CN1157635A - Alpha-lactalbumin gene constructs - Google Patents
Alpha-lactalbumin gene constructs Download PDFInfo
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- CN1157635A CN1157635A CN95194129A CN95194129A CN1157635A CN 1157635 A CN1157635 A CN 1157635A CN 95194129 A CN95194129 A CN 95194129A CN 95194129 A CN95194129 A CN 95194129A CN 1157635 A CN1157635 A CN 1157635A
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- lactalbumin
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Abstract
The present invention provides recombinant gene constructs for expressing the protein alpha -lactalbumin, especially human alpha -lactalbumin, in bovine cells. Novel genetic constructs, vectors and transformed cells are provided as well as transgenic animals genetically engineered for enhanced expression of alpha -lactalbumin. Human alpha -lactalbumin has been shown to be superior for human infant nutrition and the present invention enables a cheap and effective form for production of the major whey protein in human milk.
Description
The gene constructs that the present invention relates to recombinate, this construction can be expressed alpha-lactalbumin or its function equivalent or its part.
The breast of other types such as the relative cow's milk of human milk, sheep breast, camel breast, goat breast has more superior nutritive value, helps human infant's growth.Yet many mothers think that breast hello breast is difficult or inconvenient.National greatly in the infant or baby food demand, also extremely need to provide a kind of milk-product that the human milk nutritive value is arranged.
One of key distinction of relative other Mammalss (for example ox or the sheep) breast of human milk is that it contains alpha-lactalbumin as main whey-protein.Although alpha-lactalbumin also is present in the Ruzhong of other kinds, its concentration is relatively low, and main whey-protein is a beta-lactoglobulin.The alpha-lactalbumin level plant with plant between different, people's milk content is about 2.5mg/ml, cow's milk 0.5-1.0mg/ml, the newborn 0.1-0.8mg/ml of mouse.
The gene order of coding ox alpha-lactalbumin and human alpha-lactalbumin is illustrated; There is the class sequence information to deliver at Biochem J242:735-742 (1987) at Biochemie 69:609-620 (1987) and Hall etc. by Vilotte etc. respectively.
The present invention seeks to utilize genetic engineering technique to make up the gene of reorganization, makes that can produce content in the Ruzhong when this recombination construction is expressed in cells of mamma animals is higher than 1.0mg/ml, as 1.2mg/ml or higher alpha-lactalbumin.Described construction generally is suitable for especially expressing in the ox cell at inhuman zooblast.
On the one hand, the invention provides a kind of recombinant expression system that is suitable in the preferred ox cell of non-human animal's cell, expressing alpha-lactalbumin or its function equivalent or its part.Preferably, recombinant expression system of the present invention is suitable for expressing human alpha-lactalbumin or its function equivalent or its part.
Term used herein " expression system " is meant and comprises protein-coding region and be attached to all gene orders to the hereditary signal of protein expression necessity.Randomly, expression system also comprises such as regulatory elements such as promotor, enhansers to strengthen transcribing and/or translating of encoding histone zone, perhaps control expression.Regulatory element can be positioned at the upstream or the downstream in encoding histone zone, perhaps is arranged in the intron (non-encoding part) of protein-coding region.It also is possible that encoding histone zone sequence self has regulating power.
Term " function equivalent " refers to derivative any and reference sequences or protein function essentially equivalent." function equivalent " comprises that especially those Nucleotide and/or aminoacid sequence are inserted into, lack or replace but biological function especially galactopoiesis biological function do not had the derivative of significant adverse influence.
Genetically engineered all is strong technology to research or to commercial purpose.Use genetic engineering technique (to see Maniatis etc., the molecular cloning laboratory manual, cold spring harbor laboratory, cold spring port, New York, 1982 and " Principle of Genetic Engineening ", Old and Primrose, the 5th edition, 1994, two Wen Jun draw be document) can change exogenous genetic material over to host cell, duplicate therein and/or express by this exogenous genetic material encoded protein or polypeptide.For easy and simple to handle, genetic engineering is done the host with prokaryotic micro-organisms such as E.coli etc. usually, but also uses eukaryote especially as enzyme mother, algae etc.Also use eukaryotic cell to cultivate in some applications.
Brinster etc. are at Cell27:223-231, have described the caused mammals gene alteration of the unicellular embryo's protokaryon of the micro-injection of gene in 1981.Here exogenous genetic material is introduced in the fertilised non-human eggs, and this zygote further develops into the embryo with normal way after in implanting foster mother's body.Real transgenic animal all contain the foreign DNA copy in each cell.
In case the genetic material that injects successfully is incorporated into host chromosome, this animal promptly is called the gene of " transgenic animal " and transfer with the heredity of normal Mendelian's mode, but it is lower that transgenosis is operable to power, especially to large-scale feeding animals such as pig, sheep, ox etc.So far also can not control the position that the gene integration that shifts enters host chromosome.
The donor animal that only produces " mosaic " under the certain condition has been very sufficient for purpose of the present invention.The gene integration that shifts this moment is in the karyomit(e) of certain organ only.The mosaic animal generally obtains by foreign DNA is introduced the embryo who grows than late period.
One of application of the genetically modified tool prospect of livestock is to utilize the Mammals breast to produce as " bio-reactor " and contain breast medicinal and the nutritive value recombinant protein.For expressing foreign protein, the Mammals breast has much magnetism because of it with secretion mode mass production albumen.Recombinant DNA technology can be used to change the albumen composition of the cow's milk that is used for human or animal's consumption, can increase its nutritive value as infant or baby food as expressing human milk-protein in cow's milk.(Ladisch and Boser edits, american chemical association, 38-21 page or leaf (1992) for Strijker etc., Harnessing Biotechnology for the 21st Century).Thereby breeding voluminous animal with common and senior breeding technology, to increase more cheaply that output uses above-mentioned recombinant technology be useful.
The first step in Mammals breast express transgenic is the gene of clone's proteins of interest.In order to make protein expression, generally use the promotor of expressing main milk protein gene in the Ruzhong to the Ruzhong.Milk protein gene is subjected to strict the adjusting, does not express in non-breast tissue, so that organize the possibility of negative impact to drop to minimum to other.Be used for that the proteic regulatory gene of expressing heterologous has α-S1-casein (Strijker et. in breast, 1992, above-mentioned document), beta-lactoglobulin (Wright etc., progress report (1993)) and beta-casein (Ebert Schindler biotechnology 9:831-834 (1991)), whey acidic protein (Ebert and Schindler, transgenosis agricultural animal:, 1993, above-mentioned document).
New gene constructs is general the check at transgenic mice earlier before being used for ox.The breast that derives from transgenic mice is done the recombinant protein quantitative analysis, if the breast amount is sufficient, separable albumen is checked its constitutional features and biological activity.The selection of the particular build thing that is used for ox is mainly considered expression level and the really degree of the recombinant protein that produced.
The gene constructs that the transgenosis of ox starts from the hundreds of copies of injection usually in two protokaryons of zygote.Zygote is taken from uterine tube (Roschlau etc., Arch Tierz, Berlin31:3-8 (1988) in the body; Roschlau etc., breeding and fertilization magazine (Suppl38), the cytobiology of mammalian ovum operation, Greve etc. edit (1989); Hill etc., Theriogenology37:222 (1992); Bowen et al. biological self reproducing 50:664-448 (1994)) or to the ovum of maturation in vitro make (Krimpenforth etc., biotechnology 9:844-847 (1991) in vitro fertilization; Hill etc., 1992, above-mentioned document; Bowen etc., 1993 above-mentioned documents).The zygote of ox needs therefrom to remove opaque lipid in centrifugal several minutes at 15,000 * g, so that use phase microscope, Nomarski and Hoffman interfere phase microscope to observe protokaryon.The damping fluid that 2-4pl is contained hundreds of DNA construction copies injects protokaryon.Transgenosis is considered to be integrated into the breakpoint at random of chromosomal DNA, and this breakpoint is produced by mechanical lysis in the microinjection.It would be desirable that transgenosis is duplicated preceding integration at zygote stage D NA, guarantee that each cell contains transgenosis in the adult.General genetically modified several " copies " become wire to link, and are incorporated into the single site of individual chromosome.Integration site is at random.Integration may take place behind first round dna replication dna, also may (Walland Seidel, 1992) take place evening to 2 or 4 cell stages, produces the animal of transgenosis mosaic.High to 30% can somatic tissue be checked through genetically modified animal not to the offspring transmit this transgenosis (or transport be lower than expection 50%).
After the microinjection, the embryo directly transfer in the uterine tube of acceptor or cultivate a couple of days after transfer to the intrauterine of recipient cattle.Southern engram analysis by to newborn calf's tissue sample proves genetically modified integration.And genetically modified expression is measured by the gene product of analyzing in the suitable tissue, and the present invention is a gene product amount of analyzing the Ruzhong.Embryo survival after the microinjection, the transgenosis integrating frequency, expression frequency and level, gamete system transmits frequency, all different and different because of the amount of DNA construction of injection and matter, employed mouse species (Brinster etc., Proc Natl Acad Sci USA82:4438-4442 (9185)) and operator's technique and skill.This basic skills has been used for production Transgenic Sheep (Wright etc., 1991, above-mentioned document), transgenic goat (Ebertand Schinder, 1993, above-mentioned document), transgenic pig (Rexroad and Purcel, the 11st animal reproduction international conference A.I.Dublin5:29-35 (1988)) and transgenic cattle (Krimpenfort etc., 1991, above-mentioned document; Hill etc., 1992, above-mentioned document; Bowen etc., 1994, above-mentioned document).
WO-A-88/01648 (Immunex company), WO-A-88/00239 and WO-A-90/05188 (being pharmaceutical protein company limited owns) are also as a reference, it has described the recombination structure, the transgenic animal of integrating recombination produce, and express proteins encoded and express suitable technique and the method that is adopted in growing up the female mammal breast lactation.These and above-mentioned document all are incorporated herein for referencial use.
Stacey etc. also draw at the article of molecule and cytobiology 14 (2): 1009-1016 (in February, 1994) and are that reference, this article described the mouse alpha-lactalbumin gene by the displaced knock-out experiment of human alpha-lactalbumin gene.Yet this paper is not reported the expression of alpha-lactalbumin.
In one embodiment, the invention provides a kind of expression system, this system produces with being different from natural other technology of the technology of gene that exists of replacement.
