CN100335632C - Seven kinds of yak milk protein gene sequence - Google Patents

Abstract

The present inventio discloses a complete sequence and a partial sequence of 7 kinds of milk protein genes of yaks, namely a complete sequence of alpha-lactoalbumin genes, a 5' flanking sequence of alpha-lactoalbumin, a 5' flanking sequence and a 3' terminal sequence of beta-lactoglobulin genes, a 5' flanking sequence and a 3' terminal sequence of alpha S1-casein genes, a 5' flanking sequence of alpha S2-casein genes, a 5' flanking sequence and a 3' terminal sequence of beta-casein genes, a 5' flanking sequence and a 3' terminal sequence of kappa-casein genes, and a 5' flanking sequence of lactoferrin genes.

Description

Seven kinds of milk protein gene sequences of yak

The present invention relates to bioengineering field, especially relate to the complete sequence or the partial sequence of seven kinds of milk protein genes of yak.

Animal bioreactor is one of focus of transgenic research in the present world wide, not only national governments' investment, and some private financial groups do not stint yet and drop into substantial contribution and researched and developed.

With similar transgenic animal production process, obtaining the most probable approach of exogenous medicinal protein on one's body from animal has several, is mainly blood plasma, seminal fluid and milk etc.From transgenic animal blood plasma, extract recombinant protein, this is a kind of feasible way, it is that blood sampling volume is restricted on the one hand that blood sampling does not need kill animals, the shortcoming of this method, and the protein secreting of some biologically actives advances blood plasma on the other hand has certain influence to animal health.And expression alien gene brings detrimentally affect for the fertility of transgenic animal through regular meeting in the seminal fluid.By contrast, lactation is a kind of physiological activity of animal, to not influence of animal health, mammary gland picked-up in addition, synthetic, secretory protein very capable, and can carry out multiple translation post-treatment to recombinant protein, comprise beta-hydroxyization, glycosylation, γ hydroxylations etc., the while can be folded into recombinant protein the conformation of function.Therefore mammary gland becomes the desirable organ of generally acknowledged production recombinant protein.Use mammary gland and produce recombinant protein, the promotor of the employed expression vector of general requirement has the tissue in the expression and the specificity of organ and developmental stage.The human protein of at present existing multiple biologically active can obtain from the milk of transgenic animal, and these animals comprise mouse, rat, and rabbit, pig, sheep, goat etc., the recombinant protein that obtains comprises tPA, α ₁-AT, PROTEIN C, hSA, bGH, hAFP, LAtPA etc., making up mammary gland specific expression vector uses more promotor to have the globin upstream of ox, sheep, goat to transfer dead zone, bovine beta-lactoglobulin gene promoter (bBLG), sheep beta-lactoglobulin gene promoter (oBLG), mouse whey acidic protein promotor (mWAP), rat whey acidic protein promotor (rWAP) and rabbit whey acidic protein promotor (rWAP).The dead zone is transferred in more above-mentioned several upstream, and its characteristics drive downstream gene for the beta-globin gene promoter expression has nothing to do with transgenic insert locus, the copy number positive correlation of expression level and integration, but the expression specificity of its regulation and control occur in the red corpuscle; As if bBLG, oBLG have the ability of stronger driving downstream gene expression, and the expression that is driven does not show position effect yet; Mouse, rat and rabbit WAP gene have the ability of very strong driving downstream gene expression, expression amount in every milliliter of milk can reach several milligrams even more than ten milligram, but its shortcoming is that the specificity of expressing is relatively poor, except milk, the quite expression of level is also arranged in the blood, may have some bad influence animal.It is exactly that most of transgene expression level is extremely low that Study on Transgenic Animal also exists one and headachy problem simultaneously, and few part transgene expression level is too high.Because the influence of position effect, most of transgenosis (especially cDNA) expression level is low as to be difficult to detect, and indivedual genetically modified expression level is too high.Commentaries on classics h α as making such as Wright (1991) ₁AT gene sheep, wherein transgenic sheep after lambing during lactation, h α in the milk ₁The level of AT is up to 60g/L, the h α of lactation milk in mid-term ₁The AT level still maintains 35g/L, and this high level expression of foreign gene is that host animal is difficult to bear.

Therefore the research that the expression regulation of milk protein gene is carried out extensively and profoundly is very important.Research method to gene expression regulation is a lot, and one of them is exactly that control region composition to the genetic expression of the species with different expression efficiencies is analyzed, and therefrom obtains Useful Information.About 5.5 grams of existing bibliographical information yak milk proem content/100 gram dry-matteies, wherein 61% alpha-whey albumin of casein and milk proem total amount accounts for 6.08%, beta-lactoglobulin accounts for 16.76%, other albumen accounts for 16.16% (KattsinA, E.V.1993) and the milk proem content of ox be about 3.7 the gram/100 the gram dry-matteies, wherein casein accounts for 80% of total protein content, beta-lactoglobulin accounts for 4 ~ 5% of total protein content, alpha-whey albumin accounts for 12% of total protein content, and other albumen accounts for 3.5%.Therefore from the shared ratio of each composition of the total content milk proem of milk proem all be have very big-difference other.The clonal analysis mistake and the control region of the milk protein gene of yak is also had no talent.The clone measures seven kinds of milk proem gene orders of yak (full gene or partial sequence), analyzes the regulation and control composition that its control region comprises, and compares with the corresponding construction of ox, may can obtain some Useful Informations.

In addition, yak (Bos grunniens) is species in the Niu Yake Bos ox subgenus, mainly be distributed in highlands, the Central Asia, it is generally acknowledged that it is to be tamed by Bos mutus, it is a kind of instrument of mainly packing of extremely frigid zones, its colory meat and milk are the highlands people's main food sources, and fur is again the superfine product of resistance to colds.The quantity of China yak accounts for more than 90% of world's yak sum; mainly be distributed in the Qinghai-Tibet Platean; it is the important species resource of China; yet at present yak quantity descends just with surprising rapidity and the danger of final disappearance is arranged; therefore; for protection and the development and use of strengthening the yak resource, imperative to the research that yak carries out extensively and profoundly.

