Method for promoting secretion of protein

Abstract

The present invention provides: a method for promoting secretion of a target protein that involves causing a construct that encodes a fusion protein of ovomucoid or a portion thereof and the target protein to be expressed in avian cells and causing the fusion protein to be secreted from the avian cells; a construct that encodes a fusion protein of ovomucoid or a portion thereof and the target protein; a bird into which the construct has been introduced by knock-in; eggs from the bird; and a method for producing the target protein that involves acquiring the target protein from the eggs.

Description

Method for promoting protein secretion

[0001] RELATED APPLICATIONS This application claims the benefit of priority from Application No. 2024-028936, filed with the Japan Patent Office on February 28, 2024. The priority application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for promoting protein secretion.

[0003] In recent years, so-called "animal factories," which use genetic engineering techniques to produce useful substances in animals, have been attracting attention. One such technology, the egg bioreactor, is a technology for mass-producing foreign proteins in egg white by genetically modifying chickens (Patent Document 1). In female genetically modified chickens, a foreign protein is expressed in the cells of the oviduct tissue, which is secreted extracellularly in the same way as endogenous egg white proteins and accumulates together with egg white proteins in the lumen of the oviduct tissue, ultimately obtaining the recombinant protein in the egg white of chicken eggs. The greater the amount of foreign protein accumulated in the egg white, the more useful the production system. Therefore, it is important not only to simply express the foreign protein in the cells of the oviduct tissue, but also to efficiently secrete it from the oviduct cells.

[0004] Generally, when foreign proteins are expressed in animal cells, the foreign proteins are secreted as secretory proteins with a signal peptide. For proteins secreted via the endoplasmic reticulum-Golgi pathway, known as "conventional protein secretion," efficient secretion is often possible simply by expressing them in cells. Furthermore, the secretion yield of foreign proteins can sometimes be further improved by replacing the signal peptide with a different signal peptide rather than the original signal peptide of the foreign protein. A signal peptide commonly used in mammals is the mouse immunoglobulin kappa chain signal sequence (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 58)). However, depending on the type of cell in which the protein is expressed and the combination of the signal peptide and the foreign protein, the secretion yield may not be improved. No consistent rule has been found for determining which signal peptide will increase secretion yield. Furthermore, it has been reported that the secretion yield differs between mouse and human cells even when the same signal peptide is used, suggesting the existence of species-specific secretion control mechanisms. On the other hand, secretory proteins that do not have a signal peptide and do not pass through the endoplasmic reticulum-Golgi pathway are called "unconventional protein secretion," and proteins that exhibit this secretory mode (e.g., FGF2 and IL-1β) are difficult to secrete efficiently simply by expressing them as foreign proteins. Furthermore, proteins that are not naturally secreted, such as nuclear proteins and cytoplasmic proteins, are rarely or completely secreted outside the cell simply by expressing them as foreign proteins.

[0005] To achieve highly efficient secretion and expression of foreign proteins in microorganisms, a technique is used in which a signal peptide or a portion of a readily secreted microbial protein is placed at the N-terminus of the foreign protein to allow secretion (Non-Patent Document 1). It has also been reported that signal peptide replacement in mammalian cells can improve the secretion efficiency of foreign proteins (Non-Patent Document 2). However, methods for highly efficient secretion and expression of foreign proteins in avian cells have not been thoroughly studied. Meanwhile, advances in egg bioreactor technology have made the development of efficient foreign protein secretion from avian cells important. In egg bioreactors, signal peptides such as ovotransferrin and lysozyme have been used as foreign protein signal peptides. Furthermore, research on mammalian cells has shown promising promise for improving secretion efficiency by experimenting with various signal peptides already in use. However, more efficient secretion methods using techniques other than signal peptides have yet to be elucidated, and new techniques that can significantly improve secretion efficiency are needed. Efficient secretion-promoting techniques for poorly secreted proteins are considered particularly important.

[0006] International Application Publication WO2017 / 111144A1

[0007] Appl Environ Microbiol. 2003 Jan;69(1):358-366. doi: 10.1128 / AEM.69.1.358-366.2003.Acta Biochim Biophys Sin (Shanghai). 2016 Apr; 48(4): 391-394. doi: 10.1093 / abbs / gmw007Nature. 2006 Jun 8;441(7094):766-769. doi: 10.1038 / nature04831.

