Gene transplanted plant secreting desired protein in milk

[Detailed Description of the Invention]

3 Detailed description of the invention] (Field of the Invention) The present invention relates to a transgenic animal. (PRIOR ART) It is possible to be inserted in the genome of the animal which inserting Foreign gene in the embryo of Vertebral animal and the gene make the object. How (for example, Husser (Huszar) et al., 1985 Py N A varnish (P, N, A.)) to use a method (the microphone loin Jekshishin method (minute injection method)) and a retroviral vector with mechanical insertion of foreign genes S) It is carried out on Knee varnish A (U, S, A), and 82-volume 8587 pages. oh obtained by this Hill work -- "TRANS GENIC (iffff Teshiba transplant J] -- a snout -- " -- called.) In sexual reproduction, these foreign genes are transmitted to a next generation, and are often revealed in that animal. 0 revealed in m Weave with specific protein currently coded by foreign genes in some examples, for example, a metalaw Thionein promotor, in liver of a transgenic mouse, other examples used for ordering it growth hormone gene expression of a rat (Palmi Tarr et al. (palmite -- "), 1982, Nature, 300 volumes, 611 pages) are elastase promotors. Moreover it was used for ordering it revelation by the pancreas of foreign genes (Ornitz et al. (OrniLz), 1985, NaLure, 313 volumes, 600 pages), in a transgenic animal, it also became possible to control gene expression embryologically. 0 by which I!p Chi and foreign genes can also be transferred only in specific Mlla at a certain specific time -- for example In Magnum (Magnam) et al. and 1985, controlling embryologically Nature, 315 volumes, and 338 pages of genes on the basis of a grotesque bottle promotor's instructions will be shown, and he is Krummallaf et al. (Krumlauf) (1985, Mol.), Ce11.Biol, five volumes, and 1639 pages of same results are shown using the mini gene of alpha fetoprotein. (Constitution of an invention) Generally the present invention is characterized by DNA arrangement containing a gene which codes a certain protein. The gene has received in nature transfer system ?J of a milk protein promotor of the mammals which do not control the transfer. Although the promotor in whom the DNA arrangement includes the arrangement which is coding DNA!p Chi which makes secretion of the protein possible, and the secretion signal which exists between the gene concerned and promotor again may be a promotor of milk serum protein, or a promotor of casein protein, The promotor of milk serum protein is more preferred so that it may explain in detail later. (In this specification, "5 A word" means both genome DNA arrangement and cDNA arrangement). The maintenance of the present invention is easy and stable, and it makes it possible to produce [ to wish ] of arbitrary protein in the cultivation system which can move. Ip Chi and a cultivation system of the present invention are live livestock. It not only produces protein to wish to have, but it may transmit the same capability as female posterity. By secreting the protein in the milk of a host's animal, a closed model is easy and it makes unnecessary removal operation of the culture-medium additive which has a constituent of blood and that poisonous and it is carcinogenicity. It is proteinic yield of a much more important thing is high, and the production has high cost efficiency, and effective. Other features and advantages of the present invention are clear from the following description about the desirable example of the present invention, and the statement of a claim. better -- Dragon r pL-PA which is a middle vector [ in / in Drawing 1 / the present invention ] It is a manufacturing process figure of VPI-LP gas (K), and is Drawing 2, pWAP which is a middle vector in the present invention It is a manufacturing process figure of (Hs) and is Drawing 3, pWAP-L-PA which is a middle vector in the present invention pWAP-Hb s which it is a manufacturing process figure of (S), and Drawing 4 is a manufacturing process figure of pHbssVA which is a middle vector in the present invention, and is a middle vector [ in / in Drawing 5 / the present invention ] It is a manufacturing process figure of (S). DNA Arrangement 1 A time bifurcated D As for a milk protein promotor, the thing from any Tits animal species is (as long as it is a promotor of the protein usually secreted into the milk of Milk in a nature, what kind of thing may be used.). -Milk protein is classified into casein, and it is defined as being the milk protein which exists in milk by the shape of micell here by C, and is removed from skim milk by the solidification by rennet. Milk serum protein is defined as non-casein milk protein here. It is the main ingredients of a whey protein and milk serum protein, and the protein called whey acid protein is contained in Rodent neck. Whey acid protein (rWAPJ) was named based on the acid isoelectric point (Pirec (FileLz)). Other examples of milk serum protein given in 1981 (J, B i o 1. Chem), 256 volumes, and 11509-page zero literature, It is alpha-lactalbumin (about mouse WAP, they are Uzen (Henntnghausen), and Syros Pell (Sippe]), 1982, Eur, J and Biocbem to Henning.). 