On the other hand, the invention provides a kind of transgene mammal, the cellular integration of this animal be suitable for expressing the recombinant expression system of alpha-lactalbumin (preferred human alpha-lactalbumin) or its function equivalent or its part.Recombinant expression system generally is integrated in the genome of transgenic animal, can heredity, therefore so its offspring is also contained transgenosis and is also included within the present invention.Suitable transgenic animal include, but is not limited to sheep, pig, ox, goat, especially preferred ox.
In addition, the present invention includes the carrier that contains this recombinant expression system, also comprise with this recombinant expression system transformed host cells (randomly transforming) with carrier format.
On the other hand, the invention provides the alpha-lactalbumin of express producing by recombinant expression system, hope be in transgene mammal, to produce this alpha-lactalbumin.Alpha-lactalbumin gene activated in the female mammal breast naturally in lactation, so recombinant expression system expressed proteins of the present invention will be produced and secrete as composition of milk during this period.Also can induce the breast secretion to produce protein of interest by hormone or additive method.Alpha-lactoalbumin containing finished milk-product also within the scope of the present invention.
In a preferred embodiment, recombinant expression system comprises following construction, called after pHA1, pHA2, pBBHA, pOBHA, pBAHA, pBova-A or pBova-B.Construction pHA1, pHA2, pBBHA, pOBHA and pBAHA expressing human alpha-lactalbumin are therefore preferred, especially preferred pHA2.Construction pHA2 is preserved in NCI MB, preserving number NCI MB 40709 February 15 nineteen ninety-five.
The similar transgene mammal that contains above-listed particular build thing is preferred.
Find that further the increase of alpha-lactalbumin concentration, milk production also increases the breast that the present invention obtains in per unit volume.This discovery is a unanticipated, and therefore, the construction and the transgenic animal (particularly ox) that contain human alpha-lactalbumin gene (or function equivalent or its part) are the preferred embodiments of the invention.
Although we are not wishing to be bound by theory, it is believed that only natural expression has partial action to alpha-lactalbumin enhanced in human body for the promoter region of human alpha-lactalbumin gene.It is believed that and strengthen to express and to obtain by protein-coding region 3 ' and/or 5 ' flanking sequence are included in the recombinant expression system of the present invention.
3 ' and 5 ' flanking sequence of people α-1ac gene protein coding region checks order first.The partial sequence of 3 ' flanking region (Nucleotide 1-264,1331-2131,2259-2496,2519-2680 and 3481-3952) is shown in SEQ ID NOS.16 to 20, and the complete sequence of 5 ' side is shown in SEQ ID NO.21 simultaneously.Observe in the experiment, comprise arbitrary or both sequences all can make the expression level of alpha-lactalbumin increase greatly.The increase of expressing also can be observed when the encoding histone zone is non-human alpha-lactalbumin.
Sequence SEQ ID NOS.16-20 and 21 it is believed that useful to alpha-lactalbumin high level expression in human milk, therefore forms another aspect of the present invention.
On the other hand, the invention provides a kind of polynucleotide, have with SEQ ID NOS.16-20 in any or SEQ ID NO.21 or its part or the essentially identical sequence of its function equivalent.
This polynucleotide various informative (as DNA or RNA, two strands or strand), but general double-stranded DNA is the most convenient.The part that polynucleotide of the present invention can be used as the recombination construction exists, and the latter is included among the carrier (as expression vector) or is integrated in the karyomit(e) of transgenic animal.Any carrier that comprises above-mentioned polynucleotide or transgenic animal constitute of the present invention one other aspect.
From more on the one hand, the invention provides a kind of recombinant expression system (preferably being suitable for expressing alpha-lactalbumin (preferred human alpha-lactalbumin) or its part or its function equivalent), described recombinant expression system comprises the EcoRI that is selected from the wild-type alpha-lactalbumin gene and BamHI and the polynucleotide of the polynucleotide between the EcoRI site, perhaps its part or its function equivalent of polynucleotide between the XhoI restriction site and wild-type human alpha-lactalbumin gene.
In an embodiment preferred, recombinant expressed sequence of the present invention comprises above-mentioned polynucleotide, its part and its function equivalent.
The present invention also comprises the carrier that contains above-mentioned recombinant expression system and with this carrier cell transformed.The present invention further comprises transgenic animal, and transgenosis wherein comprises recombinant expression system.
Fig. 1 as described in example 1 above, illustrates ox alpha-lactalbumin PCR primer sequence.
Fig. 2 as described in embodiment 1 and embodiment 4, illustrates the position and the product thereof of ox alpha-lactalbumin PCR primer.
Fig. 3 as described in embodiment 2, shows two eclipsed genome λ clones' of human alpha-lactalbumin gene restriction map (pHA-2 and pHA-1).
Fig. 4 as described in embodiment 3, shows three eclipsed genome λ clones' of bovine beta-lactoglobulin gene restriction map.
Fig. 5 as described in embodiment 4, illustrates the SDS-PAGE that the skimming milk of the transgenic mice that changes the ox alpha-lactalbumin gene over to and the breast comparative analysis of non-transgenic mouse are done.
Fig. 6 as described in embodiment 5, illustrates the human alpha-lactalbumin transgenosis construct.
Fig. 7 as described in embodiment 5, illustrates the SDS-PAGE that the skimming milk of the transgenic mice that changes the human alpha-lactalbumin gene over to and the breast comparative analysis of non-transgenic mouse are done.
Fig. 8, as described in embodiment 5, the Western that human alpha-lactalbumin transgenic mice breast is shown analyzes, and compares with the human alpha-lactalbumin standard.
Fig. 9 as described in embodiment 6, illustrates the PCR clone strategy of transgenosis construct PKU1 to PKU4.
Figure 10 as described in embodiment 6, illustrates the sequence of PKU primer 1 to 10.
Figure 11 illustrates invalid and the allelic structure of humanized alpha-lactalbumin.
Figure 12 be the alpha-lactalbumin defective lactation mammary gland the Northern of total RNA analyze.
Figure 13, the Western that the alpha-lactalbumin of target mouse is shown analyzes.
Figure 14 is the histologic analysis of wild-type α-lac-mammary gland lactation.
Figure 15 A illustrates RNase protection and analyzes, in order to the mRNA of the alpha-lactalbumin of the replacement type alpha-lactalbumin mRNA that distinguishes the people and mouse.Figure 15 B illustrates the RNase protection of mouse and people's replacement type alpha-lactalbumin mRNA and analyzes.
Figure 16 illustrates with hydrophobic interaction chromatography quantitative analysis alpha-lactalbumin.
SEQ ID NOS16-20 provides from the BamHI site to the carrier restriction site partial sequence of (comprising the EcoRI site), and it is positioned at 3 ' one side in the encoding histone zone of endogenous human alpha-lactalbumin made gene, and is as follows:
SEQ ID NO 16: nucleotides 1 to 264 (comprising two-end-point)
SEQ ID NO 17: nucleotides 1331 to 2131 (comprising two-end-point)
SEQ ID NO 18: nucleotides 2259 to 2496 (comprising two-end-point)
SEQ ID NO 19: nucleotides 2519 to 2680 (comprising two-end-point)
SEQ ID NO 20: nucleotides 3481 to 3952 (comprising two-end-point)
SEQ ID NO.21 has provided the sequence from the EcoRI site to the XhoI site, is positioned at encoding histone zone 5 ' one side of endogenous human alpha-lactalbumin made gene.
Particularly, Figure 11 top submeter understands the site of wild-type mice alpha lactalbumin, and the position of transcriptional domain and direction are by shown in the arrow. Translation termination site and RNA polyadenylic acid site indicate that also mid portion shows invalid allelic structure. The striped district has shown that HPRT can select box (Cassette). A bottom submeter person of good sense's the allelic structure of replacement. The grid zone shows the human alpha-lactalbumin made fragment. Transcription initiation, translation termination and polyadenylic acid site are all indicated. Shown in restriction enzyme site be: Hind III (H); BamHI (B); XbaI (X).
Two autoradiographs are end user's alpha lactalbumin probe shown in Figure 12, use rat beta-casein probe same filter membrane to be made the result of recross thereupon. Used probe is indicated under each autoradiograph. The source mark explanation thereon of RNA in each bar road.
The A road contains the human alpha-lactalbumin made of purifying among Figure 13. B to F road is the newborn sample from the target mouse, and genotype is indicated above it. G and H road are the short time exposure thing in C and D road.
The photo of light microscopic shown in Figure 14 is the haematine/eosin stained slice (original multiplication factor 1 00X) of breast tissue. Each mammary gland-specific gene type shows.
Among Figure 15 A, mouse and people DNA are at α-lachAllelic 3 ' binding is positioned between translation termination site and polyadenylic acid signal. The untranslated end of human alpha-lactalbumin made mRNA3 ' contains the mouse sequence of 120b p. The alpha lactalbumin mRNA of mouse and people's replacement passes through hybridization check with the mouse rna probe, and is distinguished by the RNA clip size that the rnase digestion protection produces. People's sequence is shown that by grid the mouse sequence is shown by the shadow region. Shown in restriction site be Hind III (H); BaI (B); XbaI (X).
Autoradiograph shown in Figure 15 B is 5% polyacrylamide urea shallow layer gel. The source of RNA is indicated above each road. The A road illustrates the wild type rna with the hybridization of mouse rna probe, and this probe is not by rnase digestion. D road to J road illustrates from α-lacm/ α-lachThe RNA sample of heterozygote; Numeral shows concrete mouse, is the source that institute's specified rate is estimated among Figure 15. The prediction size of protected fragment is also indicated.
Figure 16 top branch provides the Phenyl-Sepharose elution curve of three kinds of newborn samples. 1, α-lach/α-lac
hAutozygote (mouse #22); 2, α-lacm/α
-lac
hHeterozygote (mouse #76); 3, α-lacm/α-lac
mWild type. Lower part illustrates the calibration curve of integration peak district being done with known human alpha-lactalbumin made amount.
The present invention is further described with reference to following non-limiting example.