[0008] A new expression control system for foreign proteins is needed to efficiently secrete not only proteins that are secreted via the endoplasmic reticulum-Golgi pathway, but also proteins that are secreted without the endoplasmic reticulum-Golgi pathway and proteins that are not originally secreted, particularly in poultry cells such as chickens, with the aim of highly efficient production in egg bioreactors.

[0009] The present inventors have conducted extensive research to solve the above problems and have found that by placing ovomucoid protein, or a part of it, which is secreted by oviduct cells and accumulated in egg white, at the N-terminus of a foreign protein, the foreign protein can be secreted with extremely high efficiency, thereby completing the present invention.

[0010] That is, the present invention provides the following: [Item 1] A method for promoting secretion of a target protein, comprising expressing in poultry cells a construct encoding a fusion protein of ovomucoid or a portion thereof with a target protein and secreting the fusion protein from the poultry cells, wherein the construct comprises a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a portion thereof, and a polynucleotide encoding the target protein, and the polynucleotide encoding the signal peptide, the polynucleotide encoding ovomucoid or a portion thereof, and the polynucleotide encoding the target protein are arranged in the construct from the 5' to the 3' end. [Item 2] The method of Item 1, wherein the portion of ovomucoid comprises the first domain of ovomucoid or a portion thereof. [Item 3] The method of Item 1 or 2, wherein the portion of ovomucoid comprises an amino acid sequence selected from the group consisting of amino acids 1 to 69, 1 to 31, 1 to 22, and 1 to 15, counting from the N-terminus of mature ovomucoid. [Item 4] The method of any one of Items 1 to 3, wherein the portion of ovomucoid comprises AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMN (SEQ ID NO: 64), AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (SEQ ID NO: 65), AEVDCSRFPNATDKEGKDVLVC (SEQ ID NO: 66), or AEVDCSRFPNATDKE (SEQ ID NO: 67). [Item 5] The method of any one of Items 1 to 4, wherein the target protein is a poorly secreted protein. [Item 6] The method of any one of Items 1 to 5, wherein a polynucleotide encoding a linker is positioned between the polynucleotide encoding ovomucoid or a portion thereof and the polynucleotide encoding the target protein.[Item 7] A construct comprising a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a portion thereof, and a polynucleotide encoding a protein of interest, wherein the polynucleotide encoding the signal peptide, the polynucleotide encoding ovomucoid or a portion thereof, and the polynucleotide encoding the protein of interest are arranged in the 5' to 3' direction. [Item 8] The construct of Item 7, wherein the portion of ovomucoid comprises the first domain of ovomucoid or a portion thereof. [Item 9] The construct of Item 7 or 8, wherein the portion of ovomucoid comprises an amino acid sequence selected from the group consisting of amino acids 1 to 69, 1 to 31, 1 to 22, and 1 to 15, counting from the N-terminus of mature ovomucoid. [Item 10] The construct according to any one of Items 7 to 9, wherein the portion of ovomucoid comprises AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMN (SEQ ID NO: 64), AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (SEQ ID NO: 65), AEVDCSRFPNATDKEGKDVLVC (SEQ ID NO: 66), or AEVDCSRFPNATDKE (SEQ ID NO: 67). [Item 11] The construct according to any one of Items 7 to 10, wherein the protein of interest is a poorly secreted protein. [Item 12] The construct according to any one of Items 7 to 11, wherein a polynucleotide encoding a linker is positioned between the polynucleotide encoding ovomucoid or a portion thereof and the polynucleotide encoding the protein of interest. [Item 13] A poultry cell comprising the construct according to any one of Items 7 to 12. [Item 14] A vector comprising the construct according to any one of Items 7 to 12. [Item 15] A poultry cell comprising the vector according to Item 14. [Item 16] A method for producing a target protein, comprising culturing the cell according to Item 15. [Item 17] A poultry into which the construct according to any one of Items 7 to 12 has been knocked in.[Item 18] A method for producing poultry according to Item 17, comprising knocking in the construct according to any one of Items 7 to 12 by arranging it in-frame into an avian oviduct-specific gene locus. [Item 19] An egg laid by the poultry according to Item 17. [Item 20] A method for producing a target protein, comprising collecting the target protein from the egg according to Item 19.

