Transgenic animals secreting desired proteins into milk

Description: TECHNICAL FIELD [Detailed description of the invention]

(Industrial application field) The present invention relates to transgenic animals. (Conventional technology) It is possible to insert a foreign gene into a vertebrate embryo and into the genome of the animal of interest. The insertion of foreign genes is performed by a mechanical method (microinjection method (microinjection method)) and a method using a retrovirus vector (for example, Huszar et al., 1985 PNAS (PNAS) U.S.A. SA (USA), Vol. 82, p. 8587). The animals obtained by this operation are called "transgenic (genetically modified) animals". This foreign gene is transmitted sexually to later generations and is often expressed in the animal. In some examples, proteins encoded by foreign genes are expressed in specific tissues. For example, the metallothionein promoter was used to direct the expression of rat growth hormone genes in the liver of transgenic mice (Palmiter et al., 1982, Nature, 300, p. 611). Another example is the elastase promoter, which was used to direct the expression of foreign genes in the pancreas (Ornitz et al., 1985, Nature, 313, 600). It has also become possible to control gene expression developmentally in transgenic animals. That is, the foreign gene can be transcribed only in a specific tissue at a specific time. For example, Magnum et al., 1985, Nature, Vol. 315, p. 338) show that genes are regulated developmentally under the direction of the globin promoter, Krumlauf et al., ( 1985, Mol.Cell.Biol.5, p. 1639) shows similar results using the alpha-fetoprotein minigene. (Construction of the invention) In general, the invention is characterized by a DNA sequence containing a gene encoding a protein, which is naturally subject to transcriptional regulation of a mammalian milk protein promoter that does not regulate its transcription. The DNA sequence also includes the DNA that enables the secretion of the protein, that is, the sequence encoding the secretory signal that exists between the gene and the promoter. The promoter can be a whey protein promoter or a casein protein promoter, but a whey protein promoter is preferred, as will be described in detail later. (In the present specification, "gene" means both a genomic DNA sequence and a cDNA sequence). The present invention allows the production of any desired protein in a culture system that is easy to maintain, stable and mobile. That is, the culture system of the present invention is a living livestock and can not only produce the desired protein but also transmit the same ability to female offspring. Secreting the protein into the milk of the host animal facilitates purification and eliminates the need for operations to remove blood components and media additives that may be toxic or carcinogenic. More importantly, the yield of protein is high and its production is cost effective and effective. Other features and advantages of the invention are evident from the following description of preferred embodiments of the invention and the claims. Description of preferred embodiments FIG. 1 is a manufacturing process diagram of pt-PA VP1-LP (K), which is an intermediate vector in the present invention. FIG. 2 shows pWAP (H), which is an intermediate vector in the present invention.₃) Is a manufacturing process diagram. FIG. 3 is a manufacturing process diagram of pWAP-t-PA (S), which is an intermediate vector in the present invention. FIG. 4 is a manufacturing process diagram of pHbsSVA, which is an intermediate vector in the present invention. FIG. 5 is a manufacturing process diagram of pWAP-Hbs (S), which is an intermediate vector in the present invention. Components of DNA sequence promoter The milk protein promoter can be from any mammalian species and can be any promoter of a protein normally secreted in mammalian milk in nature. In general, milk proteins are classified as casein, which are defined here as micelle-like milk proteins present in milk and are removed from skim milk by coagulation with rennet. Whey protein is defined herein as non-caseinous milk protein. It is the main component of whey protein and whey protein, and in rodents, it contains a protein called whey acidic protein. Whey acid protein (WAP) was named after its acidic isoelectric point (Piletz, 1981 (J. Biol. Chem.), Vol. 256, p. 11509). Another example of a whey protein described in the literature is α-lactalbumin (for mouse WAP, Henninghausen and Sippel, 1982, Eur.J. Biochem., Vol. 125, 131). See page). Milk proteins are described in detail in Walstra and Jenness, Dairy Chemistry and Physics, (John Wiley &amp; Sons, 1984). Casein is usually produced in female animals not only after childbirth but also during pregnancy, whereas WAP appears only during the lactation period after childbirth. Therefore, in the present invention, the whey protein promoter is generally the casein. Preferable over promoters. This