim 1, it is characterized in that: to the modifier alpha antitrypsin gene of pronuclear-stage embryos microinjection, the construction step of its transgene carrier is: the 5 ' end that 5 ' control region of sheep β casein gene is connected in mAAT structure gene, insert the caseic 3 ' control region of β then, be connected on the T carrier, with Not I endonuclease digestion recombinant plasmid, reclaim the external source fragment of 14kb, obtain transgene carrier.

7, the method for production people anti-trypsin according to claim 1, it is characterized in that: the acceptor of described donor or spontaneous estrus is given in described bright embryonic implantation of having injected the modifier alpha antitrypsin gene.

8, the method for production people anti-trypsin according to claim 1, it is characterized in that: the method for judging described transgenic sheep is, extract total DNA of the livings lamb of institute, with the detection of PCR-Southern method change foreign gene coding region N-end over to and 3 ' control region partial sequence whether positive.