The clone of 1 N of alpha-lactalbumin gene of embodiment
Known have three kinds of different ox alpha-lactalbumins, and wherein Type B is the most general.A type from Bos (Bos) nomadicus f.d.indicus is different from Type B, shows that No. 10 position residue: A are Glu, then replaces for Arg among the B.Its sequence difference of C type from Bos (Bibos) javanicus it be unclear that (Mc Kenzie ﹠amp; White, Advances in Protein Chemistry 41,173-315 (1991).The PCR primer of indicating among the available Fig. 1 of ox alpha-lactalbumin gene (coding Type B) is from genomic dna cloning.Primer has provided following sequence number:
Ba-2?SEQ?ID?NO?1
Ba-7?SEQ?ID?NO?2
Ba-8?SEQ?ID?NO?3
Ba-9?SEQ?ID?NO?4
DNA source in all PCR reactions is the blood from the Holstein-Friesian ox.
Use primer Ba-9 to be 0.72kb in conjunction with the promoter region length of primer Ba-8 amplification.This-the BamHI/EcoRI fragment cloning is to Bluescript (pBA-P0.7).
Use primer Ba-7 to be 2.05kb in conjunction with the promoter region length of primer Ba-8 amplification.This-the BamHI/EcoRI fragment cloning is to Bluescript (pBA-P2).
Whole ox alpha-lactalbumin gene comprises the flanking sequence district of 5 ' 0.72kb and 3 ' 0.3kb, and available primer Ba-9 is increased in conjunction with primer Ba-2.These primers comprise the BamHI restriction enzyme recognition site, can be with the direct subclone of 3kb fragment of amplification to the BamHI sites of pUCI8, and generation pBova-A construction (see figure 2).
To link the EcoRV/BamHI fragment of pBOVA-a from the BamHI/EcoRV fragment of clone pBA-P2, produce construction pBOVA-b (see figure 2).
Because the TAQ polymerase lacks proofreading activity, therefore whether the ox alpha-lactalbumin DNA of check amplification and the ox alpha-lactalbumin gene of delivering be consistent is necessary.Sequential analysis is carried out in all exons and two primer fragments.Compare the result that ox alpha-lactalbumin exon and Vilotte deliver, shown 3 changes:
(i) exon I becomes A at+759 by C; Become 5 ' non-coding region
(ii) exon I becomes CTG at+792 by CTA; The leucine of all encoding
(iii) exon II becomes ACG at+1231 by GCG; L-Ala becomes leucine
This shows that the protein B type is more common.
Mispronounce although can not get rid of in the pcr amplification sequence, above mispairing may be because ox DNA source is different.
The clone of embodiment 2 human alpha-lactalbumin genes
The dna sequence dna of human alpha-lactalbumin is delivered (Hall et al., Biochem.J., 242:735-742, (1987)).End user's sequences Design PCR primer is to clone two small segments from the human gene group DNA, and one of them is positioned at 5 ' end of gene, and another is at 3 ' end.They to the pUC18 carrier, are purchased (Stratageme) λ genomic library as probe in detecting by subclone.Two recombinant bacteria phages that contain alpha-lactalbumin gene, promptly 4a and 5b.1 separate (Sa mbook et al, molecular cloning laboratory manual, the 2nd edition, Cold Spring Har bor Laboratory (1989)) with ready-made method.Two clones of restriction endonuclease map proof all contain the complete encoding sequence of people α-lac gene, but 5 ' and the 3 ' terminal sequence (Fig. 3) different in size that exists.Clone's 5b.1 exon sequence is analyzed and clone 4a exon and 5 ' side areas The sequencing results show them and to deliver sequence consistent.
The some parts of 3 ' sequence is shown in SEQ ID NOS.16 to 20.5 ' sequence is shown in SEQ ID NO.21.
The dna sequence dna of ox BLG (bBLG) is delivered (Jamieson et al; Gene, 61; 85-90, (1987); Wagner, unpublished, E MBL Data Library:BTBLACEX (1991)).Use ox sequences Design PCR primer with the fragment of clened cows BLG gene since 5 ' end.This fragment subclone uses as probe to the pUC18 carrier, checks ox (Stratagene) the λ genomic library that is purchased.Separate 3 genome λ clones and made the qualitative (see figure 4) of restriction analysis.Wherein clone (BB13 for two, BB17) contain complete bBLG coding region, add the side areas of different length, and clone BB25 lacks coding region, be made up of 5 ' side areas fully, sequential analysis shows that this clone's terminal point is positioned at the upstream 12bp of ATG translation starting point.The Sal fragment subclone that comprises whole BB13 and BB17 is to pUC18, from the EcoRI fragment cloning of clone BB25 to pBluescript (Fig. 4).
The assembling and the expression of 4 Ns of alpha-lactalbumin constructions of embodiment
Transgenosis construct (Fig. 2)
PBova-A is by ox alpha-lactalbumin coding region, and the 5 ' side of 0.72kb and the 3 ' side areas of 0.3kb are formed, as the BamHI fragment cloning of 3Kb to Bluescript vector.
PBova-B contains 3 fragments:
1, from the BamHI-EcoRV fragment of 1.47kb size of clone pBA-P2.
2, from the EcoRV-BamHI fragment of 2.78kb size of clone pBova-A.
3, the cloning vector Bluescript of BamHI digestion.
The expression of ox alpha-lactalbumin in transgenic mice
Two construction pBova-A and pBova-B (Fig. 2) are injected mice embryonic, produce transgenic animal.Compare in conjunction with Coomassie blue stain analysis breast and with the alpha-lactalbumin normal content by the SDS-PAGE electrophoresis, the change conditions that has shown the alpha-lactalbumin expression level, wherein PBova-A detect less than, PBova-B expression level height is to 0.5-1mg/ml (seeing Fig. 5 and table 1).
The expression of ox alpha-lactalbumin in table 1 transgenic mice
The mouse Coomassie blue stain
244.12?BOVA-a????????????????????-
244.14?BOVA-a????????????????????-
244.15?BOVA-a????????????????????-
245.23?BOVA-b????????????????????-
245.8??BOVA-b????????????????????-
245.4??BOVA-b????????????????????-
245.7??BOVA-b????????????????????+
245.21?BOVA-b????????????????????-
245.13?BOVA-b????????????????????+
249.13?BOVA-b????????????????????-
246.15?BOVA-b????????????????????-
249.18?BOVA-b????????????????????++
249.23.1?BOVA-b??????????????????++
249.23.5?BOVA-b??????????????????++
249.25.3?BOVA-b??????????????????-
249.25.7?BOVA-b??????????????????-
249.30.3?BOVA-b??????????????????-???????-＝＜0.5mg/ml
249.30.4?BOVA-b??????????????????-???????+＝＝0.5-1mg/ml
249.33.2?BOVA-b?????????????????+/++????++＝＝1-2mg/ml
249.33.3?BOVA-b?????????????????+/++
Table 1 shows the level relatively of transgenic mice Ruzhong ox alpha-lactalbumin, and protein standard estimation obtains on the glue of Coomassie blue stain by comparing.
The assembling and the expression of embodiment 5 human alpha-lactalbumin gene constructs
Alpha-lactalbumin is the main whey-protein of people, and the main whey-protein of sheep and ox is a beta-lactoglobulin.The expression level kind of alpha-lactalbumin is different with kind, and human milk contains about 2.5mg/ml, and cow's milk contains 0.5-1.0mg/ml, and the mouse breast contains 0.1-0.8mg/ml.In order to understand fully the sequence that makes the alpha-lactalbumin gene high level expression, designed different constructions.They contain a) 5 ' and the 3 ' side areas from the human alpha-lactalbumin locus of different lengths.B) from the 5 ' side areas in ox alpha-lactalbumin site, or c) from 5 ' side areas of ox or bird beta-lactoglobulin gene.Bird beta-lactoglobulin gene promoter has been successfully used to efficiently express in the mouse Ruzhong (＞10mg/ml) heterologous gene.
Transgenosis construct (Fig. 6)
PHA-1 forms by the human alpha-lactalbumin coding region with from the 5 ' side of about 1.8kb of λ clone 5b.1 and the 3 ' side areas of 3kb, its as the EcoRI/SalI fragment cloning of 7kb to pUC18.
PHA-2 forms by the human alpha-lactalbumin coding region with from the 5 ' side of about 3.7kb of λ clone 4a and the 3 ' side areas of 13kb, its as the SalI fragment cloning of about 19kb to pUC18.
POBHA (bird beta-lactoglobulin, human alpha-lactalbumin) is implemented in 4 dna fragmentations:
1, the SalI/ECoRV fragment of one 4.2 kb comprises bird beta-lactoglobulin promotor (seeing WO-A-90/05188);
2, the synthetic oligonucleotide of a 74bp corresponding to the BClI joint of a 8bp and the 15-77 bit base of human alpha-lactalbumin sequence, uses as a flat end/BglI fragment;
3, the BglI/PstI human alpha-lactalbumin fragment of a 6.2kb, it is derived from λ clone 4a, comprises the zone that is positioned between 77 bit base BglI sites and 3 ' side XhoI site.
4, the pSL1180 (Phamacia) that cuts with PstI and SalI enzyme.
PBBHA (bovine beta-lactoglobulin, human alpha-lactalbumin) is implemented in following 4 dna fragmentations:
1, the ECoRI fragment of a 3.0kb contains the bovine beta-lactoglobulin promotor from clone BB25-3, uses as the EcoRI/EcoRV fragment;
2, the synthetic oligonucleotide of a 74bp corresponding to BClI joint and the human alpha-lactalbumin sequence 15-77 bit base of a 8bp, uses as flat end/BglI fragment;
3, the BglI/PstI human alpha-lactalbumin fragment of a 6.2kb from clone 4a, comprises the zone that is positioned between 77 bit base BglI sites and 3 ' side XhoI site;
4, the Bluescript vector of cutting with EcoRI and PstI enzyme.
PBAHA (bovine beta-lactoglobulin, human alpha-lactalbumin) is implemented in following 4 dna fragmentations:
1, the BamHI-StuI fragment of a 0.72kb contains the ox alpha-lactalbumin promotor from clone pBA-P0.7;
2, the synthetic oligonucleotide of a 62bp corresponding to human alpha-lactalbumin sequence 15-77 bit base, uses as flat end/BglI fragment;
3, the human alpha-lactalbumin gene BglI/PstI fragment of a 6.2kb from λ clone 4a, contains the sequence that is positioned between 77 bit base BglI sites and 3 ' side XhoI site;
4, the Bluescript vector of cutting with BamHI and PstI enzyme.