[0011] According to the present invention, foreign proteins that are secreted in a non-traditional manner or proteins that are not originally secreted can be secreted highly efficiently from poultry cells. Specifically, by locating ovomucoid protein, or a portion thereof, which is secreted in oviduct tissue and accumulated in egg white, at the N-terminus of the target protein, the target protein can be secreted with extremely high efficiency. Furthermore, by enabling the expression of foreign proteins in poultry oviduct tissue cells in this manner, highly efficient extracellular expression of the target protein and its large-scale secretion into egg white in individual chickens becomes possible.

[0012] FIG. 1 is a schematic diagram of the FGF2 expression construct used in the experiment. OVTsp+4aa represents a polynucleotide encoding a peptide with four amino acid residues added to the C-terminus of the ovotransferrin signal peptide (amino acid sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 59)). 4aa represents APPK (SEQ ID NO: 60). OM1st represents a polynucleotide encoding an amino acid sequence corresponding to the first domain of ovomucoid. FIG. 2 is a Western blot showing the effect of the type of fusion partner on FGF2 secretion. A schematic diagram of the construct used in the experiment is shown below the Western blot (see also FIG. 1). The left panel of FIG. 3 is a Western blot showing the effect of ovomucoid on FGF2 secretion. The right panel of FIG. 3 shows a schematic diagram of the construct used in the experiment. The left panel of FIG. 4 is a Western blot showing the effect of a partial peptide of ovomucoid on FGF2 secretion. The right panel of Figure 4 shows a schematic diagram of the construct used in the experiment. Figure 5 is a schematic diagram of a construct in which a GS linker (GGGGS (SEQ ID NO: 61)) was inserted between the first domain of the ovomucoid mature protein and FGF2. Figure 6 shows the results of Western blotting examining the expression of the construct shown in Figure 5 in the culture medium. Figure 7 is a graph showing the results of examining the specific activity of FGF2 expressed from the construct shown in Figure 5. Figure 8 is a schematic diagram of a donor construct prepared for chicken genetic recombination (gene knock-in). Figure 9a is a schematic diagram showing the donor construct positioned in exon 2 of the ovalbumin gene. Figure 9b shows the results of PCR confirming knock-in of the donor construct into the ovalbumin locus. Figure 10 is a schematic diagram of a PAC expression construct. Figure 11 is a Western blot showing the effect of the type of fusion partner on PAC secretion when expressed in DF-1 cells. A schematic representation of the constructs used in the experiments is shown below the Western blot (see also Figure 10). Figure 12 is a schematic representation of the CRE expression construct.The left panel of Figure 13 shows the results of Western blotting when CRE, OA-CRE, LZ-CRE, OM-CRE, and OM1D-CRE were expressed in DF-1 cells. The right panel of Figure 13 shows the expression levels of CRE, OA-CRE, LZ-CRE, OM-CRE, and OM1D-CRE, when the expression level of CRE was set to 1. The bottom panel of Figure 13 is a schematic diagram of the construct used in the experiment. The left panel of Figure 14 is a Western blot showing the effect of ovomucoid on PAC secretion when the signal peptide was a lysozyme signal peptide (amino acid sequence MRSLLILVLCFLPLAALG (SEQ ID NO: 62)) with four amino acid residues (APPK (SEQ ID NO: 60)) added to its C-terminus, and the fusion partner was PAC. The right panel of Figure 14 shows a schematic diagram of the construct used in the experiment. The left panel of Fig. 15 is a Western blot showing the effect of a partial sequence within the first domain of ovomucoid on PAC secretion. The right panel of Fig. 15 is a schematic diagram of the construct used in the experiment. Fig. 16 is a Western blot showing the effect on PAC secretion when ovomucoid is used as a fusion partner when expressed in poultry oviduct cells.

[0013] In one aspect, the present invention provides a method for promoting secretion of a target protein, comprising expressing in avian cells a construct containing a polynucleotide encoding a fusion protein of ovomucoid or a portion thereof with a target protein, and secreting the fusion protein from the poultry cells.

[0014] The present inventors have found that not all proteins secreted by oviduct cells and accumulated in egg white promote the secretion of a target protein. When proteins other than ovomucoid or parts thereof, such as ovalbumin or lysozyme, are used, the secretion-promoting effect of the target protein is not sufficient, or secretion is not observed at all. The present inventors have discovered for the first time the superiority of ovomucoid or parts thereof in promoting the secretion of target proteins, particularly poorly secreted proteins.