difference is potentially important for two reasons. First, the prenatal production of the desired protein under the transcriptional control of the casein promoter is wasteful in that it cannot be isolated until it is secreted into milk after delivery. is there. Second, when toxic in the presence of large amounts of the desired protein (eg, human tissue plasminogen activator (t-PA)), the production of the protein in the tissue prior to lactation is a host animal. It is also harmful to your health. Another advantage of whey promoters, such as the WAP promoter, is that they can be strong promoters, as evidenced by the large amount of whey protein present in milk, which is a single promoter, including the WAP promoter. For milk protein genes that can be sourced, Henning Hansen and Shippel, supra, and Campbell et al., 1984, Nucleic Acids. It can be obtained in the same way that the WAP gene was isolated, as described by Reaearch, Vol. 12, p. 8685. In the method of the present invention, mRNA is generally isolated from a milk-secreting mammary gland, a cDNA library is prepared from this mRNA, the cDNA of a specific milk protein to be sought is screened from the library, and the cDNA is cloned into a vector. It consists of the operation of using a suitable cDNA as a probe for isolating a genomic clone from a library of genomes. The sequence upstream from the transcription start site in the genome clone is considered to constitute the desired "promoter". That is, it is a genomic sequence that exists prior to the target gene and is considered to be involved in its regulation. The promoters of the present invention are isolated by restriction endonuclease digestion and subcloning processes. The promoter does not have to be of a particular length and does not have to directly exhibit any regulatory properties. The mouse WAP promoter was isolated as a 2.6 kb EcoR1-Kpn I fragment just beside the 5'end of the WAP signal sequence. Desired protein According to the present invention, any desired protein can be produced. Preferred proteins are useful in the treatment, prevention and / or diagnosis of human diseases, such as t-PA and hepatitis B surface antigens. In particular, the present invention is useful for proteins that must be produced in large quantities and economically, such as industrial oxygen and animal proteins. Signal sequence In order to secrete the desired protein into the milk of the host animal, the DNA sequence containing the gene for the desired protein must contain the DNA that secretes the protein from the mammary gland tissue into the milk when translated. is there. Without such a sequence, the desired protein would remain in the mammary gland tissue, making protein purification difficult and necessitating the need to kill the host animal. This DNA encodes a hydrophobic secretory signal sequence that is cleaved during the secretory process. If the desired protein is normally secreted (eg, t-PA), the signal sequence may be one that is naturally associated with the desired protein. Alternatively, the signal sequence of the milk protein containing the promoter can be used as the sequence encoding the signal. That is, the milk protein gene is digested, the promoter is isolated, and a DNA fragment containing both the promoter and the sequence encoding the signal immediately downstream from the promoter is selected. Another alternative is to use a sequence that encodes a signal derived from another secreted protein that is neither the milk protein normally expressed by the promoter nor this desired protein. Stop site It is desirable that the stop site be present in the desired gene or downstream of the 3'end. This site may be provided as a sequence present within the gene, but otherwise it needs to be added. If that sequence is added, the preferred sequence is provided by the polyadenylation sequence of the SV40 virus, as described in detail below. Genetic engineering In general, all DNA manipulations used in the gene construction of the present invention are customary, as described, for example, in the Molecular Cloning Manual (Cold Spring Harbor Laboratory, 1982) by Maniatis et al. It is carried out using technology. Introduction of DNA into the embryo Once gene construction is achieved in a vector such as a plasmid, the DNA fragment consisting of the promoter-signal sequence-desired protein-stop sequence is cleaved and introduced into the desired mammalian embryo. This introduction can be done, for example, by a retrovirus, or by Kraemer et al. (1985), edited by Costantini and Jaenisch, Genetic Manipulation of the Early Mammalian. Embryo), Cold Spring Harbor Laboratory (Bovine Embryo Microinjection); Hammer et al. (1985), Nature, Vol. 315, p. 680 (Rabbit, Sheep, and Pig Embryo Microinjection); and Gordon And Ruddle, (1984), Methods in This can be done by the standard microinjection method described in Enzymology, Vol. 101, p. 411 (Mouse Embryo Microinjection). Microinjection is designed