The expression of human alpha-lactalbumin in transgenic mice
5 kinds of constructions are injected mice embryonic, produce transgenic animal.All constructions are equal expressing human alpha-lactalbumin in the mouse Ruzhong.Contain the pHA-1 of side areas of human alpha-lactalbumin gene and different length and pHA-2 in most of animals expression level between 1 to 18mg/ml (213.5pHA-2).The pOBHA and the pBBHA that contain the human alpha-lactalbumin gene that is started by bird or ox BLG promotor have low slightly expression level.The pBAHA that contains by the human alpha-lactalbumin gene of 0.72kb ox alpha-lactalbumin promoters driven has the expression level similar to pHA-1 or pHA-2, but it is lower to express the percentage ratio that can examine the proteic transgenic animal of level.This discovery is surprising, because same ox promoter sequence drives the ox alpha-lactalbumin gene, the result shows extreme difference and (sees embodiment 4 and Vilotteet al; FEBS, Vol.197,1.2.13-18 (1992)).
Table 2 sums up the transgene protein relative quantity.From the skimming milks of these animals with SDS-PAGE in conjunction with Coomassie blue stain, isoelectric focusing, Western trace (the anti-human alpha-lactalbumin antibody (Sigma) that use is purchased carries out) and chromatofocusing technical Analysis.The result of these analyses shows, compares with human alpha-lactalbumin standard (Sigma), and the molecular weight of transgene protein, iso-electric point and antigenicity are correct.
Table 2
α- Western205.19 pHA1--204.10 pHA1 ++ ++204.7 pHA1 +++ +++230.15.3 pHA1 +++ n.d.230.15.5 pHA1 +++ n.d.230.15.6 pHA1 +++ n.d.230.21.5 pHA1 +++ n.d.230.21.1 pHA1 ++ n.d.211.18 pHA2 + ++211.17 pHA2--211.16 pHA2 +++ +++212.11 pHA2 + n.d.213.5 pHA2 ++++ ++++212.13 pHA2 ++ n.d.212.19 pHA2-n.d.213.4 pHA2 +++ n.d.212.7 pHA2-n.d.232.10 BBHA--233.1 BBHA--231.4 BBHA ++ ++232.9 BBHA + +231.9 BBHA-n.d.232.5 BBHA + +231.3 BBHA + +232.6 BBHA-n.d.237.6 BBHA-n.d.235.15 OBHA-n.d.235.19 OBHA ++ ++236.6 OBHA ++ ++234.1 OBHA + +234.4 OBHA ++ ++234.14 OBHA + +239.14 BAHA +++ +++239.4 BAHA-n.d.240.7 BAHA-n.d.239.3 BAHA-n.d.239.6 BAHA ++ ++239.12 BAHA-n.d.243.1 BAHA ++ n.d.242.9 BAHA +++ n.d.241.16 BAHA + n.d.234.14 BAHA-n.d.243.13 BAHA + n.d.243.10 BAHA-n.d.243.4 BAHA-n.d.
Table 2 shows the relatively level of human alpha-lactalbumin in the transgenic mice Ruzhong, and it is by obtaining with the gel and the comparison of Western trace reacting phase of standard protein in Coomassie blue stain.
-expression＜0.5mg/ml
+ expression=0.5-1mg/ml
++ expression=1-2mg/ml
+++expression=2-3mg/ml
++ ++ expression＞5mg/ml
N.d. expression fails to conclude
Result from several mouse is shown among Fig. 7 and Fig. 8.The SDS-PAGE that Fig. 7 illustrates the transgenic mice skimming milk analyzes, and is contrast with non-transgenic mouse breast; Fig. 8 shows the Western trace of the human alpha-lactalbumin of transgenosis breast, compares with people's alpha-lactalbumin standard.
It is relative higher that the genetically modified expression of human alpha-lactalbumin is compared with natural ox alpha-lactalbumin transgenosis, reflects the different of endogenous ox and human gene expression level.Because this difference may cause by the difference of 5 ' end control area, therefore, it is generation with the corresponding sequence of 5 ' zone personnel selection alpha-lactalbumin gene of ox alpha-lactalbumin transcripting start point.
Made up two kinds of constructions of PKU-5 and PKU-1H, they have comprised the aminoacid replacement that is shown in the table 3.
Following SEQ ID NOS awards used PCR primer
PKU-1?????SEQ?ID?NO.5
PKU-2????SEQ?ID?NO.6
PKU-2L???SEQ?ID?NO.7
PKU-3????SEQ?ID?NO.8
PKU-4????SEQ?ID?NO.9
PKU-5????SEQ?ID?NO.10
PKU-6????SEQ?ID?NO.11
PKU-7????SEQ?ID?NO.12
PKU-8????SEQ?ID?NO.13
PKU-9????SEQ?ID?NO.14
PKU-10???SEQ?ID?NO.15
PKU-5
The first step the clone has the EcoRI/BamHI site of three fragment subclones to pUC18:
(1) from EcoRI to the PvuI fragment (see figure 10) of using PKU-primer 7 in conjunction with the pcr amplification of primer 8;
(2) from PvuI to the BsaBI fragment (see figure 10) of using PKU-primer 9 in conjunction with the pcr amplification of primer 10; With
(3) from BsaBI to the HindIII fragment of pBA.
Final construction comprises 6 dna fragmentations:
(1) SalI to KpnI of 3.7kb fragment contains the human alpha-lactalbumin promotor (Fig. 3) from λ clone 4a;
(2) synthetic oligonucleotide of 152bp contains the KpnI site to the human alpha-lactalbumin gene order of AUG and the ox alpha-lactalbumin gene sequence from AUG to the HapI site;
(3) from HpaI to the HindIII fragment of the 1.25kb size of the first step subclone;
(4) from HindIII to the BglII fragment of the 0.95kb of pBA.
(5) from BamHI to the XhoI fragment of the 3.7kb size of the human alpha-lactalbumin gene 3 ' side of λ clone 4a (Fig. 3), use as the BamHI fragment;
(6) cut the Bluescript KS-carrier of handling with SalI and BamHI enzyme.
PKU-1H is to make up with the same mode of PKU-5, and just fragment (3) is from PKU-1.
PKU-1 makes up (see figure 9) by 6 dna fragmentations:
SstI to the HpaI fragment from pBOVA-6 of (1) 2.04kb size;
(2) one 0.46 kb sizes (the PKU-primer is to 1 and 2 from HpaI to the PvuI fragment of PCR product A; See Figure 10);
(3) 0.60kb sizes (primer is to 3 and 4 from PvuI to the BsaBI fragment of PCR product B; See Figure 10);
BsaBI to the HindIII fragment from pBOVA-6 of (4) 0.22kb sizes;
HindIII to the BglII fragment from pBOVA-6 of (5) 0.95kb sizes.
(6) the carrier pSL1180 that digests with Sst I and BglIII.
Table 3
Be present in the aminoacid replacement in the transgenosis construct
Replace people's promoter plasmid
People's 3 ' side position 9 30 53 80
Tyr，Tyr，Tyr，Tyr???????????+???????????pPKU-1H
Ser，Tyr，Leu，Leu???????????+???????????pPKU-5
Expression in the transgenic mice
Construction PKU-1H and PKU-5 have injected mice embryonic.Transgenic animal are all from the PKU-5 construction at present.Feed these animals so that analyze its breast.
Materials and methods
Mouse species
Carry the alpha-lactalbumin amorphs and the humanization alpha-lactalbumin is replaced allelic mouse, be to obtain (Fitzgerald et al.J.Biol.Chem245:2103-2108) as previously mentioned by raising respectively from target embryonic stem cells clone M2 and F6 and Balb/c mating gained mosaic.Raise in these strain processes, the genotype of alpha-lactalbumin determines by the genomic dna for preparing in the afterbody living tissue being done the Southern analysis.
RNA analyzes
Total RNA makes (Eur.J.Biochem 107:303-14) with the method for Auffray and Rougeon from the female mice belly mammary gland in 506 days postpartum.Northern detects and carries out (Sambook et al, Molecular Cloning) according to standard program.The probe that is used to hybridize is: the BamHI fragment that contains complete mouse alpha-lactalbumin gene of a 3.5kb size and the rat beta-casein cDNA of a 1.1kb size (Blackburn et al Nucl.Acids Res10:2295-2307).
RNAse protects analysis
32The sense-rna of P-CTP mark is cloned to transcribe in the HindIII-BalI mouse alpha-lactalbumin gene fragment of the 455bp of Bluescript KS from one with t7 rna polymerase (Promega) and is obtained (Figure 15 A), and the condition of responsive transcription, solution hybridization and RNAse digestion all recommends to carry out by Promega.Protected fragment is separated with polyacrylamide gel electrophoresis, autoradiography observation.
Breast component and volume analysis
The breast sample was collected under Hypnorm (Roche)/Hypnovel (Janssen) narcosis 3-7 days lactation.Inject the pitocin (Intervet) of 150mU and massage crowded sending out gently by peritonaeum.Dairy fats content calculates (J.Physiol 175:15) with Fleet and Linzell method.The breast of degrease is used for analyzing proteins (Bradford Analyt.Biochem 72:248-54), lactose detects with enzyme process, with newborn sample successively with beta-galactosidase enzymes (Boehringer), glucose oxidase and peroxidase (Sigma) insulation, this method is from Bergmeyer and Bernt (Methods in Enzyme Analysis 3:1205-1212).
Milk production is with titration water technique computes, as (Comp.Biochem.Physiol 84A:127-133) as described in the Knight etc., measure generally be selected in mouse lactation 3 to 6 days lactations and finished 48 hours after.
The analysis of Ruzhong alpha-lactalbumin and quantitative
The breast sample is gone up at 16% SDS-PAGE gel (Novex) and is analyzed, and transfers to the ImmobilonP film and do the reaction of Wester trace.Earlier with antiserum(antisera) (Dako) absorption of the anti-human alpha-lactalbumin of rabbit, then with the IgG peroxidase antibody coupling matter absorption of goat-anti rabbit, and with the enhanced chemiluminescence system observe (Amersham) thus the detection alpha-lactalbumin.