[0015] The construct comprises a polynucleotide encoding a fusion protein of ovomucoid or a portion thereof with a protein of interest. Specifically, the construct comprises a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a portion thereof, and a polynucleotide encoding the protein of interest. In the construct, the polynucleotide encoding the signal peptide, the polynucleotide encoding ovomucoid or a portion thereof, and the polynucleotide encoding the protein of interest are arranged from the 5' end to the 3' end.

[0016] As used herein, a construct refers to an artificially produced polynucleotide that consists of or contains polynucleotides that encode two or more proteins (polypeptides, peptides).

[0017] As used herein, a polynucleotide encoding a protein (polypeptide, peptide) includes a polynucleotide encoding an amino acid sequence consisting of the amino acid sequence of the protein (polypeptide, peptide), and a polynucleotide encoding an amino acid sequence including the amino acid sequence of the protein (polypeptide, peptide).

[0018] As used herein, the term "polynucleotide" encompasses DNA, RNA, or a mixture thereof. The polynucleotide may include a complementary sequence. The polynucleotide may include a degenerate sequence.

[0019] As used herein, the term "target protein" refers to a protein whose secretion from cells is desired to be promoted. The target protein may be a protein derived from any organism and may be any type of protein. The target protein encompasses not only poorly secreted proteins but also secreted proteins. The target protein is typically a poorly secreted protein. As used herein, the term "poorly secreted protein" refers to proteins other than easily secreted proteins that have a signal peptide and are secreted outside the cell via the endoplasmic reticulum-Golgi pathway, including proteins that are secreted without passing through the endoplasmic reticulum-Golgi pathway, as well as proteins that are not originally secreted, such as nuclear proteins and cytoplasmic proteins. Examples of poorly secreted proteins include, but are not limited to, fibroblast growth factor 2 (FGF2), CRE recombinase (CRE), puromycin N-acetyltransferase (PAC), DNA polymerase, RNA polymerase, reverse transcriptase, nuclease, transcriptional regulator, nucleic acid modifying enzyme, intracellular enzymes such as intracellular protein kinase / phosphatase, and intracellular signal transduction factors. The above-described method of the present invention may be used to promote the secretion of a poorly secreted protein that is not easily secreted extracellularly, or to cause a poorly secreted protein that is not secreted extracellularly to be secreted extracellularly. The above-described method of the present invention may be used to improve the secretion of a secretory protein. The target protein may be a naturally occurring protein or a non-naturally occurring protein. The target protein may be a wild-type protein or a mutant thereof. The target protein may be artificially designed or modified. The artificially designed or modified target protein may be a naturally occurring protein to which amino acid substitution, addition, insertion, or deletion has been performed. For example, a histidine tag may be attached to the N-terminus of the target protein to facilitate purification and isolation of the target protein. The target protein may be one type or two or more types. A polynucleotide encoding a target protein can be obtained using known methods. Codon usage may be modified to facilitate expression in avian cells.

[0020] Ovomucoid is a protein contained in egg white. Its amino acid sequence and the nucleotide sequence encoding it are publicly known. It is known that ovomucoid contains domains 1 to 3. Ovomucoid may be derived from any bird, for example, poultry. Examples of poultry include chickens, quail, turkeys, ducks, geese, cockroaches, bantams, pigeons, ostriches, pheasants, and guineafowl, and preferably include, but are not limited to, birds of the Phasianidae family, such as chickens and quails. Ovomucoid may be wild-type or a mutant thereof. For example, a histidine tag may be added to the N-terminus of ovomucoid to facilitate purification and isolation of the fusion protein. A polynucleotide encoding ovomucoid can be obtained using publicly known methods. Unless otherwise specified, "ovomucoid" herein refers to the mature ovomucoid protein.