As ruminating animal, the galactopoiesis proterties is important economic characters, the milk of yak has some common oxen characteristics can't be obtained, has higher anti-microbial activity as its milk, and protein content is than common cow's milk height, therefore but the milk yield of yak is very low, and improving its milk yield, to keep its good milk quality again simultaneously be an important topic of yak breeding.Realize that above-mentioned target developing and the yak relevant genetic marker of performance of giving milk is an important prerequisite, and exploitation is the milk proem gene with yak relevant its first-selected candidate gene of genetic marker of performance of giving milk, yet owing to some reasons of the residing special geographical position of yak and other are also very limited to the research of yak, the research of molecular level is all the more so, still have nothing to do in the relevant report of yak milk proem gene studies at present, so we clone seven kinds of milk proem gene orders of yak of mensuration for laying a good foundation with the give milk exploitation of the relevant genetic marker of performance of yak.

One of purpose of the present invention has systematically been carried out the cloning and sequencing work of seven kinds of milk proem genes of yak, has obtained seven kinds of milk protein gene complete sequences or the partial sequence of yak.Another object of the present invention is the research and the exploitation of making dna vaccination that is transgenic animal, gene therapy and provide to overlap brand-new controlling element and dna sequence dna information with the give milk work of aspects such as exploitation and application of performance-relevant genetic marker of yak.

The present invention is according to realization as described below:

It is as follows that the present invention has designed and synthesized 15 pairs of its primer sequences of primer according to seven kinds of milk proem gene corresponding sequence of the common ox of having delivered:

The primer amplification scope

(annotate: according to the known array design of ox and determine)

The ALA primer

No. 1 F1 5 ' TATTTAGTGGTATTGGTGGTTGG3 ' comprises 5 ' flanking region 698nt of this gene from 68nt to 1292nt

F2 5 ' TATTCTGTGCTGTCATTGTTTTG 3 ' and first exon, first intron and part second exon.

No. 2 F3 5 ' TCGTCTTTCTTTCAGGGGTC3 ' comprises this gene second exon, portion from 1205nt to 1655nt

F4 5 ' AACTTCATCAACCAACTTAG 3 ' minute second intron.

No. 3 F5 5 ' GACCAGAACCCTCACTCAAGC 3 ' comprise that from 1332nt to 2519nt this Gene Partial second is outer apparent

F6 5 ' CAGAAGCAGCAAAGACAGCAG 3 ', second intron, second exon, the 3rd intron, the 4th

Exon.

No. 4 F7 5 ' CCATAAAGCACTCTGTTCTG 3 ' comprise this gene from 2437nt to 3070nt part all round

F8 5 ' CCTTGTGGGCTACCGTCTAT 3 ' shows son and comprises 3 ' flanking region of tailing signal.

The beta-lactoglobulin primer

No. 5 F9 5 ' TGTCCAGCCTCCCTCCCATAG 3 ' is positioned at gene 5 ' flanking region upstream from 14nt to 1200nt

F10 5′GGGTAGGGGAACAGGAGCAAG 3′

No. 6 F11 5 ' CCCCGCTATCAGGAGACACC 3 ' are positioned at gene 5 ' flanking region and draw for C number from 1155nt to 2182nt

F12 5 ' AGGGAGTGGAGGGCTGAGAC 3 ' thing downstream, 5 ' end and overlapping 45 bases of C fragment, 3 ' end comprises

Preceding 11 bases of this gene first exon

No. 7 F13 5 ' CGGCTCTCCCTCCCCCACAG3 ' has comprised part the 5th intron from 6259nt to 7355nt,

F14 5 ' CCCCTTCCAACCGTGACCTC3 ' 6-7 exon and this gene 3 ' flanking region partial sequence

The beta-casein primer

No. 8 F15 5 ' GCTTTCATTTTCTTCTTCC 3 ' from-991nt to-9nt is 5 ' flanking region of this gene

F16 5 ' ATCTGATTTTGTGGTTGTTT 3 ' sequence

No. 9 F17 5 ' AGAGGATTTCAATGTGAATGC3 ' have comprised this gene the 8th exon from 7412nt to 8578nt

F18 5 ' ATGCCTAAGGGTTAATTTATT3 ', the 8th intron and the 9th exon full sequence.

Kappa-casein primer

No. 10 F19 5 ' ATTACTTCATACTCAGGTTCTT3 ' comprise the part of this gene from-397nt to 41nt

F20 5 ' GCTTGGCAGTAGGTTCAGTTGG3 ' intron and 5 ' flanking region sequence.

No. 11 F21 5 ' CGCTGTGAGAAAGATGAAAGATTC3 ' comprise the 4th exon of this gene from 1530nt to 11310nt

F225 ' AGATTCAAGGAGTATACCAATTGTTG3 ' and part the 4th intron sequences

α-s1 casein primer

No. 12 F23 5 ' ATGGAAAATCAGAGTTATGGTT3 ' comprised from-806nt to 103nt this gene first outside show

F24 5 ' ATGATGAGACAGAGGAAAAT3 ', part first intron and 5 ' flanking region partial sequence.

No. 13 F25 5 ' GATTTTGTCTTTCTTCTTTTC3 ' has comprised from 19596nt to 20577nt outside this gene the 19th

F26 5 ' TTGTGTTTTCCTGATTCTTTG3 ' shows sub 43 sequences and 3 ' flank section sub-sequence.

α-S2 casein primer

No. 14 F27 5 ' TAAGCAATAGAAGATAAACTGG3 ' have comprised in this Gene Partial first to 82nt from-902nt

F28 5 ' AGGTACATAGAATAACAAAAAG3 ' contains son, whole first exons and 5 ' flank section sub-sequence.

The lactoferrin primer

No. 15 F29 5 ' CACAAAACAACACAAGGGGTAG3 ' comprise this Gene Partial from-707nt to 66nt

F30 5 ' CAGCAGGGCGGGGACGAAGAG3 ' exon and part 5 ' flanking sequence.

The method of application PCR is that template is carried out pcr amplification with the genomic dna of yak, to obtain 5 ' flanking sequence of each gene.Its PCR reaction conditions is as follows:

The loop parameter of table 1PCR reaction

94℃	5 minutes	1 circulation
			94 ℃ X ℃ (actual temp sees attached list 2)	1 minute 1 minute	30 circulations

72℃	2 minutes
			4℃	20 minutes	1 circulation

The annealing temperature of the different primer PCR reactions of table 2

The primer sequence number	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
																Annealing temperature (℃)	61	60	68	61	72	68	68	61	61	61	58	61	61	61	68

The PCR reaction system:

1～No. 14 the primer PCR reaction system is: 2.5 μ l, 10 * Taq dna polymerase buffer liquid (500mmol/L KCl, 100mmol/L TrisCl, 20mmol/L MgCl ₂, 0.01% gelatin), 2 μ l dNTPs (2.5mmol/L for each), 1 μ l primer I (50pmol/ μ l), primer I I (50pmol/ μ l), 1U Taq archaeal dna polymerase, template DNA 200ng, end reaction volume are 25 μ l.