Alpha-lactalbumin is with improve one's methods quantitatively (the Analyt Biochem 140:394-402) of the method for Lindahl etc. in the breast sample, and this method is with the phenyl-agarose chromatography purifying alpha-lactalbumin that relies on calcium.The breast sample dilutes by 1: 10 with the ammonium sulfate of 27% (W/V), and is room temperature insulation 10 minutes, centrifugal then.Supernatant and equal-volume 100mM Tris/Cl, pH7.5,70mMEDTA mixes, and is loaded on and uses 50mM Tris/Cl, and pH7.5 is on phenyl-agarose (Pharmacia) post of 1m ME DTA pre-equilibration (200 μ l packing volume).Wash pillar with same damping fluid, alpha-lactalbumin 50m MTris/Cl, pH7.5,1m MCaCl
2Wash-out goes out.Detect the photoabsorption of pillar, and the peak area of alpha-lactalbumin part is quadratured at 280nm.Use the human alpha-lactalbumin of the purifying of known quantity to set up typical curve (Figure 16) from zero to 2.46mg/ml.
Histologic analysis
The young baby was in female mouse of lactation from the 6th day postpartum removes, put to death female mouse after two hours, decompose chest mammary gland, be stored in the formalin of neutral buffered, dye with phenodin/eosin with paraffin embedding and with standard method.
The result
Mouse alpha-lactalbumin gene disappearance
As Stacey etc. (1994, above-mentioned document) described, set up a strain mouse species, it comprises the 2.7kb fragment of complete mouse alpha-lactalbumin coding region and the promotor of 0.57kb lacks, and replaces (seeing Figure 11) with hypoxanthine phosphoribosyltransferase (HPRT) selectable marker gene that contains of 2.7kb.Carry this allelic animal called after α-lac-.Wild-type mice alpha-lactalbumin allelotrope called after α-lac
m
The Northern analysis revealed that the mammary gland RNA that takes from the 5th day lactation is done, there be not (seeing Figure 12) in alpha-lactalbumin mRNA in α-lac-/α-lac-autozygote, the proof alpha-lactalbumin gene is removed, does not have other alpha-lactalbumin mRNA source and exists.Same RNA and sample beta-casein RNA hybridization (seeing Figure 12) in all samples.
The alpha-lactalbumin defective except that to lactation to not significantly influence in mouse other period.α-the lac-of two kinds of sexes/α-lac-autozygote and α-lac
m/ α-lac-heterozygote appearance, behavior and breeding are all normal.Yet α-lac-/α-female mouse of lac-autozygote can not successfully feed the young baby, and its young baby dies in 5-10 days after birth.The offspring of the female mouse α-lac-of autozygote/α-lac-is transferred to the female mouse of wild-type and feeds and can normally survive.On the contrary, the offspring of the female mouse of wild-type is transferred to the female mouse of autozygote α-lac-/α-lac-and feeds, and can not survive.The young baby that table 4 explanation α-lac-/α-the female mouse of lac-is fed approximately is α-lac
m/ α-lac
mWild-type mice is fed half of young baby's weight.The calculation result of milk production is consistent therewith.α-lac
m/ α-lac-heterozygote milk production is close with wild-type, but the milk production of α-lac-/α-lac-autozygote sharply descends (table 4).
Table 4
In the target mouse species, milk-content, young baby's weight, breast tissue weight and milk production
| Genotype | α-lac m/α-lac m | α-lac m/α-lac- | α-lac-/α-lac- | α-lac m/α-lac h | α-lac h/α-lac h |
| Fat (% v/v) | 28.23±1.65(7) | 29.6±1.3(6) | 45.25±2.15(6) *** | 25.25±1.36(7) | 21.2±0.23(4) * |
| Protein (mg/ml) | 87.52±5.82(7) | 95.81±9.5(5) | 164.63±13.92(8) *** | 94.51±5.97(7) | 77.07±1.05(4) |
| Lactose (mM) | 62.44±9.27(7) | 42.7±4.2(6) | 0.7±0.34(3) *** | 42.40±1.93(7) | 56.85±3.8(4) |
| Single young baby's weight (g) | 2.82±0.25(8) | 3.14±0.1(7) | 1.52±0.12(10) *** | 2.9±0.15(8) | 3.4±0.75(4) |
| Every young baby's breast tissue weight (g) | 0.34±0.06(7) | 0.4±0.1(7) | 0.35±0.05(8) | 0.31±0.04(6) | 0.51±0.09(4) |
| Milk production (g/ days) | 7.51±0.44(4) | 6.7±0.38(6) | 1.37±0.48(4) *** | n.t. | 9.94±0.65(5) * |
The t-test statistics is not analyzed in pairs,
*P＜0.05;
*P＜0.01;
* *P＜0.001;
Data are mean value ± SE
Numeral shows female mouse number of analysis in the bracket
N.t., do not detect
Breast is obtained by each genotype by artificial milking, and key component has wherein been done analysis.Take from α-lac
mThe breast of/α-lac-heterozygote and wild-type breast outward appearance does not have different, yet similar (table 4) of fat, protein content and wild-type.Although lactose-content is at α-lac
m/ α-lac-heterozygote part omitted shades few, and the statistical results show difference is also not obvious.On the contrary, take from the newborn thickness of α-lac-/α-lac-autozygote, be difficult to extrude and forming obviously differently with wild-type from nipple, the fat content ratio wild-type is high about 60%, and protein content is high about 88%, and lactose obviously lacks.Detected 0.7mM lactose apparent concentration is represented the Ruzhong glucose content in the female mouse of α-lac-/α-lac-, because used lactose analytical method relates to the enzymatic conversion method of lactose to glucose.The direct analysis of the glucose of wild-type breast shows that its concentration is 1.8mM.
From the Ruzhong of α-lac-/α-lac-autozygote, the Western of milk-protein analyzes and is not checked through alpha-lactalbumin (seeing Figure 13, the F road).Phenyl-agarose chromatography has further proved this point.This technology can special discriminating alpha-lactalbumin, is suitable for newborn alpha-lactalbumin content quantitative is calculated (table 5; Also see Figure 16).When being used to take from α-lac-/α-lac-autozygote newborn, do not detect alpha-lactalbumin.And α-lac
mThe Ruzhong alpha-lactalbumin concentration of/α-lac-heterozygote is 0.043mg/ml, is half (table 5) of wild-type approximately.
Table 5
Breast alpha-lactalbumin content
Source alpha-lactalbumin (mg/ml)
The people 2.9 ± 0.1 (2)
α-lac
m/ α-lac
mMouse 0.09 ± 0.005 (2)
α-lac-/α-lac-mouse 0 (3)
α-lac
m/ α-la
c-mouse 0.043 ± 0.004 (5)
α-lac
m/ α-lac
hMouse 0.65 ± 0.07 (4)
α-lac
h/ α-lac
hMouse 1.38 ± 0.12 (5)
Alpha-lactalbumin content is by phenyl-agarose chromatography estimation in the breast sample
Numerical value is mean value ± SE
Numeral shows female mouse number of analysis in the bracket.
The alpha-lactalbumin defective does not have obvious influence to mammogenesis.Table 4 explanation wild-type, heterozygote α-lac
m/ α-lac-does not have obvious different with the mammary gland gross weight of autozygote α-lac-/α-lac-mouse lactation period.The spectroscopic analysis of mammary gland (Figure 14) shows that the mammary gland of heterozygote and autozygote is normal on histology.But the vesicle of autozygote mammary gland and mammary duct ectasia are full of fat and material such as drip.
The human alpha-lactalbumin gene is replaced the mouse alpha-lactalbumin gene
We have obtained the mouse at mouse alpha-lactalbumin gene site carrier's alpha-lactalbumin gene.2.7kb mouse alpha-lactalbumin gene fragment in α-lac-amorphs disappearance is replaced by the fragment that contains whole person's alpha-lactalbumin coding region and 5 ' lateral order row of 2.97kb.This people's gene fragment extends to the EcoRI site at 3 ' end 136bp place, translation termination site from people's transcription initiation site upstream 0.77kb, occur in the BamHI site at 0.57kb place, mouse transcription initiation site upstream with the binding of mouse sequence and Stacey et al (is seen in the XbaI site at 3 ' end 147kbp place, mouse translation termination site, 1994, above-mentioned document, also see Figure 11), we have described carrying this equipotential gene is α-lac
hThe analysis of mouse.
Mouse alpha-lactalbumin gene disappearance shows that it is synthetic that the alpha-lactalbumin defective hinders lactose, the havoc milk production.We use α-lac
hAllelotrope detects human alpha-lactalbumin and recover the ability of galactopoiesis when the mouse alpha-lactalbumin lacks.α-lac
m/ α-lac
hHeterozygote and α-lac
h/ α-lac
hAutozygote mouse appearance, reproductivity, behavior are all normal.
Compare α-lac-/α-lac-mouse, α-lac
h/ α-lac
hThe female mouse galactopoiesis of autozygote is apparent normal, can successfully bring up the offspring.Table 4 shows by α-lac
m/ α-lac
hHeterozygote and α-lac
h/ α-lac
hThe young baby that the female mouse of autozygote is brought up and the wild-type mother mouse young weight of raising an infant is similar.We observe these animal capables and fully normally feed the little son continuously, obtain proof thus.These evidences clearly show that people's gene can have the gene of replacing mouse functionally.Breast proximate analysis (table 4) shows lactose concn all similar in all genotype.Although α-lac
h/ α-lac
hAlbumen and fatty consistency decrease in the autozygote animal, but only the fat minimizing is proved significantly by statistical paired t-test.Compare wild-type, these animal milk productions risings (table 4).
The relative quantification of people and mouse alpha-lactalbumin RNA
Human milk contains the alpha-lactalbumin (2.5mg/ml) of Duoing relatively than mouse breast (0.1mg/ml).We wish whether the alpha-lactalbumin gene fragment of determining the people keeps high-caliber expression in the mouse site, or also lower than mouse genetic expression.α-lac
m/ α-lac
hThe heterozygote mouse provides desirable approach for this problem is described, wherein the expression of people's gene can directly be compared with mouse genetic expression in same animal.
Figure 15 A has illustrated the method that compares mouse and human alpha-lactalbumin mRNA level.Because the binding of people and mouse alpha-lactalbumin gene sequence is positioned at the upstream in polyadenylic acid site, so α-lac
mRNA contains the mouse sequence of not translating of 120 bases that are positioned at 3 ' end.Unified radiolabeled mouse rna probe is used for rnase protection analysis, distinguishes people and mouse alpha-lactalbumin mRNA in the same RNA sample to detect.Every kind of mRNA relative abundance is calculated by the segmental labelled amount that is subjected to people and mouse mRNA protection.