125 volumes and 131 pages -- 3 -- Wl(s). Milk protein is Warstra (WalsLra) and '; Engineering 7 Kness (Jenness) i and Da iry. ChemrsLry and It is indicated in detail in Physics and (Jigon Wiley & Suns and 1984). While casein is usually produced not only after childbirth but during pregnancy in [animal, since WAP appears only in the lactation period after childbirth, generally in the present invention, it is more preferred [ the milk-serum-protein promotor ] than a casein promotor. This difference is potentially important for two reasons. Until being produced before the protein wished to have in the first place under a casein promotor's transfer Min giving birth comes to be secreted in milk after this protein's giving birth n# It is useless at the point that it cannot do.! It is harmful to the health of a host animal that come toxicity more when the protein which Ambition to the second exists in a large door (for example, human &l11a plasminogen activator (t-PA)), and the protein is built all over an organization in advance of breast-feeding. Re since, as for the other strong points which a whey promotor like a WAP promotor has, a whey protein exists in large quantities in milk -- it is that are, and they are strong light promotors and get. Milk protein '?7m which can be made into 11 sources of promotors including a WAP promotor (Kimiko) Henning Hansen, Syros Pell and ml Bd, one to key 7 bells (campbell), 1984, Nuclejc. AcIds Reaearch. The method of the 0 present invention which can be obtained by the method same as described by 12 volumes and 8685 pages as having carried out the monolayer of the WAP gene, -Carry out the monolayer of the mRNA to a from the mammary gland which is secreting milk, and create a cDNA library from this mRNA, cDNA of the specific milk protein for which it asks is screened from a library, and it consists of Comparative work of using suitable cDNA as a probe for a genome clone being isolated from the library of; which carries out the cloning of the cDNA to a vector, and a genome. It is thought that upstream arrangement constitutes the "promotor" who asks from the transfer starting point in a genome clone. That is, it exists in advance of the target gene, and the promotor of the 0 present invention which is a genome sequence considered to participate in the ElilgB isolates by restriction and nuclease digestion, and a subcloning process. The WAP promotor of a mouse who the promotor does not need to be a thing of specific length and does not need to show the character of a certain regulation directly is 2 *6kb of immediately a side to the 5-degree end of a WAP signal sequence. The monolayer was carried out as an EcoRl-Kpnl fragment. Rare! J3d11pi According to the present invention, the arbitrary protein to wish to have is producible. Desirable protein is useful for medical treatment, prevention, and/or diagnosis of a human illness. For example, they are L-PA and a hepatitis B surface antigen. The present invention is especially useful about the protein which must be produced in large quantities and economically, for example like the enzyme for industry, or animal protein. l[ this ] ± Stop ■ victory In order to secrete the protein wished to have into the milk of a host animal, the DNA arrangement containing the gene of the protein to wish to have needs to include DNA which secretes the protein into milk from a mammary gland organization, when translated. If there is no such arrangement, the necessity of the protein to wish to have remaining during a mammary gland organization, and a proteinic closed model becoming difficult, and murdering a host animal will be produced. This DNA codes the hydrophobic secretion signal arrangement cut in a secretion process. If the protein to wish to have is usually secreted (for example, t-pA), as the signal sequence, what relates to this protein to wish to have in the nature can be used. As an exception method, the signal sequence of milk firefly white matter including a promotor can be used as arrangement which is coding the signal. That is, a milk protein gene is digested, a promotor is