The PCR reaction system of the 15th pair of primer is: 2.5 μ l 10 * no magnesium ion Taq dna polymerase buffer liquid (500mmol/L KCl, 100mmol/L TrisCl, 0.01% gelatin), 1.5 μ l MgCl ₂(25mol/ μ l) 2 μ l dNTPs (2.5mmol/L for each), 1 μ l primer I (50pmol/ μ l), primer I I (50pmol/ μ l), 1U Taq archaeal dna polymerase, template DNA 200ng, end reaction volume are 25 μ l.

With the pCR of PCR product cloning to the production of Invitrogen company ^TMOn the II T-carrier, carry out sequential analysis, repeat the accuracy of 3 row that checked order to guarantee with the ABI377DNA sequenator.

Carrying out sequential analysis with sequence analysis softwares such as GENTYP-MAX8.5 uses Http: //www.rw Cp.or.jp/lab/pdappl/papia.htmlThe TFSEARCH:DNA Transcription Factor Binding Site Prediction software of website carries out the prediction of transcriptional regulator binding site.

The effect of invention

The present invention clones and has measured 7 kinds of milk proem gene (alpha-lactalbumin gene complete sequences of yak, beta-lactoglobulin gene 5 ' and 3 ' terminal sequence, αS1-Lao Danbaijiyin 5 ' and 3 ' terminal sequence, α S2-casein gene 5 ' and sequence, one section sequence of beta-casein gene 5 ' terminal sequence and the 8th to the 9th exon thereof, kappa-casein gene 5 ' flanking sequence and the 4th exon thereof and the 4th intron sequences and bovine lactoferrin gene 5 ' terminal sequence etc.) sequence, and the binding site of the transcriptional regulator of the control region sequence of these genes predicted, prove the alpha-lactalbumin that we clone, beta-lactoglobulin, αS1-Lao Danbai, α S2-casein, 5 ' flanking sequence of beta-casein gene has comprised research and the making that the efficient specific expressed needed most of transcriptional regulator binding site sequence of mammary gland can be directly used in transgenic animal, the promoter region sequence that the kappa-casein that we cloned and 5 ' flanking sequence of bovine lactoferrin gene have comprised this gene complete can merge the making that the chimeric 5 ' regulating and controlling sequence of formation is used for transgenic animal with the tissue specificity correlated series of other genes, the research and development of gene therapy and dna vaccination, and the alpha-lactalbumin that we cloned, beta-lactoglobulin, αS1-Lao Danbai, isogenic 3 ' the terminal sequence of kappa-casein and beta-casein can provide good PolyA tailing signal structure for the structure of mammalian expression vector.

The all sequences of the above-mentioned yak milk proem gene that we cloned in addition is for exploitation and the yak performance-relevant molecule marker of giving milk provide indispensable reference information.

Brief description of drawings:

Fig. 1. be yak alpha-whey albumin gene 5 ' flanking sequence, it has the TATA box of two potential functions to lay respectively at-25 ~-33 and-239 ~-247 position through scanning discovery, agrees with sequence and is respectively CAAATAAAA and TAAATAAA.The binding site of its mammary tissue idiosyncratic transcription factor (MGF) may be positioned at-454 ~-467 position, nucleotides sequence is classified CACTTCTTTTGTTT as, this section sequence almost all is (the Maritien a.M.1993) that guards in all milk proem genes, another kind of mammary gland-specific transcription factor is called milk proem binding factor (MPBF), at first from lactoglobulin gene promoter district, find, the binding site of this factor may be positioned at-267 ~-279 position.The binding site (Maritien a.M.1993) that a more typical PMF (conceived specificity nf) is arranged in-263 ~-635 position in addition, binding site (the Yue Junming that a glucocorticoid receptor is arranged on-583 ~-588 position, 1995), locational this section sequence-55 ~-67, with the sequence of the protein-bonded recognition site of TGGCA in the lysozyme gene sequence 85% homology is arranged, may be relevant with the transcriptional control of alpha-whey albumin.This site plays a part transcriptional control (Jean Lac Vilotte, 1987) in N,O-Diacetylmuramidase.

Above-mentioned analytical results shows this section control region sequence of the alpha-whey albumin of the yak that we clone, substantially possessed the various control region compositions that specific efficient is expressed in mammary gland, but it carries out the experimental study of transgenic animal as the control region of foreign gene.Compare with the corresponding regulatory factor recognition site of ox, have only some variation slightly of AGF recognition site, promptly comparative result is listed in this:

Yak CACTTCTTTTGTTT (454 ~-467)

***** ********

Ox CACTTGTTTTGTTT (454 ~-467)

Mode sequences-RNTTCYTRGAAYY

As if the sequence that the pattern recognition sequence of the MPBF/MGF that is summed up with Martien A.M. in 1993 etc. is compared yak is more better than ox, and this species diversity has nonsensically actually certainly, also needs further experiment confirm.

Fig. 2. be yak beta-lactoglobulin 5 ' flanking sequence, through scanning discovery, it has two potential TATA frames, lay respectively at-25 ~-29 and-690 ~ 696, their sequence is respectively TATAA and TTTAAAA fails to find comparatively typical C AAT structure near-75 districts, is GTCAATTG and in-915 ~-922 position its sequence of comparison typical C AAT structure is arranged.We have also found 5 more typical MPBF recognition sites simultaneously, they lay respectively at-193 ~ 205,-653 ~ 664,-1300 ~-1312 ,-1967 ~-1979 and-2045 ~ 2056, its sequence is respectively CCTACCAGGAACC, GGGTCCTGTAGG, TGTTGCAAGAATT, GCAGCTTGGACGT, GTTCCTGGAAGT.There are two MGF recognition sites to lay respectively at-205 ~-216 and-923 ~ 935.Their sequence is: CACTTCTGGGGC and AGTTGCTTAGAAT.It is a kind of trans regulator that a factor recognition site factor is arranged on-1956 ~-1964 position, the effect of its inhibition of gene expression is disengaged (Yue Junming etc. 1998) by functions of hormones, a TRE (pth receptor) recognition site is arranged on-582 ~-597 position, and its sequence is CGCAGGGTCAGGTCA.At-1870 ~-1875 places a glucocorticoid receptor binding site is arranged.Compare with the corresponding sequence of ox (following only listed discrepant sequence comparative result)