Rnase protection analysis the results are shown in Figure 15B.The A road illustrates indigested 455 base probes, and the K road shows that yeast tRNA does not protect any fragment.Wild-type mice RNA has protected the RNA fragment of one 305 base, this fragment consistent with endogenous mouse alpha-lactalbumin RNA (seeing the B road).Homology α-lac
h/ α-lac
hBody of gland RNA protects a less band, the RNA consistent (seeing the C road) of 120 bases of being protected with human alpha-lactalbumin mRNA.The D-J road shows and serial heterozygote α-lac
m/ α-lac
hThe corresponding to result of animal.Every kind of sample is medium and small to show that with big protected fragment people and mouse alpha-lactalbumin mRNA all exist.Protected fragment is downcut from glue, measures emitting isotope content and complies with the adjustment that varies in size, and estimated the ratio of people and mouse alpha-lactalbumin mRNA.Table 6 illustrates 305 bases of downcutting in from Figure 15 B D to J road and the emitting isotope amount of 120 base fragments of being present in, from and heterozygote α-lac
m/ α-lac
hThe ratio of middle people and mouse alpha-lactalbumin mRNA.Although fluctuation but human alpha-lactalbumin mRNA is obviously abundanter than mouse mRNA content to some extent between the Different Individual mouse.Average 7 α-lac
m/ α-lac
hHeterozygote, human alpha-lactalbumin mRNA expresses high 15 times than mouse.
Table 6
α-lac
m/ α-lac
hPeople and mouse alpha-lactalbumin mRNA relative quantity swimming lane mouse number 120 bases, 305 base people/mouse in the mammary gland
Fragment
aFragment
aThe RNA ratio
bD 2 5,957 1,000 15: each swimming lane name of 1 E, 3 5,770 547 26: 1 F, 4 4,825 810 15: 1 G, 76 6,018 1,077 14: 1 H, 98 5,206 1,452 9: 1 I, 99 5,481 1,117 12: 1 J 79 26,858 3,561 19: 1 show the source of protected fragment and corresponding a. arithemetic unit with Figure 15 B be count per minute (c.p.m) b.120 the c.p.m of base fragment multiply by the ratio of 2.54 (varying in size with adjustment) and the c.p.m of 305 base fragments
Human alpha-lactalbumin is expressed
Alpha-lactalbumin in the target mouse species has been done the Western analysis.Human alpha-lactalbumin can distinguish (seeing A, B road) with the mouse alpha-lactalbumin because of its swimming rate fast.α-lac
h/ α-lac
hAutozygote and α-lac
m/ α-lac
hA tangible lower molecular weight band can be observed (seeing C, D, G, H road) in the heterozygote mouse, and it is corresponding to the position of human alpha-lactalbumin standard, and only can observe in the mouse of expressing human alpha-lactalbumin.This mouse obtains (data not shown goes out) by gene targeting or protokaryon microinjection.This point is confirmed by the peptide analysis (data not shown goes out) of phenyl-agarose chromatography (seeing Figure 16) and cyanogen bromide decomposition release.The band of low swimming rate is arranged, similar to the mouse alpha-lactalbumin, also be the human alpha-lactalbumin gene product, its character the unknown, it is with α-lac
hGene dosage difference and intensity changes (seeing G, H road) to some extent also is present in the human alpha-lactalbumin transgenic mice Ruzhong that produces by the protokaryon microinjection.
The alpha-lactalbumin content of breast sample is done quantitatively with phenyl-agarose chromatography.Figure 16 illustrates three kinds of shown among Figure 13 newborn samples, comprises α-lac
m/ α-lac
hHeterozygote and α-lac
h/ α-lac
hThe overlapping light absorption value curve of the post wash-out of autozygote.Peak corresponding to the alpha-lactalbumin of wash-out marks.Alpha-lactalbumin content is by relatively the peak area and the estimation of human alpha-lactalbumin typical curve of integration obtain.Linear between the peak area of integration and the alpha-lactalbumin amount, can highly repeat.The estimation of alpha-lactalbumin content is as follows in the sample among Figure 16: α-lac
m/ α-lac
mWild-type, 0.1mg/ml; α-lac
m/ α-lac
hHeterozygote #76,0.45mg/ml; α-lac
h/ α-lac
hAutozygote #22,0.9mg/ml.Table 5 illustrates from alpha-lactalbumin concentration in target mouse species and lactating women's the newborn sample.Fairly obvious, Ruzhong alpha-lactalbumin concentration is directly related with gene dosage, as α-lac
m/ α-lac
hHeterozygote only is half of wild-type alpha-lactalbumin concentration.Because the milk production of these mouse similar (table 4), so the concentration of alpha-lactalbumin provides a reasonable index that shows its synthetic quantity.Heterozygote α-lac
m/ α-lac
hThe alpha-lactalbumin amount that the relative proportion of people from Ruzhong and mouse alpha-lactalbumin component is expressed from single mouse allelotrope by supposition is 0.043mg/ml, and all the other representative's alpha-lactalbumins.This point with by α-Lac
mThe alpha-lactalbumin amount that/α-Lac-heterozygote and wild-type mice are expressed is consistent.Therefore, α-lac
m/ α-lac-heterozygote estimates to express 0.61mg/ml human alpha-lactalbumin and 0.043mg/ml mouse alpha-lactalbumin.Therefore, α-lac
m/ α-lac
hHeterozygote Ruzhong human alpha-lactalbumin is 14 times of mouse alpha-lactalbumin approximately.This and relative mRNA ratio unanimity.
The enhancing of embodiment 8 heterologous genes is expressed
These digital proofs, be included in upstream promoter district in the pHA-2 construction (AUG to-3.7kb) strengthened expression of heterologous genes.Table 7 illustrates the newborn analytical results from the female mouse of the pHA-2 transgenosis person of foundation.Expressed high-caliber people α-lac for 6 in 10 female mouse, but 3 female mouse fail to express the people α-lac (being less than 0.2mg/ml in this analyzes) of detection level, can not transmit transgenosis.We can not affirm that they are low expressers or can not transgenosis.
Table 8: construction PKU-0 to BALT-B all contains ox α-lac promotor (about 2kb), and construction PKU-1H to PKU-16 all contains people α-lac promotor (3.7kb).End user α-lac promotor increases transgene expression to almost 100%.
Data show end user α-lac promotor, than with ox promoter expression efficient height, and can be in more animal body abduction delivering.
Table 7
| Construction | Mouse | Sex | ??C | The mg/ml breast is analyzed | TRANS. | MI?FREQ. |
| ??pHA2 | 210-17 | M | - | - | 1/21＝5％ | |
| 211-12 | F | ＜1 | - | - | 14/54 ＝26％ | |
| 211-16 | | 10 | 5 | 3/4 | ||
| 211-17 | F | ＞＞10 | | 0/8 | ||
| 211-24，27， 29，31，36， 37，42，46， 47，54 | M | |||||
| 212-7 | | 10 | | 0/5 | 8/77＝10％ | |
| 212-11 | F | ＜10 | 1 | 0/4 | ||
| 212/13 | F | ＞1 | 1-5 | 2/4 | ||
| 212-19 | F | ＞10 | | 0/8 | ||
| 212-36，44， 45，46 | M | |||||
| 213-4 | F | ＜10 | 5 | 0/2 | 2/14＝14％ | |
| 213-5 | F | ＞10 | 10 | 2/8 |
Total integrating frequency 25/166=15%
ND=does not measure
TBA=is to be analyzed
TBO=continues to raise
The C=copy number
TRANS.=transmits
MI FREQ.=integrating frequency
Table 8
| Construction | Transgenosis person | The expresser | The maximum expression |
| PKU-0 | ??6F/5M | ??3/5 | ??500 |
| PKU-1 | ??18F/23M | ??5/18 | ??200 |
| PKU-2 | ??8F/7M | ??3/8 | ??400 * |
| PKU-3 | ??6F/5M | ??2/6 | ??100 * |
| PKU-4 | ??7F/3M | ??5/18 | ??300 * |
| BALT-A | ??34F/24M | ??n.t. | ??n.t. |
| BALT-B | ??10F/18M | ??n.t. | ??n.t. |
| PKU-1H | ??6F/5M | ??4/5 | ??100 |
| PKU-5 | ??13F/14M | ??13/13 | ??800 |
| PKU-6 | ??3F/4M | ??2/2 | ??100 |
| PKU-7 | ??4F/11M | ??1/1 | ??＜20 |
| PKU-16 | ??13F/12M | ??n.t. | ??n.t. |
| HαPKU-1 | ??n.t. | ??n.t. | ??n.t. |
| HαPKU-2 | ??n.t. | ??n.t. | ??n.t. |
N.t.=does not test
* estimation
Sequence table (1) general information:
(i) applicant:
(A) title: PPL Therapeutics (Scotland) Ltd.