isolated and a DNA fragment including both of the arrangement which codes the signal which is downstream immediately is chosen from that promotor and this promotor. Other exception methods are using the arrangement which codes the signal originating in another secretory protein which is not the milk protein usually revealed from a promotor, or this protein to wish to have, either. Stopped region the inside of the gene to wish -- or -- although this part with desirable a stopped region existing downstream from 3" end may be provided as arrangement which exists in the gene -- otherwise, -- coming -- it is necessary to add When the arrangement is added, desirable arrangement is provided by the Pori Ade Nils-ized arrangement of a 5V4O virus so that it may explain in detail below. Mold soil'=lk= Work -All the DNAa works used for gene construction of the present invention C, For example, it carries out using commonly used art which is indicated in the More error cloning manual (Molecujar Cloning Manual) (Cold Spring Harbor Laboratory) of Maniatis and others, and 1982. DNA - Once gene construction is attained, for example in a vector like a plasmid, the DNA fragment which consists of protein-stop arrangement wishing promoter signal sequence-will be cut, and will be introduced to the embryo of a mammal to wish. Whether a retrovirus performs this introduction, for example, or Kramer (Kraemer) et al., (1985), Costantini (cosLantini), and Genetic(Jaenisch) W Genetic Manicure racing object early Mam Marian embryo (Genepic Manipulation or the Early Ma organization alian Embryo), Cold Spring Harbor Laboratory (cow Embryo microinjection); A 1 A 7- et al. (Ha organization ar), (1985), NaLure, 315 volumes, 680 pages (a rabbit, Sheep) and Buta -- an embryo -- microinjection; and Gorton (Gordon), and Radl (Ruddie), (1984), Methods in It can make by Enzymology, 101 volumes, and the standard microinjection technique indicated to 411 pages (mouse embryo microinjection). As for a microinjection, being made to the embryo of - cell term is preferred so that the frequency where poured-in DNA is taken into all the cells of the animal containing Weave in mammary gland &[, and the DNA may be taken also into a productive cell and the frequency where the posterity of the animal is also TRANS GENIC may become the maximum. A microinjection is art which consists of pouring in the buffer solution which isolates a fertilized ovum, catches a front core within a view, holds an egg and contains DNA using a pipette with a sharp tip about 1.5 micrometers in diameter with a pipette with a blunt tip about 50 micrometers in diameter to a front core if it explains briefly. Ripen a transgenic female animal sexually, it is made to copulate following a microinjection, and milk is collected after childbirth. That by which the breeding was carried out so that a lot of milk might be produced as a host mammal, for example, a cow, Sheep, a goat, and a pig are preferred. The 1st class of one rainbow 2" no The gene which codes human womb L-PA include the arrangement which codes a signal receives a mouse WAP promotor's transfer control, and it explains construction of plasmid DNA by which the polyadenylation site of SV40 is connected with the 3 * end. This DNA is made from two middle plasmids, and one of these is bearing a mouse WAP promotor. Another side is bearing a signal sequence, a structured array, and SV40 polyadenylation site of t-PA. The WAP promotor containing plasmid pWAP-CAT (from Mr. Rota- Henninghausen (Lother Henninghausen) of NIH to Drawing 2 and a help), Henninghausen, Syros Pell (1982), Eur, J and Biochemo, 125 volumes, 131 pages, Campbell et al., Nucleic Ac1ds Re5earch. It originates in 12 volumes and the plasmid created [ 8685-page ] by the method of the statement. CAT which is a gene in which pWAP-CAT does not have a relation for the present invention This also containing a gene (chloramphenicol Acetyl transformer Ferrase) does not constitute a part of NA arrangement by the last target in which a microinjection is carried out. If reference is furthermore made about Drawing 2, he will be Klenow (Klen.) at least about one copy of EcoR of pWAP-CAT. It changed at least into 11 copies of Hind using W) and a Hindnl linker. Plasmid pL-PA containing Mu-PA VPI-LP gas (K) and (Drawing 1), It originates in pt-PAVPl-LP gas including a t-PA gene (the arrangement which codes the signal of L-PA is included), and SV40 polyadenylation site, A Kpn linker is added at least to one copy of unique Nco in the 5-degree end of a t-PA gene using NcoT and nuclease, and Klenow, and at least one copy of Kpn is created. Drawing 3 is explained. pt-PA