Yak MPBF1 GCAGCTTGGACT-1967 ~-1979

Ox MPBF1 GCAGCTCGGACGT-1967 ~-1979

Yak MPBF2 CCTACCAGGAACC-193 ~-205

Ox MPBF2 CGTACCAGGAACC-193 ~ 205

Yak MGF AGTTGCTTAGAAT-923 ~-935

Ox MGF ACTTGCTTAGAAT-923 ~-935

Mode sequences RNTTCYTRGAAYY (Li Ning, 1997)

Annotate: R represents that purine Y represents pyrimidine

Comparative result shows that they are identical with the degree of conformity of mode sequences, so they may not have difference in function.

Fig. 3. be yak αS1-Lao Danbai 5 ' flanking sequence, through scanning as can be seen, it has two TATA boxes with potential function and lays respectively at-23 ~ 29 and-83 ~-89.Its sequence all is TTTAAAT.The binding site of its mammary tissue idiosyncratic transcription factor (MGF) is positioned at-87 ~-100 position, nucleotides sequence is classified the another kind of mammary gland-specific transcription factor of AATTCTTAGAATT-milk proem binding factor (MPBF) as, the possible binding site of base has two to lay respectively at-11 ~-24 and-333 ~-346 position, and its sequence is respectively ATAGCTTGGAAGCA and TTGTCCCAAGAATT.But its locational sequence TCTCCTTAGAATTTCTTGGG of-139 ~-158 is called " milk box " (milk box), is the binding site of the active general nuclear factor of a kind of CTF/NF-1 of being similar to.And the core binding site of another kind of general nuclear factor oct-1 on yak α S1 casein gene sequence may be positioned at-47 ~-54 position, and sequence then is AATTAGCA.Therefore mammary gland-specific transcription factor binding site point and some other important regulating and controlling elements of yak α S1 casein gene all are positioned between-400 ~-10, one section responsive albumen consensus sequence of calcium at-109 ~-125 places.Its sequence is that AGAACATTGATTTCCTA compares with the corresponding controlling element of ox, does not find any difference.

Fig. 4. for yak α S2-casein gene 5 ' flanking sequence,, on-24 ~-30 positions a TATA box through scanning discovery.Its sequence is TATAAT.The responsive caseic consensus sequence of calcium on-46 ~-66 positions, it can be categorized as two portions, last partial sequence CAAACCAC strengthens one section very similar sequence of electronic publishing system to SV40, and it is a 0cc 1 factor recognition factor that the rear portion is divided into AATTACCATAT.Another general nuclear factor 0ct 1 also has three recognition sites, lays respectively at-208 ~-218 ,-257 ~ 267 and-469 ~-478 position.The recognition site that a mammary gland-specific transcription factor (MGF) is arranged in-87 ~-100 position has the recognition sequence of a special feelings transcription factor of another kind of mammary gland in-330 ~-344 position, its sequence is TCGTGACTAGAAGAG.On-232 ~-246 position, there is the sequence homology of the above estrogen receptor binding site of vitellogenin gene II of one section sequence and chicken very high.Its sequence is GAGCTACACAAACAT.May be relevant to the expression regulation of milk proem with oestrogenic hormon.At-176 ~-181 locational TGTTCT are parts of glucocorticoid receptor, may be relevant to the milk proem expression regulation with glucocorticosteroid.On-109 ~-123 position, the responsive proteic consensus sequence of three kinds of calcium is arranged, its sequence is AGAAAA GCAATTCTAA, compares with the corresponding transcription factor recognition site of ox, two mammary gland-specifics are transcribed recognition site, and to have some differences to be listed as now as follows:

Yak MPBF TCGTGAC-TAGAA

Cattle MPBF TCGTGACGTAGAA

Yak MPBF2 CCTACCAGGAACC

Yak MGF GACTTCTTAGGAT

Cattle MGF GACTTCTTAGGAT

Fig. 5. for yak beta-casein gene 5 ' flanking sequence, through scanning discovery, be the TATA box of a tool potential function-26 ~-35, beta-casein 5 ' flanking region lacks the typical C CAAT box.In-45 ~-65 districts is α S1, α S2, one section consensus sequence of beta-casein, its sequence is CAAACCACAAAATTAGCATGC, it can be divided into two parts, its 5 ' end parts CAAACCAC is very similar to the enhanser structure of SV40, and 3 ' end parts ATTAGCAT is the binding site of a 0ct-1.It is GATTTCTAGGAATT that its sequence of binding site of a MGF is arranged in-90 ~-103 position.On-111 ~-127 position, also have one section total sequence of another one α S1, α S2 and beta-casein, concrete function is not clear, and its sequence is AAGAATTCATTTTCTAA.In-142 ~-160 position " milk box " structure is arranged, its sequence is GCTCCCCAGAATTTTTGGG.The first half of this sequence is that GCTCCCCAGAATT is the binding site of MPBF.Agree with sequence with the corresponding transcription factor of ox and compare, do not find any difference.

Fig. 6. be yak κ-casein gene 5 ' flanking sequence, because the fragment of being cloned too weak point fails to find more transcription factor binding site point, the binding site that an activator (AP-1) is arranged at-47 ~-53 places, its sequence is TGACGCA, and the binding site of a trans regulatory factor D is arranged on-268 ~-275 position.The TATA box is positioned at-25 ~-32 position, and its sequence is: TTTAATTA.A CAAT box is arranged on-75 ~-78 position.This shows that the transcriptional regulator of yak κ casein gene mainly is distributed in the far-end of 5 ' flanking region, thus this section sequence that we cloned, be not suitable for use in control region, come expression alien gene.

Fig. 7. be yak lactoferrin 5 ' flanking sequence, only find two SP-1 protein binding sites to lay respectively on-57 ~-64 and-191 ~-196 the position, fail to find the binding site of mammary gland-specific transcription factor by scanning us.Its TATA box is positioned at-24 ~-28 position, and a complementary CAAT box is positioned at-71 ~-75 position.This shows that we clone this fragment and are not suitable for coming expression alien gene as controlling element.