(B) street: Luo Siling
(C) city: Edinburg
(E) name of the country: Britain
(F) postcode: EH259PP
(ii) denomination of invention: alpha-lactalbumin gene constructs
(iii) sequence number: 21
(iv) computer-reader form
(A) media types; Floppy disk
(B) computer: IBP PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: Patent In Release#1.30
(EPO) information of version (2) sequence SEQ ID No:1:
(i) sequence signature:
(A) length: 27 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:1GCGGATCCAC AACTGAAGTG ACTTAGC 27 (2) sequence SEQ ID No:2:
(i) sequence signature:
(A) length: 35 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:2GATGGATCCT GGGTGGTCAT TGAAAGGACT GATGC 35 (2) sequence SEQ ID No:3:
(i) sequence signature:
(A) length: 43 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:3GCAGGCGAAT TCCTCAAGAT TCTGAAATGG GGTCACCACA CTG 43 (2) sequence SEQ ID No:4: (i) sequence signature:
(A) length: 33 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:4GAGGATCCAA TGTGGTATCT GGCTATTTAG TGG 33 (2) sequence SEQ ID No:5:
(i) sequence signature:
(A) length: 46 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:5GCTGAATTCG TTAACAAAAT GTGAGGTGTA TCGGGAGCTGAAAGAC 46 (2) sequence SEQ ID No:6:
(i) sequence signature:
(A) length: 58 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:6CCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGGT ACGCGGTACA GACCCTG 58 (2) sequence SEQ ID No:7:
(i) sequence signature:
(A) length: 58 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:7GCGGATCCGA TCGCTTGTCT GTCATAACCA CTGCTATGGA GCGCGGTACA GACCCCTG 58 (2) sequence SEQ ID No:8:
(i) sequence signature:
(A) length: 69 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:8GCGGATCCGA TCGTACAAAA CAATGACAGC ACAGAATATG GACTCTACCA GATAAATAAT 60AAAATTTGG 69 (2) sequence SEQ ID No:9:
(i) sequence signature:
(A) length: 34 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:9GCTCTAGATC ATCATCCAGG TACTCTGGCA GGAG 34 (2) sequence SEQ ID No:10:
(i) sequence signature:
(A) length: 24 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:10GCTGAAGCTT CACTTACTTC ACTC 24 (2) sequence SEQ ID No:11:
(i) sequence signature:
(A) length: 65 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:11GCGGATCCAA AGACAGCAGG TGTTCACCGT CGACGACGCC TACGTAACTT CTCACAGAGC 60CACTG 65 (2) sequence SEQ ID No:12:
(i) sequence signature:
(A) length: 46 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:12GCTGAATTCG TTAACAAAAT GTGAGGTGAG CCGGGAGCTG AAAGAC 46 (2) sequence SEQ ID No:13:
(i) sequence signature:
(A) length: 54 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:13GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGAT ACGCGGTACA GACC 54 (2) sequence SEQ ID No:14:
(i) sequence signature:
(A) length: 69 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:14GCGGATCCGA TCGTACAAAA CAATGACAGC ACAGAATATG GACTCCTCCA GATAAATAAT 60AAAATTTGG 69 (2) sequence SEQ ID No:15:
(i) sequence signature:
(A) length: 34 base pairs
(B) type; Nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:15GCTCTAGATC ATCATCCAGC AGCTCTGGCA GGAG 34 (2) sequence SEQ ID No:16:
(i) sequence signature:
(A) length: 264 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:16GGATCCAAAG TTGGCTAAAC ACTGGCCGGG TGCAGTGCTT CCACCTGTAA TTCCAGCACT 60TTGGAAGGCT GAGGTGGGCA GATTGCTTGA GGTCAGGAGT TTGAGACCAG CTTGGCTAAC 120AGCAAAACCC TGTCTCTACC AAAAGTACAA AAATTATCTG GGTGTGGTGG CAGGCGCCTG 180TAATCCCAGC TACTCGGGAG GCTGAGGCAG AAGAATTGTT TGAACCTGGG AGGCAGAGGT 240TGTAGTGAGC TGAGATCGCT CATT 264 (2) sequence SEQ ID No:17:
(i) sequence signature:
(A) length: 803 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
( xi ) ：SEQ ID No：17TCTTTTTCAA TTATTCATTT GTTACAGTGG GTTATGATAC AAATGTTTAT AGATGCCTAC 60TCTGTACTAG TACTACAGAG CAGTTTTTCT GTGTTTATAT TCAGTTCAAT TGTAGTGTGT 120TGAGTTGTAT WWTAATCCAT GTATTAAATC AAATAAACAA ACAAAATGCC ATGTTCTTTG 180GTACAAGCAA CACTCACCAA AGGCATTTGG GGTCTGCATT TGGAATTCTC AGGCAAACTC 240TCTCTTGTTC CTAGTCTGTA CTTATTTTCC CCACACTAGC TTATGTATAT ATATTTTTGA 300GATTGGAGTT GCCCTTGTTG CCCAGGCTGG AGTGCAGTGG CACGATTCTT GGCTCACGAG 360ACCTCCACGT CTTGGGTTAA AGCGTTTCTC CTGCCTCAGC CTCCTGACTA CTGGGATTAC 420AGGCGCCTGC CACCATGCCC GGCTAATTTT TGTATTTTTA GTAGAGATGG GCTTTCACCA 480TGTTGCTCAG GCTGGTCTTG AAACTCCCCA CCTCGGCCCT TCCCAAATGC GCTGGGATTA 540CAGGTGTGAG CCACAGTGCC TGGCCTGTAC ATTTTTTAAA TTTCAATGTC TAATATGGTG 600TCCACTGAAT TAAGAATTCT TTTGAGAAAA TGAATCAATA AATCTATACA CTGCCTCCTT 660TATCCAGTGA GGTATGGCTG GATCAGCTTC ATGACATACA TGCCAGTAGT TCTCCTCCTC 720CTCCTTTTTT ACAAATAAAA ATTGTATATG TTGAAGGTGT ACAACTTGAT GTTTGTTATA 780TGTATACACT TAAATGTCAC CAC 903
(i) sequence signature:
(A) length: 238 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID No:18TTTGGGTAGA AGCAGACAGT AAACTTGCTG TTCTCTTCCT GAGATCTTTT GTTGAGATGC 60TGAATAGGAG GCAGCATGGC AGCTGAGCTA TCTGTTCTGC TTTCTCTACC TCTGTCTCTT 120TCCCTTAGGC CTAAAATGAA GCTCTAAGCC AAGCAAAGGT CTGAAGTCAT CCAGACTAAT 180TGGGAAGCGG GTAGGCTCCA GGGAGTGGCT CTCAGAGAGC AGACCATTTA CTGAGCTC 238, (2) information of sequence SEQ ID No:19:
(i) sequence signature:
(A) length: 162 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: the information of SEQ ID No:19AATACAGAGT TTTGTTCTCT ACTCTTATCC TGCTTTCTCC TCCCTGCTAC TTTTCCCTGA 60CACCTATCTT GTTGTGAAGA CAGGAATTGC ATTAGATAAA ATCAAATCTT TTTTATTTTT 120TTTTGAGATG GAATCTTGCT CTGTTTCCAG GCTGGAGTGC TG 162 (2) sequence SEQ ID No:20:
(i) sequence signature:
(A) length: 472 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
( xi ) ：SEQ ID No：20ATCTGGTCAG CAGTGAAGCT CAGTGTACAC ATTCATTCCT TCCTTCACTG CTTGATTTGT 60CACCAAGTGG TTATTGAGGA TATGCTGTTT GCTAGGTACT ACTTTACTTA TTTATTTGTT 120TATTTAGAGA TGGGGTCTCA CAATGTTGCC CAGTCTACAG GACAGTGGCT ATTCACAGGT 180GTGAGCACAG CACACTACAG CCTCAAACTC CTGAGTTCAA GAGATCCTCC TGCCTCAGTC 240TCTCGAGTAG CTGGGACTAC AGGGATGTGC CACCACACAT GGCTTAGGCT CTACTTTAGC 300TGCTACTTGA AGGATGAAGA TAGGAGGAGA CACTCTTATT TTATTTGATT TCTTTTTTTT 360TTTTTTTTTT TTGACAGAGT TTTGCTCTGT TGCCAGGCTG GAGTGCTCAC TGCAACCTCC 420ACCTCCAGGT CAAGCAATTC TCCTGCTCAG CCTCCGAGTA GTCGGACCAA GG 472 ( 2 ) SEQ ID No：21：
(i) sequence signature:
(A) length: 2119 base pairs
(B) type; Nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
( xi ) ：SEQ ID No：21GGAATTCCCA TTCCTGTCGT GTACCCTTGC AGTGCCTCTG GGTGGAATGC GGAGAAATGG 60AGTGGCTCCA CTTCTGTTGT GTTTCTGAAC ATGTATCTCT TGCTATCAGA ACTTTCTGCT 120CATCCCTTCT GGCACACCAA GATCCTCCAC ATTCCCTTCA CTCATGCCAC TTCATATACT 180GGTTATCCAT GGTACAGAAG ACAGGATTTA ACTGAGAGGA CTTTTCCCTG ACTCTGAATA 240CATGTAGGAG ATAACGATAT GGAAGACCTT CAGTATGTAA GTCTTAAATA GATTGGTTGG 300GATAAATGTT CCCTGAAACA TAAGAAACAG CGCAGCGGCT CCTGTCTGTA ATCTAGCACT 360TTGGGAGGGC CGAGGCCAGG CAGGCAAATT GCCTGAGCTC AGAAGTTTGA GACCAGCCTG 420GCCAACATGC AGAAACTCCG TCTCTACTAA AAATACATAA ATTAACCGGG CATGGTAACA 480CGTGCCTGTA GTCCCAGCTA CTCGGGAGGC TGAGGCAGGA GAATCACTTG AGCCTGGGAG 540GCAGAGGTTG CAGTGAGCCA AGATCGCGCC ACTGCATTCC AGCCTGGGCA ACAGAGTGAG 600ACTTGGTCAA AAAAAAAAAA AAAAAAAAAA AAAAGGAAGA AGAAGAAGAA ATCAGGTTTA 660GAGATGAGGA CAAAGAAGAC GAATCGGTGG CATGAAGGAG CTAAGAGCTA CTTGTCACCA 720TGACATGAAG CTTCATGCCA GCAAATTAAA GGAGCTATTC AGAACTAGTA TCCTCAACTC 780TACTTGCTCA GGGGCACTGA CCTTATAGAG ATTCCAGACA TAAGCTTGTT CAGCCTTAAG 840TCCAATCTTT CCACTGGCTT GGTCCTTCCC ACTTTCTGTG GCCAACTCTG AGGTTGTCTA 900CAAGTTATTG GTCTTAGATT TATGTAATGT CTCAATGCCA GTGTAGTATT TGGTTATTTA 960CGGTAGGAGT GGTTAGGGGT GGGGAATCTG ATAATAGCTC GTAGGATAGC TAGATTCTTT 1020TTTTTTTTTT TTTTTTTTAA AGATAGGGTC TCACTTTGTC TCCCAGGATG GATGGATGGA 1080GTGCAGTGGA GTGAACATGG CTCACTGCAG CCTCGACCTC CTGTGCTCAA GTGTTCCTCC 1140TGCCTCAGCC CCTCAAGTAG CTGGGACTAC AGGCACATGT CACCATGCCC AGCTAATTTT 1200TTTTGTAGAG ATGGGATTTT ACCATGTTGC CCAGGCTGGT CTCGAGCTCC TGGGCTCAAG 1260TGATCCACCA GACTCGGCCT CCCAAAATGC CGGGATTACA GGTGTGAGCC ACTGTGCCTG 1320GCCTAGATGC TTTCATACAG GCTTTTCAAT TATGCATTTT CCTTAAGTAG GAAGTCTTAA 1380GATCCAAGTT ATATCGGATT GTTGTAGTCT ACGTTCCCAT ATTCTATTCC TATTTCTGAG 1440CCTTCAGTCA TGAGCTACCA TATTAAAGAA CTAATTCTGG GCCTTGTTAC ATGGCTGGAT 1500TGGTTGGACA AGTGCCAGCT CTGATCCTGG GACTGTGGCA TGTGATGACA TACACCCCCT 1560CTCCACATTC TGCATGTCTC TAGGGGGGAA GGGGGAAGCT CGGTATAGAA CCTTTATTGT 1620ATTTTCTGAT TGCCTCACTT CTTATATTCC CCCCATGCCC TTCTTTGTTC CTCAAGTAAC 1680CAGAGACAGT GCTTCCCAGA ACCAACCCTA CAAGAAACAA AGGGCTAAAC AAAGCCAAAT 1740GGGAAGCAGG ATCATGGTTT GAACTCTTTC TGGCCAGAGA ACAATACCTG CTATGGACTA 1800GATACTGGGA GAGGGAAAGG AAAAGTAGGG TGAATTATGG AAGGAAGCTG GCAGGCTCAG 1860CGTTTCTGTC TTGGCATGAC CAGTCTCTCT TCATTCTCTT CCTAGATGTA GGGCTTGGTA 1920CCAGAGCCCC TGAGGCTTTC TGCATGAATA TAAATAAATG AAACTGAGTG ATGCTTCCAT 1980TTCAGGTTCT TGGGGGTAGC CAAAATGAGG TTCTTTGTCC CTCTGTTCCT GGTGGGCATC 2040CTGTTCCCTG CCATCCTGGC CAAGCAATTC ACAAAATGTG AGCTGTCCCA GCTGCTCAAA 2100GACATAGATG GTTATGGAG 2119
| Clear people in Shen or attorney docket international application no PT/GB95 01651 |
Explanation about microbial preservation
(detailed rules and regulations 13 two)
| A. to specification sheets the _ _ _ _ _ _ _ _ page or leaf, the _ _ _ _ _ _ _ _ _ _ _ _ explanation of the described microorganism of row | |
| B. the preservation item other be deposited in the supplementary page | |
| NATIONAL COLLECTIONS OF INDUSTRIAL AND MARINE BACTERIA LTD depositary institution's title industry and preservation company limited of marine bacteria country | |
| Depositary institution address (comprising postcode and name of the country) 23 ST MAchar DRIVE ABERDEEN ABZ IRY | |
| 15 days February nineteen ninety-five of preservation date | Deposit number NCIMB40709 |
| C. have supplementary page in supplementary notes (in case of necessity) this column | |
| The bacillus coli | |
| D. this explanation is done (if explanation is not done for all designated states) for following designated state | |
| All designated states | |
| E. remark additionally (in case of necessity) | |
| Following explanation will provide (writing out the classification of explanation, for example: " numbering of preservation ") preserving number to international office subsequently: 40709 preservation days: the construction of preservation on the 15th in February nineteen ninety-five: pHA-2 | |
PCT/RO/134 shows (in July, 1992)
Claims (34)
1, a kind of recombination construction, this construction are suitable for expressing alpha-lactalbumin or its function equivalent or its part in the ox cell.