[0021] In the present invention, a polynucleotide encoding a portion of ovomucoid may be used. Unless otherwise specified, the term "portion of ovomucoid" refers to a portion of the mature ovomucoid protein. The portion of ovomucoid may be derived from any sequence of ovomucoid. Preferably, the portion of ovomucoid comprises the first domain of ovomucoid or a portion thereof. As used herein, an amino acid sequence corresponding to the first domain of ovomucoid refers to an amino acid sequence comprising the first domain of ovomucoid, an amino acid sequence consisting of the first domain of ovomucoid, an amino acid sequence comprising a portion of the first domain of ovomucoid, or an amino acid sequence consisting of a portion of the first domain of ovomucoid. The first domains of ovomucoid from various birds are known. In the case of chicken ovomucoid, examples of polypeptides comprising the first domain or a portion thereof include, but are not limited to, polypeptides from any of the amino acids 1 to 10 to any of the amino acids 60 to 70 counting from the N-terminus of mature ovomucoid. For example, in the case of chicken ovomucoid, examples of a polypeptide comprising the first domain or a portion thereof include the 1st to 69th amino acids, the 1st to 31st amino acids, the 1st to 22nd amino acids, or the 1st to 15th amino acids counting from the N-terminus of mature ovomucoid. In one embodiment, a polypeptide comprising the first domain or a portion thereof includes AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLLCAYSIEFGTNISKEHDGECKETVPMN (SEQ ID NO: 64), AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (SEQ ID NO: 65), AEVDCSRFPNATDKEGKDVLVC (SEQ ID NO: 66), or AEVDCSRFPNATDKE (SEQ ID NO: 67). One or more of these may be used as a portion of ovomucoid, as long as they contain these sequences. The amino acid sequence of a portion of ovomucoid may be wild-type or a mutant thereof. For example, a histidine tag may be added to the N-terminus of a portion of ovomucoid to facilitate purification and isolation of the fusion protein. A polynucleotide encoding a portion of ovomucoid can be obtained using known methods.

[0022] Various signal peptides are known. A signal peptide is positioned at the N-terminus of a target protein to secrete it. In the present invention, any type of signal peptide may be used, including, but not limited to, the signal peptide of a mammalian immunoglobulin kappa chain, the signal peptide of tissue-type plasminogen activator, the signal peptide of growth hormone, the signal peptide of prolactin, the signal peptide of avian ovotransferrin, and the signal peptide of lysozyme. The signal peptide may be a wild-type or a mutant thereof. The signal peptide may be naturally occurring or non-naturally occurring. In the present invention, a polynucleotide encoding a signal peptide and a portion of the protein to which it is bound may be used. A polynucleotide encoding a signal peptide can be obtained using a known method.

[0023] In the construct, a polynucleotide encoding a linker may be positioned between the polynucleotide encoding ovomucoid or a portion thereof and the polynucleotide encoding the target protein. Various linkers are known, including, but not limited to, GS linkers, polyglycine linkers, EV linkers, and leucine zipper linkers. For example, a linker that can be degraded by proteases may be used to facilitate the purification and isolation of the target protein secreted as a fusion protein. The linker may be one or more types. The type of linker may also be one or more types.

[0024] A construct in which a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a part thereof, and a polynucleotide encoding a target protein are arranged in the 5'- to 3'-direction can be produced using known methods.

[0025] Avian cells may be derived from any part of any bird. They may be used in the form of cultured cells in a medium, or may be cells present in a living avian organism. Avian cells may be derived from, for example, poultry. Examples of poultry include chickens, quails, turkeys, ducks, geese, cockroaches, bantams, pigeons, ostriches, pheasants, and guinea fowl, with chickens and quails being preferred, but are not limited to these. Avian cells may be fibroblasts, epithelial cells, blood cells, primordial germ cells, oviduct cells, etc., or primary cultured cells derived from embryos or tissues, or cells in embryos or individual tissues. For example, if a target protein can be secreted from an established fibroblast or epithelial cell line, the target protein can be obtained from the cell culture medium of the cell. If a target protein can be secreted from oviduct cells, the target protein can be obtained from the egg white of eggs laid by genetically modified birds. To produce a genetically modified bird that can secrete a target protein from oviduct cells, the desired genetic modification can be carried out using primordial germ cells, etc. The target protein may be expressed and secreted in tissues and cells throughout the body of the bird, without being limited to being secreted only in oviduct cells, etc.

[0026] The above construct can be expressed in avian cells, and a fusion protein of ovomucoid or a portion thereof and a target protein can be secreted extracellularly as an expression product. Such expression and secretion can be achieved by culturing avian cells containing the above construct under appropriate conditions. Such culture conditions are known. The secreted fusion protein comprises or consists of a fusion protein of ovomucoid or a portion thereof and a target protein. The ovomucoid or a portion thereof is located at the N-terminus of the target protein. The ovomucoid or a portion thereof and the target protein may be fused via a linker. The target protein can be obtained by excising the ovomucoid or a portion thereof (or the linker, if present). The linker may be cleaved or removed by a protease, peptidase, or drug. The target protein may be used as a fusion protein as is.