In sum, we clone and have measured 7 kinds of milk proem gene (alpha-lactalbumins of yak, beta-lactoglobulin, αS1-Lao Danbai, α S2-casein, beta-casein, kappa-casein and bovine lactoferrin gene etc.) 5 ' flanking sequence, and the binding site of the transcriptional regulator of these sequences predicted, prove the alpha-lactalbumin that we clone, beta-lactoglobulin, αS1-Lao Danbai, α S2-casein, 5 ' flanking sequence of beta-casein gene has comprised research and the making that the efficient specific expressed needed most of transcriptional regulator binding site sequence of mammary gland can be directly used in transgenic animal, and the promoter region sequence that the kappa-casein that we cloned and 5 ' flanking sequence of bovine lactoferrin gene have comprised this gene complete can merge with the tissue specificity correlated series of other genes and form the making that chimeric 5 ' regulating and controlling sequence be used for transgenic animal, the research and development of gene therapy and dna vaccination.

Fig. 8. the aminoacid sequence of yak alpha-lactalbumin gene sequence and prediction is annotated: capitalizing the sequence that does not add frame among the figure is exon sequence, the little write sequence of letter is flanking sequence or intron sequences, the sequence that the Italic capitals edged is rectified is various expression regulation factor recognition site sequences, italic underscore sequence is to be palindrome in the sequence of ox, but in the sequence of yak, interrupted and no longer be palindrome by base mutation (mutating alkali yl is write with black matrix), block letter underscore sequence is a palindrome all in the sequence of yak and common ox, and small letter black matrix sequence is that intron is sheared recognition site among the figure.

The sequencing results shows that it promptly all is by 5 ' flanking region that the alpha-lactalbumin gene of yak has the gene structure similar to common ox, four exons, three introns and 3 ' flanking region constitute, comparison shows that by corresponding sequence this gene complete sequence homology of yak alpha-lactalbumin gene complete sequence and ox is 98% with ox (Vilotte J.L.et al.1987), 5 ' flanking sequence wherein, the coding region of exon and 3 ' flanking region homology are 99%, its coded amino acid sequence homology also is 99%, the homology of intron and 3 ' non-coding region is that the homology of 98%, 5 ' non-coding region is 93%.

Detailed sequence comparative result shows that in this gene non-coding sequence of ox, the palindrome of some of existence 5 to 10 bases disappears (as shown in Figure 4) because of base mutation in the sequence of yak.Especially wherein-1～-10 and+16～+ 25 sequence is palindrome in the sequence of ox, and it is destroyed because of the variation of two bases in the sequence of yak, because regulation and control have certain effect the palindrome of non-coding sequence to expression of gene in the gene structure, and the alpha-whey albumin in yak Ruzhong accounts for 6.08% (Wang Yong etc., 1998 of total milk proem; Wang Yusong etc., 1995), but only accounting for 3.6% (Wang Yusong etc., 1995) common ox, the destruction that therefore can infer these palindromes is the reason that this gene has higher level to express in yak.

Fig. 9 yak αS1-Lao Danbaijiyin 3 ' terminal sequence

It exon that comprises partly is black matrix capitalization part, and underscore partly is a PolyA tailing signal structure.

Figure 10 yak beta-lactoglobulin gene 3 ' terminal sequence

The letter small letter partly is intron sequences or 3 ' flanking sequence, and the capitalization part is an exon sequence, and underscore partly is the PolyA tailing signal.

Figure 11 yak kappa-casein gene 3 ' terminal sequence

Capitalization partly is apparent all round subdivision, and capitalization and italicized item are the coding region part, and non-italicized item is 3 ' non-coding region, and lowercase partly is 3 ' flanking sequence, and underscore partly is a PolyA tailing signal part.

Figure 12 yak beta-casein gene 3 ' terminal sequence

Capitalization partly is the exon part, and lowercase partly is the intron sequences part, and underscore partly is a PolyA tailing signal structure division

3 ' the terminal sequence that can find out yak alpha-lactalbumin gene, β-lactoglobulin gene, αs1-caseingene, kappa-casein gene and the beta-casein gene of our institute's colony assay from Fig. 8-12 has all possessed the needed PolyA tailing signal of making mammalian expression vector structure, the structure of available and mammalian expression vector, thus be used for making, gene therapy and the dna vaccination etc. of transgenic animals.

Embodiment

Materials and methods

One, material

1. 1. milk cow blood sample:

China Holstein milk cow blood sample adopts doube bridge cattle farm, doube bridge farm, Beijing.

2. yak blood sample: Part of Qinghai Plateau type yak, pick up from logical greatly cattle farm, Qinghai Province.Yak is organized sample: wherein 30 are picked up from the Xining, Qinghai Province, are responsible for collection by Mr. He Chengji of Qinghai Hai Bei herding Science Institute.Other 30 Mr.s Luo Xiaolin by Qinghai animal and veterinary institute provide these 60 yaks all to belong to the Part of Qinghai Plateau type.

2. bacterial classification:

Bacillus coli DH 5 alpha, intestinal bacteria JM101 and e. coli jm109 are this chamber and preserve.

3. plasmid

PCR ^TMII is available from Invertrogen company.

4. enzyme and reagent:

Various restriction enzymes, RNase, Proteinase K, IPTG, X Gal, alkaline phosphatase, T 4 dna ligases etc. are available from Huamei Bio-Engrg Co., and U.S. Promega company.The Taq enzyme provides for this chamber.All the other chemical reagent are homemade, import packing or import.

5. test kit:

GENECLEANII Kit is U.S. Bio 101 company's products.

Dyeprimer sequencing Kit is available from PE company

Two, solution preparation

The preparation of substratum, reagent all is solvent with the redistilled water if having no special requirements, and the autoclaving condition is 15lbf/in 2 (1.034 * 10 ⁵Pa) vapor sterilization is 20 minutes.

1. substratum preparation

(1) LB liquid nutrient medium: peptone 10g, yeast powder 5g, NaCl 10g is dissolved in the 800ml water, regulates pH value to 7.5, is settled to 1000ml, autoclaving.

(2) LB solid medium: peptone 10g, yeast powder 5g, NaCl 10g is dissolved in the 800ml water, adds agar powder 15g,

Regulate pH value to 7.5, be settled to 1000ml, autoclaving.

2.STE damping fluid: 0.1mol/L NaCl, 10mmol/L TrisCl (pH8.0), 1mmol/L EDTA (pH8.0), autoclaving.