including a t-PA gene and SV40 arrangement The Kpn I-BamHI fragment of VPI-LP gas (K) is separated, It connects with BamHI-Kpnl processing pWAP (H3), and is pWAP-L PA. This which builds (S) is E and coli further. It was rearranged into the TET-susceptibility guidance stock of MC1061. This recombination stock has plasmid DNA in which a Hindlll-BamHI fragment contains the arrangement to which a L-PA signal is coded under a WAP promotor's transfer 2II'4B, and the t-PA gene which contains 1<5v40 polyadenylation site in it. This recombinant was deposited with American Type Culture Collection (ATCC) on March 13, 1986, and was able to give ATCC deposition number No and 67032. When [ that ] an applicant assumes the duty of re-deposition when a culture becomes extinct before the expiration of a period of the published patent, and it has responsibility about giving notice of issue of this patent to ATCC, generally deposition is exhibited and the help of it becomes possible. Until then, the United States patent agency director general is available in this deposition cell by regulation of United States patent method 37CFRIJ1.14 and 35USC cloudy weather 112. For 30 years after the patent term of validity when this specified culture was accepted, and the day of deposition, or after patent issue, an applicant agrees to maintain over the latest period, while of one for five years, after having the part V& claim of the last of deposition. Production of the milk in which L-PA is secreted cuts down the Hindll-BamHI fragment from the deposited stock, and is made in accordance with the conventional method described above by transplanting to a mammalian 1 thin blastula using a microinjection or other methods preferably. The embryoes which may carry out four fragments cut by the whole plasmid or the restriction enzyme to an embryo although it is not necessarily desirable as an exception method are after that and in. It is made to grow up by vivo. Thus, the animal borne by the embryo carried out a works has it screened whether DNA introduced into the genome exists, and it is screened about the female of the transgenic lactation period whether t-PA is revealed in milk. L-PA is assayed with the conventional technique to the protein in the milk of the female animal of the lactation period which ripened. Production of Bmicro =-side r pWAP-Hb s to which pWAP-CAT which is a middle vector, and pHBs5VA have a polyadenylation site of SV40 after the gene of the hepatitis B surface antigen under a WAP promotor's transfer control if Drawing 5 is explained It was used for building (S). It mentioned above about plasmid pWAP-CAT. Plasmid pHbssVA was built to Drawing 4 as the statement. They are coR1, Sac+, and Bg about p CL Hs A including SV40 Pori Ade Nils-ized arrangement! ■ decomposed restrictively. Although pBSBam contained the gene of the hepatitis B surface antigen, EcoRl, BamHI, and Pvul cut it. Two mixtures were connected and pHbssVA was obtained. Here, SV40 arrangement is located in 3" end of a Hbs gene on a BamHI-Bgl[I fragment. Subsequently, this fragment is connected to PWAP-CAT processed by BamHI and the alkaline phosphatase of the microorganism, and they are Drawing 5 of To, E, and coli. It transformed into 061 shares of MCl(s), and isolated plasmid pWAP-Hbs (S). WAP-Hb s B a m HI -E c o R1 fragment of (S) was cut, and as mentioned above, it was used for producing a hepatitis B surface antigen. Although it is not desirable as an exception method, the fragment by the whole plasmid or other restriction enzymes may also be introduced to an embryo. An embryo is in after that. It raises by vivo. It screens whether DNA introduced into the genome per [ which was borne by such a Work(ed) embryo ] animal exists, and the hepatitis B surface antigen is revealed in the milk about the female animal of the transgenic lactation period, or it screens. pWAP-Hbs (S) was deposited with American Type Culture Collection (ATCC) on March 13, 1986, and was able to give ATCC deposition number No and 67033. When [ that ] an applicant assumes the duty of re-deposition when a culture becomes extinct before the expiration of a period of the published patent, and it has responsibility about giving notice of issue of this patent to ATCC, generally deposition is exhibited and becomes available. Until then, the United States patent agency director general is available in this deposition cell by regulation of United States patent method 37CFRIJ1.14 and 35USCJ112. For 30 years after the patent term of validity when this specified culture was accepted, and the day of deposition, or after patent issue, an applicant agrees to maintain over the latest