to maximize the frequency with which the injected DNA is taken up by all cells of the animal, including mammary tissue, and the frequency with which the DNA is also taken up by germ cells and the offspring of that animal are also transgenic. It is preferably done for embryos at the cellular stage. For microinjection, simply explain, a fertilized egg is isolated, the pronucleus is captured in the field of view, the egg is held with a blunt pipette with a diameter of about 50 μm, and a pipette with a sharp tip with a diameter of about 1.5 μm is used. This is a technique consisting of injecting a buffer solution containing DNA into the pronucleus. Following microinjection, transgenic females are sexually matured, mated, and after birth, milk is collected. Host mammals are preferably those bred to produce large amounts of milk, such as cows, sheep, goats and pigs. t-PA production The construction of a plasmid DNA in which a gene encoding human uterine t-PA containing a signal-encoding sequence is subject to transcriptional regulation of the mouse WAP promoter and a polyadenylation site of SV40 is linked to its 3'end is described. .. This DNA is made up of two intermediate plasmids, one carrying the mouse WAP promoter and the other carrying the signal and structural sequences of t-PA as well as the SV40 polyadenylation site. WAP promoters containing the plasmid pWAP-CAT (Fig. 2, obtained from Lother Henninghausen of NIH) are Henningenhausen and Shippel (1982), Eur.J.Biochem., Vol. 125, 131. Derived from the plasmid prepared by the method described in Page and Campbell et al., Nucleic Acids Research, Vol. 12, p. 8685. pWAP-CAT also includes a CAT (chloramphenicol acetyltransferase) gene, which is a gene unrelated to the present invention. It does not form part of the final microinjected DNA sequence. Furthermore, referring to Fig. 2, the EcoR1 site of pWAP-CAT was converted to the Hind III site using Klenow and a Hind III linker. The plasmid pt-PA VP1-LP (K) containing t-PA (Fig. 1) contains the t-PA gene (containing the sequence encoding the t-PA signal) and the SV40 polyadenylation site pt-PAVP1- It is derived from LP, and a Kpn I site was created by adding a Kpn linker to the unique Nco I site at the 5'end of the t-PA gene using Nco I endonuclease and Clenault. FIG. 3 will be described. A Kpn I-BamH I fragment of pt-PA VP1-LP (K) containing the t-PA gene and SV40 sequence was isolated and ligated to BamH I-Kpn I-treated pWAP (H3) to ligate pWAP-tPA (S). to make. It was further recombined into a TET-susceptibility inducer of E. coli MC1061. This recombinant strain is Hind III-BamH The I fragment has a plasmid DNA containing the sequence encoding the t-PA signal followed by the t-PA gene containing the SV40 polyadenylation site under the transcriptional control of the WAP promoter. This recombinant was deposited with the American Type Culture Collection (ATCC) on March 13, 1986, and was given ATCC Deposit No. 67032. The applicant is obliged to re-deposit if the culture dies before the expiration of the issued patent and is responsible for notifying the ATCC of the issuance of this patent. At that point, the deposit will be open to the public and available. In the meantime, this deposited cell is available to the Commissioner of the United States Patent Office under 35 USC § 1.14 and 35 USC § 112. The applicant is the latest of the validity period of the patent in which this explicit culture was granted, 30 years from the date of deposit, or 5 years after the issuance of the patent and the last request for sale of the deposit. Agree to maintain over. The production of milk secreted by t-PA is Hind III-BamH from the deposited strain. It is done by cutting out an I fragment and according to the conventional method described above, preferably transplanted into a mammalian single cell embryo using microinjection or other methods. Alternatively, although not preferred, the entire plasmid or fragments cleaved by limiting oxygen may be introduced into the embryo. Embryos are then grown in vivo. Animals born from embryos manipulated in this way are screened for the presence of DNA introduced into the genome and whether t-PA is expressed in milk during the transgenic lactation period. Is screened for females. T-PA assays are performed on proteins in the milk of mature lactating females by conventional methods. Production of hepatitis B surface antigen Explaining Fig. 5, the intermediate vectors pWAP-CAT and pHBsSVA are converted to pWAP-Hbs (S) having a polyadenylation site of SV40 after the gene for hepatitis B surface antigen under the transcriptional control of the WAP promoter. Used to build. The plasmid pWAP-CAT has been described above. The plasmid pHbsSVA was constructed as shown in Figure 4. PCLH containing SV40 polyadenylation sequence₃A was selectively degraded by Eco R1, Sac I, and Bgl II. Although pBSBam contains the gene for hepatitis B surface antigen, it was cleaved by EcoR1, BamHI, and Pvu