3. plasmid extracts solution I: 50mmol/L glucose, 25mmol/L TrisCl (pH8.0), 10mmol/L EDTApH8.0), 10lbf/in 2 (6.895 * 10 ⁴Pa) vapor sterilization 15 minutes under the high pressure.

4. plasmid extracts solution II: 0.2mmol/L NaOH, 1%SDS, matching while using.

5. plasmid extracts solution III: 5mol/L potassium acetate 60ml, glacial acetic acid 11.5ml, water 28.5ml, 4 ℃ of preservations.

6.4N NaOH:80g NaOH is dissolved in the 400ml water, is settled to 500ml.

7.10%SDS:100g SDS is dissolved in the 90ml water, is heated to 68 ℃ of hydrotropies, with HCl adjust pH to 7.2, is settled to 1000ml.

8.1mol/L TrisCl (pH8.0): 121.1g Tris alkali, be dissolved in the 800ml water, regulate pH value to 8.0 with HCl, be settled to 1000ml.Autoclaving.

9.0.5mol/L EDTA (pH8.0): 186.1g two water disodium ethylene diamine tetraacetate add in the 800ml water, add NaOH adjust pH to 8.0, are settled to 1000ml.Autoclaving.

10.10mol/L ammonium acetate: the 770g ammonium acetate is dissolved in the 800ml water, adds the water constant volume to the degerming of 1000ml after-filtration.

11.13% (w/v) PEG800: 0.13g PEG8000 is dissolved among the 1ml 1.6mol/L NaCl (matching while using).

12. lysozyme soln: 10mg/ml is dissolved among the 10mol/L TrisCl (pH8.0).

13.RNase solution: RNase is dissolved among the 10mol/L TrisCl (pH8.0), among the 15mmol/LNaCl, is made into the concentration of 10mg/ml,, boiled 15 minutes in 100 ℃.

14.TE damping fluid (pH8.0): 10mmol/L NaCl, 10mmol/L TrisCl (pH8.0), 1mmol/L EDTA (pH8.0), autoclaving.

15.5mol/L LiCl:LiCl2H ₂O 302.05g is settled to 1000ml after being dissolved in 800ml water fully, autoclaving.

16.50 * TAE:242g Tris alkali, the 57.1ml glacial acetic acid, 100ml 0.5mol/L EDTA (pH8.0) is settled to 1000ml after the dissolving.

17. penbritin (Amp) stock solution: the 100mg/ml sodium ampicillin is stored in-20 ℃.

18.5mol/L NaCl:292.2g NaCl is dissolved in the 800ml water, is settled to 1000ml, autoclaving.

19.IPTG solution: 1g IPTG is dissolved in the 5ml water, and filtration sterilization is distributed into 1ml/ part, is stored in-20 ℃.

20.x-gal: be made into the 20mg/ml stock solution, keep in Dark Place with dimethyl formamide dissolving 5-bromo-4-chloro-3-indoles-β-D-galactoside in-20 ℃.

21.3mol/L NaAc (pH5.2): the 24.604g anhydrous sodium acetate is dissolved in the 80ml water, regulates pH value to 5.2 with glacial acetic acid, is settled to 100ml, autoclaving.

22.1mol/L CaCl ₂: dissolving 54g CaCl in 200ml water ₂6H ₂O, filtration sterilization is distributed into every part of 10ml, stores in-20 ℃, takes out portion during the preparation competence and is diluted to 100ml, filtration sterilization, pre-cold standby.

23. ethidium bromide stock solution: add the 1g ethidium bromide in 100ml water, preserve in 4 ℃ of brown bottles the dissolving back.

24.6 * gel loading buffer: 0.25% tetrabromophenol sulfonphthalein, the blue or green FF of 0.25% dimethylbenzene, 30% aqueous glycerin solution, 4 ℃ of preservations.

25.ACD antithrombotics: the 0.48g citric acid, the 1.32g Trisodium Citrate, 1.47g glucose is dissolved in the 100ml water; Autoclaving.

26.DNA extraction extraction buffer: 1mmol/L TrisCl (pH8.0), 0.1mol/L EDTA (pH8.0), 20 μ g/ml pancreas RNase, 0.5%SDS.

27.2 * Cracking buffer:0.2N NaOH, 0.5%SDS, 20% sucrose.

28. saturated phenol: newly steam phenol, add hydroxyquinoline to final concentration 0.1%, add equal-volume 0.5mol/L TrisCl (pH8.0) and mix stirring 15 minutes, two-phase is removed the upper strata after separating, with equal-volume 0.1mol/L TrisCl (pH8.0) repeat extracting to phenol phase pH value greater than 7.8, be stored in 4 ℃ of brown bottles and be under 10mmol/L TrisCl (pH8.0) covering.

29. phenol: imitative (1: 1): equal-volume phenol, chloroform mix, and are stored in 4 ℃, in the brown bottle.

30. proteolytic enzyme (K solution): water is made into 20mg/ml ,-20 ℃ of preservations.

31. phosphoric acid buffer (PBS): dissolving 8g NaCl in 800ml distilled water, 0.2g KCl, 1.44g Na ₂HPO ₄With 0.24g KH ₂PO ₄, be used for HCl and regulate pH value to 7.4, add the water constant volume to 1000ml, autoclaving.

32.10 * Taq enzyme buffer:100mmol/L TrisCl (pH8.0), 500mmol/L KCl, 20mmol/L MgCl ₂, 0.1% gelatin.

Three, experimental procedure

1. mammalian genes group DNA extraction (blood sample)

(1) frozen blood sample (20ml): room temperature (or 55 ℃ of water-baths) is melted, and adds the equal-volume phosphoric acid buffer, mixing, and centrifugal 15 minutes of 3500g abandons supernatant, adds the resuspended precipitation of 15ml extract.

(2) 37 ℃ are incubated 1 hour.

(3) adding Proteinase K to final concentration is 100 μ g/ml, mixing.

Digest in (4) 55 ℃ of water-baths, the time is looked the solution becomes thickness till bright, shakes up reaction solution in the digestive process frequently gently.

(5) add isopyknic phenol, phenol: each extracting of chloroform once.During extracting, the gentle centrifuge tube that rotates mixes two-phase, room temperature 5000g then, and centrifugal 15 minutes, sucking-off upper strata water moved in another clean centrifuge tube.