period, while of one for five years, after having the sale-in-lots claim of the last of deposition. pWAP-Hbs Both (S) and pWAP-L-PA (S) can be used as a cassette vector which can cut the gene of a hepatitis B surface antigen, or the gene of t-PA, and can replace any genes to wish using the conventional method. By request, it is pWAP-t-PA. It is also possible to coincide a Read frame to that lower stream, and to insert in it the gene which left the arrangement which codes the signal in (S) into this vector, and is lacking such arrangement. With another method, they are pWAP-t-PA (S) or pWAP-Hbs. The signal sequence from (S) can be removed together with a structural gene, and the gene which codes the signal of a gene used for substitution can also be adopted. It is also possible to start only a WAP promotor and to insert into other desirable expression vectors. Refining and use The protein produced according to the present invention is refined from the milk in which they were secreted, and is used for the known object. the point that the hepatitis B surface antigen was transferred to these people -- although a hepatitis B virus vaccine is produced, it is useful as indicated to U.S. Serial Number 570.940 of lugs (Hstung). The contents of this U.S. patent application are also included in this specification by reference. The dissolution of a fibrin solidification lump of L-PA is useful for the medical treatment of required thrombosis as indicated to U.S. Serial Number 782.686 of way (Wei) and others transferred to these people. This patent application has also indicated the general refining process useful in the protein secreted in milk again. The contents of this U.S. patent application are also included in this specification by reference. Six sexes in the inside of milk Irrespective of existence of the protease of the versatility [ table / that is shown below / 1st ] in milk, When rearranged type L-PA is added to the milk of a raw goat and is settled by 20degreeC or 37 degreeC for 24 hours, When it measures by a stable and standard fibrin plate test (concrete data is not shown in the 1st table), or when it measures by the amide substrate decomposition activity indicated in ways, The thing which show that an active fall is not seen and which it rearranges similarly, and a hepatitis B surface antigen is stable for at least 24 hours, and is in raw goat milk was found out (concrete data is not shown). 1st Table Amide of TPA "T Degree Sexual forming time Temperature Only Unit/Ml -- <20.<20 Milk of a goat 10 -- TPA(s) O437 and 36B Milk of a goat + TPA Milk of 24-hour 20-degreeC 419,434 goat + TPA Mode besides 24 hour 37 degreeC 467.507, 0 indicated in the claim, for example, 0 which may use other milk-serum-protein promotors instead of a mouse WAP promotor and it may carry out and the thing of what kind of the mammals may be satisfactory for the promotor of whose, -- for example The WAP promotor of the rat who can use a milk-serum-protein promotor like the promotor of beta-lactoglobulin, and may use the WAP promotor of a rat instead of a mouse is indicated in above-mentioned Campbell and others. Although it is not more desirable than a milk-serum-protein promotor, what kind of important protein to wish to have may the protein in which a casein promotor is also produced using the 0 present invention which can be used similarly be on medical treatment or industry?

[Brief Description of the Drawings]

pL-PA which is a middle vector [ in / in Drawing 1 / the present invention ] It is a VPI-LP gas(K) (7) W Construction r[figure, and is Drawing 2, It is a manufacturing process figure of PWAP (H) which is a middle vector in the present invention, and is Drawing 3, PWAP-L-PA which is a middle vector in the present invention pWAP-Hb s which it is a manufacturing process figure of (S), and Drawing 4 is a manufacturing process figure of pHbssVA which is a middle vector in the present invention, and is a middle vector [ in / in Drawing 5 / the present invention ] It is a manufacturing process figure of (S). Fair copy (with no change by contents) IG of 1st page 1 ind IG 2 IG 3 Written amendment □ Showa 62 year 7 month / :3 day The Showa 62 patent application No. [ two ] 87872, the title of an invention Relation between gene transplantation animal 3 which secretes the protein to wish to have into milk, and the person incident which carries out amendment Applicant address Name integrated FTgenetics parrot Holted 4, a representative address 2-2-1, Otemachi, Chiyoda-ku, Tokyo New Otemachi Building Date of Room 206 5 and an order for amendment June 30, Showa 62 Transportation -- Japanese -- object of 6 and amendment