I. The two mixtures were ligated to give pHbs SVA. Here, the SV40 sequence is located at the 3'end of the Hbs gene on the BamH I-Bgl II fragment. This fragment was then ligated into pWAP-CAT treated with BamHI and microbial alkaline phosphatase (Fig. 5) and transformed into the E. coli MC1061 strain to give the plasmid pWAP-Hbs (S). Released. The BamH I-EcoR1 fragment of WAP-Hbs (S) was cleaved and used to produce hepatitis B surface antigen, as described above. Alternatively, although not preferred, whole plasmids or other restricted oxygen fragments can also be introduced into the embryo. The embryo is then in Grow in vivo. Animals born from such engineered embryos were screened for the presence of introduced DNA in their genomes, and transgenic lactating female animals contained hepatitis B surface antigens in their milk. Screen for expression. pWAP-Hbs (S) was deposited with the American Type Culture Collection (ATCC) on March 13, 1986 and was given ATCC Deposit No. 67033. The applicant is obliged to re-deposit if the culture dies before the expiration of the issued patent and is responsible for notifying the ATCC of the issuance of this patent. At that point, the deposit will be open to the public and available. In the meantime, this deposited cell is available to the Commissioner of the United States Patent Office under 35 USC § 1.14 and 35 USC § 112. The applicant is the latest of the validity period of the patent in which this explicit culture was granted, 30 years from the date of deposit, or 5 years after the issuance of the patent and the last request for sale of the deposit. I agree to maintain it for a period of time. Both pWAP-Hbs (S) and pWAP-t-PA (S) cleave the hepatitis B surface antigen gene or the t-PA gene and replace any desired gene using conventional methods. It can be used as a cassette vector that can be used. If desired, the sequence encoding the signal in pWAP-t-PA (S) is left in this vector, and the gene lacking such a sequence is inserted downstream thereof with a consistent reading frame. It is also possible. Alternatively, the signal sequence from pWAP-t-PA (S) or pWAP-Hbs (S) may be removed along with the structural gene to employ the gene encoding the signal of the gene used for the substitution. it can. Furthermore, it is also possible to cut out only the WAP promoter and insert it into another preferred expression vector. Purification and utilization The proteins produced according to the present invention are purified from the milk in which they are secreted and used for known purposes. Hepatitis B surface antigens are useful in producing hepatitis B vaccines, as described in US Patent Application No. 570,940 of Hsiung et al., Assigned to Applicant. The contents of this US patent application are also included herein by reference. t-PA is useful in the treatment of thrombosis requiring lysis of fibrin clots, as described in US Patent Application No. 782,686 of Wei et al., Assigned to this application. The patent application also describes common purification methods useful for proteins secreted in milk. The contents of this US patent application are also included herein by reference. Stability in milk Table 1 below shows that recombinant t-PA was added to raw goat milk and allowed to stand at 20 ° C or 37 ° C for 24 hours, despite the presence of various proteases in the milk. Decreased activity when stable and measured by a standard fibrin plate test (no specific data shown in Table 1) or by the amide substrate degradation activity described by Way et al. Indicates that no is seen. Similarly, recombinant hepatitis B surface antigens were found to be stable in raw goat milk for at least 24 hours (specific data not shown).<img file="JP2874751B2_D0001.tif" />Other aspects Other aspects are described in the claims. For example, other whey protein promoters may be used in place of the mouse WAP promoter, and the promoter may be of any mammal. For example, a whey protein promoter such as the β-lactoglobulin promoter may be used, or the rat WAP promoter may be used instead of the mouse. The rat WAP promoter is described by Campbell et al. Although less preferred than the whey protein promoter, the casein promoter can be used as well. The protein produced using the present invention may be any desired protein of therapeutic or industrial significance.

[Simple explanation of drawings]

FIG. 1 is a manufacturing process diagram of pt-PA VP1-LP (K), which is an intermediate vector in the present invention. FIG. 2 shows pWAP (H), which is an intermediate vector in the present invention.₃) Is a manufacturing process diagram. FIG. 3 is a manufacturing process diagram of pWAP-t-PA (S), which is an intermediate vector in the present invention. FIG. 4 is a manufacturing process diagram of pHbsSVA, which is an intermediate vector in the present invention. FIG. 5 is a manufacturing process diagram of pWAP-Hbs (S), which is an intermediate vector in the present invention.