(6) add the 10mol/L ammonium acetate of 0.2 times of volume and the dehydrated alcohol of 2 times of volumes, shake up gently under the room temperature.

(7) the DNA amount is chosen the oyster white flocks for a long time.When no obvious sediment forms, can be placed on-20 ℃ and spend the night, centrifugal 15 minutes of 5000g, collecting precipitation.70% alcohol is washed.

(8) vacuum is drained, but DNA can not be taken out too dried, and the alcohol that loses on the pipe end and the tube wall gets final product.

(9) add an amount of TE dissolving.

(10) detect DNA quality and concentration.

2. the extraction (high salt method) of genomic dna in the tissue:

Take a morsel after the tissue dried by air directly to press after shredding and carry out, after the tissue of 70% alcohol-pickled mistake shreds, press the step after vacuum is drained again and carry out.

(1) add 1ml DNA extract, 10 μ l Proteinase Ks (10mg/ml), in 55 ℃ of digestion 24 hours, the centre was added the equivalent Proteinase K 1 time.

(2) be packed as 400 μ l pipe, every pipe adds preheating 5M NaCl 170 μ l, and making the NaCl final concentration is 1.5M, mixing.

(3) add 570 μ l phenol: chloroform (1: 1) extracting 1 time.

(4) 12000rpm is centrifugal 15 minutes, and visible one dark egg white layer is got supernatant liquor on the interface, adds 2 times of volume dehydrated alcohol deposit D NA, and 70% ethanol is washed 2 times, drains slightly, is dissolved among the TE 4 ℃ or-20 ℃ of prolonged preservation.

3. the purifying of genomic dna (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate).

4.PCR primer design

The primer amplification scope

(annotate: according to the known array design of ox and determine)

The ALA primer

No. 1 F1 5 ' TATTTAGTGGTATTGGTGGTTGG 3 ' comprises 5 ' flanking region 698nt of this gene from 68nt to 1292nt

F2 5 ' TATTCTGTGCTGTCATTGTTTTG 3 ' and first exon, first intron and part second exon.

No. 2 F3 5 ' TCGTCTTTCTTTCAGGGGTC 3 ' comprises this gene second exon, portion from 1205nt to 1655nt

F4 5 ' AACTTCATCAACCAACTTAG 3 ' minute second intron.

No. 3 F5 5 ' GACCAGAACCCTCACTCAAGC 3 ' comprise that from 1332nt to 2519nt this Gene Partial second is outer apparent

F6 5 ' CAGAAGCAGCAAAGACAGCAG 3 ', second intron, second exon, the 3rd intron, the 4th

Exon.

No. 4 F7 5 ' CCATAAAGCACTCTGTTCTG 3 ' comprise this gene from 2437nt to 3070nt part all round

F8 5 ' CCTTGTGGGCTACCGTCTAT 3 ' shows son and comprises 3 ' flanking region of tailing signal.

The beta-lactoglobulin primer

No. 5 F9 5 ' TGTCCAGCCTCCCTCCCATAG 3 ' is positioned at gene 5 ' flanking region upstream from 14nt to 1200nt

F10 5′GGGTAGGGGAACAGGAGCAAG 3′

No. 6 F11 5 ' CCCCGCTATCAGGAGACACC 3 ' are positioned at gene 5 ' flanking region and draw for C number from 1155nt to 2182nt

F12 5 ' AGGGAGTGGAGGGCTGAGAC 3 ' thing downstream, 5 ' end and overlapping 45 bases of C fragment, 3 ' end comprises

Preceding 11 bases of this gene first exon

No. 7 F13 5 ' CGGCTCTCCCTCCCCCACAG3 ' has comprised part the 5th intron from 6259nt to 7355nt,

F14 5 ' CCCCTTCCAACCGTGACCTC3 ' 6-7 exon and this gene 3 ' flanking region partial sequence

The beta-casein primer

No. 8 F15 5 ' GCTTTCATTTTCTTCTTCC 3 ' from-991nt to-9nt is 5 ' flanking region of this gene

F16 5 ' ATCTGATTTTGTGGTTGTTT 3 ' sequence

No. 9 F17 5 ' AGAGGATTTCAATGTGAATGC3 ' have comprised this gene the 8th exon from 7412nt to 8578nt

F18 5 ' ATGCCTAAGGGTTAATTTATT3 ', the 8th intron and the 9th exon full sequence.

Kappa-casein primer

No. 10 F19 5 ' ATTACTTCATACTCAGGTTCTT3 ' comprise the part of this gene from-397nt to 41nt

F20 5 ' GCTTGGCAGTAGGTTCAGTTGG3 ' intron and 5 ' flanking region sequence.

No. 11 F215 ' CGCTGTGAGAAAGATGAAAGATTC3 ' comprise the 4th exon of this gene from 1530nt to 11310nt

F22 5 ' AGATTCAAGGAGTATACCAATTGTTG3 ' and part the 4th intron sequences

α-S1 casein primer

No. 12 F23 5 ' ATGGAAAATCAGAGTTATGGTT3 ' comprised from-806nt to 103nt this gene first outside show

F24 5 ' ATGATGAGACAGAGGAAAAT3 ', part first intron and 5 ' flanking region partial sequence.

No. 13 F25 5 ' GATTTTGTCTTTCTTCTTTTC3 ' has comprised from 19596nt to 20577nt outside this gene the 19th

F26 5 ' TTGTGTTTTCCTGATTCTTTG3 ' shows sub 43 sequences and 3 ' flank section sub-sequence.

α-S2 casein primer

No. 14 F27 5 ' TAAGCAATAGAAGATAAACTGG3 ' have comprised in this Gene Partial first to 82nt from-902nt

F28 5 ' AGGTACATAGAATAACAAAAAG3 ' contains son, whole first exons and 5 ' flank section sub-sequence.

The lactoferrin primer

No. 15 F29 5 ' CACAAAACAACACAAGGGGTAG3 ' comprise this Gene Partial from-707nt to 66nt

F30 5 ' CAGCAGGGCGGGGACGAAGAG3 ' exon and part 5 ' flanking sequence.

5. the mensuration of primer concentration

Spectrophotometric instrumentation OD260 value, primer concentration is calculated as follows:

C (μ M)=OD260 * 100 * extension rate/1.54 * N _A+ 0.74 * N _C+ 0.87 * N _T+ 0.74 * N _G

N represents the number of each base.

According to measurement result, the part primer is diluted to 50pmol/ μ l.