[0027] The construct may be introduced into cells by any method as long as the product encoded by the construct is expressed in the cells and secreted extracellularly. Various methods are known for introducing a construct into cells. For example, the construct may be incorporated into an expression vector and then introduced into the cells. Methods for introducing a vector into cells include, but are not limited to, microinjection, lipofection, electroporation, and infection with a viral vector.

[0028] The above construct may be introduced into avian cells by placing the construct in frame under the control of a promoter such as the ovalbumin promoter.

[0029] In a further aspect, the present invention provides the above-mentioned construct, a vector containing the above-mentioned construct, and an avian cell containing the above-mentioned construct or a vector containing the above-mentioned construct. Various vectors are known, and expression vectors are preferred. Examples of expression vectors include, but are not limited to, pCAG vector, pCMV vector, and pEF1α vector. Furthermore, the expression promoter does not have to be a general-purpose promoter; it may be in the form of a vector in which the above-mentioned construct is expressed under the control of a gene that is activated in the cell at an appropriate time after introduction into the cell. The construct can be incorporated into a vector using known methods. For example, a construct having a nucleotide sequence compatible with a restriction enzyme may be incorporated into a vector containing a restriction enzyme site. The avian cell containing such a vector is preferably an oviduct cell. The avian oviduct cell contains the above-mentioned construct and expresses the gene contained in the construct. The avian obtained by the avian production method described below preferably has oviduct cells containing the above-mentioned construct.

[0030] In a further aspect, the present invention provides a method for producing a target protein, comprising culturing avian cells containing the above-mentioned construct or a vector containing the above-mentioned construct. Preferably, the avian cells are cultured containing a vector containing the above-mentioned construct. Culture methods and conditions are known to those skilled in the art, and can be selected and modified as appropriate depending on the avian cells.

[0031] In a further aspect, the present invention provides birds in which the above-mentioned construct has been stably inserted into their genome, more preferably birds in which a gene has been knocked into a specific target site, more preferably birds in which the knocked-in target site is an oviduct-specific locus, and eggs laid by the birds. The birds secrete the target protein into the oviduct. The target protein is present in the eggs laid by the birds. In the birds and their eggs, the target protein is present as a fusion protein with ovomucoid or a portion thereof.

[0032] The generation of such birds is described below (see also International Application Publication WO2017 / 111144A1). The method for generating such birds is not limited to the method described below. The construct (gene) is knocked into avian cells, preferably into avian primordial germ cells. The construct is preferably placed at an oviduct-specific gene locus. The placement is preferably such that the construct is expressed in oviduct tissue under the control of the oviduct-specific gene, and the placement is preferably in-frame with the oviduct-specific gene. Another preferred placement mode may be a knock-in in which the translation start point of the construct is located 5' from the translation start point of the oviduct-specific gene. Oviduct-specific genes are known, and examples include, but are not limited to, genes encoding ovalbumin, ovomucoid, ovotransferrin, ovoglobulin, and lysozyme. For example, the construct may be inserted into exon 2 of the ovalbumin gene. This knock-in can be performed by known methods, for example, by genome editing. It is preferred that the construct include a stop codon.

[0033] The primordial germ cells thus obtained, into which the construct has been knocked in, are transplanted into recipient embryos to produce chimeric knock-in individuals, and birds in whose progeny the construct has been knocked in are obtained. In these knock-in birds, the expression product of the construct (a fusion protein of the target protein with ovomucoid or a portion thereof) is secreted from the oviduct cells instead of the expression product of the oviduct-specific gene. The secreted expression product accumulates in the oviduct lumen and is contained in the egg white. As a result, eggs laid by these birds contain the expression product of the construct.

[0034] A specific example of such birds includes, but is not limited to, birds in which the above construct is placed in frame with the ovalbumin gene under the control of the ovalbumin promoter in oviduct cells.

[0035] The genotype of the knocked-in construct (gene) may be heterozygous (+ / -) or homozygous (+ / +). Eggs laid by females with a homozygous genotype for the knocked-in gene contain more of the target protein than eggs laid by females with a heterozygous genotype for the knocked-in gene.