2, the described construction of claim 1 is suitable for expressing human alpha-lactalbumin or its function equivalent or its part.
3, the described construction of claim 1 is suitable for expressing ox alpha-lactalbumin or its function equivalent or its part.
4, a kind of carrier that contains the described recombination construction of claim 1.
5, a kind of host cell that contains the described carrier of claim 4.
6, a kind of the described construction of claim 1 is incorporated into its genomic transgenic animal.
7, the described transgenic animal of claim 6, described animal capable is passed to the offspring with construction.
8, the described transgenic cattle of claim 6.
9, a kind of production has the method for breast of the alpha-lactalbumin content of increase, and this method comprises and extracts breast in the Accessory Right requirement female transgenic animal 6 described lactation.
10, the breast of producing according to claim 9.
11, the alpha-lactalbumin of the Ruzhong extraction of Accessory Right requirement 10.
12, a kind of recombination construction that is suitable for expressing alpha-lactalbumin or its function equivalent or its part, described construction has the flanking sequence that is selected from as next group:
Any one 3 ' flanking sequence among a, the SEQ ID NOS.16 to 20;
5 ' the flanking sequence of b, SEQ ID NO.21;
The part of c, these sequences.
13, the described construction of claim 12, its 3 ' flanking sequence are selected from a sequence of one group of being made up of any sequence of SEQ ID NOS.16 to 20 and its substantial part, and its 5 ' flanking sequence is selected from SEQ ID NO.21 or its substantial part.
14, the described construction of claim 12 is suitable for expressing human alpha-lactalbumin or its function equivalent or its part.
15, the described construction of claim 12 is suitable for expressing ox alpha-lactalbumin or its function equivalent or its part.
16, a kind ofly contain the carrier that right requires 12 described recombination constructions.
17, a kind ofly contain the host cell that right requires 16 described carriers.
18, a kind of the described construction of claim 12 is incorporated into its genomic transgenic animal.
19, the described transgenic animal of claim 18, described animal capable is passed to the offspring with construction.
20, transgenic cattle as claimed in claim 18.
21, a kind of production has the method for breast of the alpha-lactalbumin content of increase, and this method comprises and extracts breast in the Accessory Right requirement female transgenic animal 18 described lactation.
22, the breast of producing according to claim 21.
23, the alpha-lactalbumin that extracts from the Ruzhong according to claim 21.
24, a kind of being selected from by pBBHA, pOBHA, pBAHA, pBova-A, pBova-B, pHA1, pHA2 and one group the recombination construction of forming by its deutero-construction.
25, a kind of the described construction of claim 24 is incorporated into its genomic transgenic animal.
26, the described transgenic animal of claim 25, described animal capable is passed to the offspring with construction.
27, the described transgenic cattle of claim 25.
28, a kind of production has the method for breast of the alpha-lactalbumin content of increase, and this method comprises that Accessory Right requirement female transgenic animal 25 described lactations extract breast.
29, the breast of producing according to claim 28.
30, the alpha-lactalbumin that extracts from the Ruzhong according to claim 28.
31, have the polynucleotide that are selected from as the sequence of next group:
The sequence of a, SEQ ID NO.16;
The sequence of b, SEQ ID NO.17; With
The part of c, above-mentioned sequence.
32, a kind ofly contain the recombination construction that right requires 31 described polynucleotide.
33, contain the transgenic animal that right requires 32 described constructions.
34, the described transgenic cattle of claim 33.
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/GB1994/001514 WO1995002692A1 (en) | 1993-07-16 | 1994-07-13 | Modified alpha-lactalbumin |
| WOPCT/GB94/0514 | 1994-07-13 | ||
| GBGB9425326.7A GB9425326D0 (en) | 1994-12-15 | 1994-12-15 | Gene constructs |
| GB9425326.7 | 1994-12-15 | ||
| US08/381,691 US5852224A (en) | 1994-12-15 | 1995-01-31 | α-lactalbumin gene constructs |
| US08/381,691 | 1995-01-31 | ||
| GB9503822.0 | 1995-02-25 | ||
| GBGB9503822.0A GB9503822D0 (en) | 1995-02-25 | 1995-02-25 | "Alpha-lactalbumin gene constructs" |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1157635A true CN1157635A (en) | 1997-08-20 |
Family
ID=56289646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95194129A Pending CN1157635A (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0765390A1 (en) |
| JP (1) | JPH10502816A (en) |
| CN (1) | CN1157635A (en) |
| AU (1) | AU700224B2 (en) |
| CA (1) | CA2193513A1 (en) |
| NZ (1) | NZ289197A (en) |
| WO (1) | WO1996002640A1 (en) |
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| CN100335632C (en) * | 2000-12-08 | 2007-09-05 | 李宁 | Seven kinds of yak milk protein gene sequence |
| CN100445379C (en) * | 2005-04-21 | 2008-12-24 | 李宁 | Human alpha-lacto albumin gene transgenic cloned macro domectic animal production method |
| CN101104635B (en) * | 2007-04-30 | 2010-11-03 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010119088A2 (en) * | 2009-04-15 | 2010-10-21 | Bodo Melnik | Milk and milk-based products modified to exhibit a reduced insulinemic index and/or reduced mitogenic activity |
| CN114736287B (en) * | 2022-04-20 | 2023-07-07 | 中国农业科学院生物技术研究所 | Hypoallergenic alpha-lactalbumin, and preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3670003B2 (en) * | 1992-06-15 | 2005-07-13 | ファーミング ビー．ブイ． | Production of recombinant polypeptides by bovine species and transgenic methods |
| GB9314802D0 (en) * | 1993-07-16 | 1993-08-25 | Pharmaceutical Proteins Ltd | Modified proteins |
| AU1454495A (en) * | 1993-12-29 | 1995-07-17 | Gene Pharming Europe Bv | Recombinant production of modified proteins lacking certain amino acids |
-
1995
- 1995-07-12 WO PCT/GB1995/001651 patent/WO1996002640A1/en not_active Application Discontinuation
- 1995-07-12 EP EP95924467A patent/EP0765390A1/en not_active Withdrawn
- 1995-07-12 JP JP8504802A patent/JPH10502816A/en active Pending
- 1995-07-12 CA CA002193513A patent/CA2193513A1/en not_active Abandoned
- 1995-07-12 AU AU28962/95A patent/AU700224B2/en not_active Ceased
- 1995-07-12 CN CN95194129A patent/CN1157635A/en active Pending
- 1995-07-12 NZ NZ289197A patent/NZ289197A/en unknown
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335632C (en) * | 2000-12-08 | 2007-09-05 | 李宁 | Seven kinds of yak milk protein gene sequence |
| CN100445379C (en) * | 2005-04-21 | 2008-12-24 | 李宁 | Human alpha-lacto albumin gene transgenic cloned macro domectic animal production method |
| CN101104635B (en) * | 2007-04-30 | 2010-11-03 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
| CN102590413B (en) * | 2012-01-18 | 2013-12-25 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
Also Published As
| Publication number | Publication date |
|---|---|
| AU700224B2 (en) | 1998-12-24 |
| EP0765390A1 (en) | 1997-04-02 |
| CA2193513A1 (en) | 1996-02-01 |
| WO1996002640A1 (en) | 1996-02-01 |
| NZ289197A (en) | 1998-09-24 |
| AU2896295A (en) | 1996-02-16 |
| JPH10502816A (en) | 1998-03-17 |
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