6.PCR reaction conditions

This time in its PCR reaction conditions of the primer except that annealing conditions, other can be identical, so for for simplicity, list a template program below earlier and see Table 3, and then the annealing temperature of each primer listed sees Table 4:

The loop parameter of table 3PCR reaction

94℃	5 minutes	1 circulation
			94 ℃ X ℃ (actual temp sees attached list 2) 72 ℃	1 minute 1 minute 2 minutes	30 circulations
4℃	20 minutes	1 circulation

The annealing temperature of the different primer PCR reactions of table 4

The primer sequence number	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
																Annealing temperature (℃)	61	60	68	61	72	68	68	61	61	61	58	61	61	61	68

The PCR reaction system:

1～No. 14 the primer PCR reaction system is: 2.5 μ l, 10 * Taq dna polymerase buffer liquid (500mmol/L KCl, 100mmol/L TrisCl, 20mmol/L MgCl ₂, 0.01% gelatin), 2 μ l dNTPs (2.5mmol/L for each), 1 μ l primer I (50pmol/ μ l), primer I I (50pmol/ μ l), 1U Taq archaeal dna polymerase, template DNA 200ng.

The PCR reaction system of the 15th pair of primer is: 2.5 μ l 10 * no magnesium ion Taq dna polymerase buffer liquid (500mmol/L KCl, 100mmol/L TrisCl, 0.01% gelatin), 1.5 μ l MgCl ₂(25mol/ μ l) 2 μ l dNTPs (2.5mmol/L for each), 1 μ l primer I (50pmol/ μ l), primer I I (50pmol/ μ l), 1U Taq archaeal dna polymerase, template DNA 200ng.

7. ligation

(1) amount of calculating external source fragment and carrier:

Size (the kb) * insertion fragment of size (kb)/carrier of insertion DNA (ng)=carrier (ng) * insertion DNA and the molecular ratio of carrier.

The general sheet segment molecule that inserts: carrier molecule=3 ~ 8: 1

(2) ligation system (10 μ l)

The 50ng carrier DNA

An amount of foreign DNA

1 μ l T ₄Dna ligase 10 * buffer

1 ~ 2U T ₄Dna ligase

Benefit adds water to 10 μ l

(3) 14 ~ 16 ℃ of incubations 8 ~ 12 hours.

(4) get 5 μ l and connect the liquid conversion, all the other-20 ℃ frozen.

8. the preparation of competent cell (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

9.DNA transform (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

10. the quick preliminary evaluation of recombinant plasmid

(1) gets one and be added with X gal, the LB flat board of IPTG and Amp.

(2) picking transforms the hickie line, chooses a locus coeruleus simultaneously and is scribed ss contrast.

Cultivated 12 hours for (3) 37 ℃.

(4) the cultured thalline of picking is suspended in the 50 μ l precooling STET solution centrifugal pipes.

Boiled expense 40 seconds in (5) 100 ℃ of water-baths.

(6) 12000rpm, centrifugal 5 minutes.

(7) will in pipe, choose by bottom settlings with the rifle head.

(8) get supernatant 10 μ l point sample electrophoresis.

(9) relatively from the DNA rate of migration of white colony and blue colonies, the plasmid that band obviously lags behind may contain the insertion fragment to ultraviolet lamp, is recombinant plasmid down.

11. the small-scale of plasmid DNA is extracted (alkaline lysis) (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

12. the extensive extraction (alkaline lysis) of plasmid DNA (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

13. plasmid DNA purification process (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

14.DNA enzyme cut (be used to prepare external source fragment, identify positive colony) (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate etc.)

(1) getting a certain amount of carrier pGEM 5zf (+) cuts with Sca I enzyme entirely.

(2) phenol: imitative extracting.

(3) ethanol sedimentation.

(4) the centrifugal supernatant of abandoning, vacuum is drained, suitably water dissolution.

(5) concentration and the purity of mensuration DNA.

15.DNA fragment electrophoresis (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

16.DNA segmental recovery (referring to " molecular cloning experiment guide ", volumes such as second edition " U.S. " J. Sa nurse Brooker, the related Sections that Quan Dongyan etc. translate)

17. order-checking: sequencing reaction is undertaken by Dyeprimer Sequencing test kit (PE company) specification sheets, and nucleotide sequence is determined to carry out on the full-automatic sequence instrument of ABI377 (ABI company).The accuracy that each clone's sequencing need are checked order to guarantee and are listed as through three repetitions.

18. sequential analysis: carry out sequential analysis with sequence analysis softwares such as GENTYP-MAX8.5 and use Http: //www.rwcp.or.jp/lab/pdappl/papia.htmlThe TFSEARCH:DNA Transcription Factor Binding Site Prediction software of website carries out the prediction of transcriptional regulator binding site.

We clone and have measured 7 kinds of milk proem genes of yak (alpha-lactalbumin by above-mentioned steps, beta-lactoglobulin, αS1-Lao Danbai, α S2-casein, beta-casein, kapp a-casein and bovine lactoferrin gene etc.) sequence (complete sequence or partial sequence), and the binding site of the transcriptional regulator of these sequence control regions part predicted, prove the alpha-lactalbumin that we clone, beta-lactoglobulin, αS1-Lao Danbai, α S2-casein, 5 ' flanking sequence of beta-casein gene has comprised research and the making that the efficient specific expressed needed most of transcriptional regulator binding site sequence of mammary gland can be directly used in transgenic animal, and the promoter region sequence that the kappa-casein that we cloned and 5 ' flanking sequence of bovine lactoferrin gene have comprised this gene complete can merge with the tissue specificity correlated series of other genes and form the making that chimeric 5 ' regulating and controlling sequence be used for transgenic animal, the research and development of gene therapy and dna vaccination.The yak alpha-lactalbumin of mensuration that we clone, beta-lactoglobulin, αS1-Lao Danbai, kappa-casein and the isogenic 3 ' terminal sequence of beta-casein have possessed the needed PolyA tailing signal of structure mammalian expression vector structure, can be used for the structure of mammalian expression vector.These expression vectors can be used for aspects such as the development, gene therapy of making, the dna vaccination of transgenic animal.Our exploitation of cloning the promising genetic marker relevant with the yak milk yield of the seven kinds of milk proem gene orders of yak that obtain simultaneously provides indispensable reference information.

Claims (1)

1, yak alpha-lactalbumin gene sequence, it is accompanying drawing 8 described sequences.

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