[0036] In a further aspect, the present invention provides a method for producing a target protein, which comprises collecting the target protein from eggs laid by the above-mentioned birds. Known methods can be used to purify and isolate the target protein from egg white. For example, the target protein can be obtained using techniques such as ultrasonication, solubilization using a solubilizing agent such as arginine hydrochloride, ammonium sulfate fractionation, chromatography such as ion chromatography, gel filtration chromatography, hydrophobic interaction chromatography, and affinity chromatography, lyophilization, crystallization, cleavage or removal of the linker with a protease, peptidase, or a drug. The target protein may be obtained as a single protein or as a fusion protein.

[0037] The present invention will be specifically and in detail explained below by showing examples, but the examples are not intended to limit the scope of the present invention.

[0038] Example 1: Improving Secretion Efficiency Using DF1 Cells as a Model for FGF2 (1) Construct of a Mammalian (Bovine) FGF2 Expression Gene FGF2 is a cell growth factor that functions by being secreted extracellularly. However, unlike typical secretory proteins, its secretory pathway exhibits atypical secretion kinetics that does not pass through the endoplasmic reticulum-Golgi pathway. For this reason, it is known that even when the FGF2 gene is introduced into cells and forced to express, very little of it is secreted extracellularly. On the other hand, it is also known that FGF2 can be secreted extracellularly by adding a signal peptide sequence of another protein to FGF2. However, the amount secreted is limited, and a technology for mass secretion is needed. In particular, there is a need to develop a technology for mass secretion in chicken cells so that it can be expressed using a chicken egg bioreactor.

[0039] A schematic diagram of the FGF2 expression construct used in the experiment is shown in Figure 1. DNA (SEQ ID NO: 1) encoding the ovotransferrin signal peptide and the four amino acids following it (amino acids 1-23 of the ovotransferrin immature protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 59)) was used as the common signal peptide. To the 3' side of this sequence were linked DNA encoding chicken ovalbumin mature protein (SEQ ID NO: 2), DNA encoding chicken lysozyme mature protein (SEQ ID NO: 3), DNA encoding chicken ovomucoid mature protein (SEQ ID NO: 4), and DNA encoding an amino acid sequence corresponding to the first domain of chicken ovomucoid mature protein (amino acids 1 to 69 counting from the N-terminus) (SEQ ID NO: 5), and further to the 3' side of these was linked DNA encoding bovine FGF2 mature protein (SEQ ID NO: 6). The overall sequences of the respective constructs are OA-FGF2 (SEQ ID NO: 7), LZ-FGF2 (SEQ ID NO: 8), OM-FGF2 (SEQ ID NO: 9), and OM1D-FGF2 (SEQ ID NO: 10). Furthermore, DNA encoding the mature protein of bovine FGF2 was ligated to the 3' end of the DNA encoding the ovotransferrin signal peptide to serve as a control for the secretion of FGF2 alone (SEQ ID NO: 11). Each gene construct was inserted into the pCAGGS vector and subjected to gene expression. Furthermore, pEGFP-N1 (SEQ ID NO: 12) was transfected as a negative control.

[0040] (2) Gene transfer into chicken DF-1 cells and expression analysis Chicken fibroblast DF-1 cells were obtained from the National Agriculture and Food Research Organization. Culture was performed using a medium prepared by adding DMEM basal medium to a final concentration of 10% fetal bovine serum, 2 mM L-glutamine, 100 Units / ml penicillin, and 100 μg / ml streptomycin. DF-1 cells were seeded at 5 x 10^5 cells / well in a 6-well plate (Corning), and gene transfer was performed the next day using Lipofectamine 2000 (Thermo Fisher). Gene transfer was performed according to the Lipofectamine 2000 instructions. 1.6 micrograms of each expression vector was suspended in 50 microliters of OPTIMEM medium (Thermo Fisher Scientific) and 3 microliters of Lipofectamine 2000 suspended in 50 microliters of OPTIMEM medium were mixed and added to the DF-1 cell culture medium. Three days after addition, the medium and cells were collected, and the recombinant proteins secreted into the medium and the recombinant proteins expressed in the cells were analyzed by Western blotting. A peroxidase-labeled antibody was used as the secondary antibody, and detection was performed using a chemiluminescence reagent (WAKO ImmunoStar). Images were captured using a WSE-6100 LuminoGraphI (Atto), and chemiluminescence was quantified using image analysis software CS Analyzer (Atto).

[0041] Figure 2 shows the results of Western blotting when OA-FGF2, LZ-FGF2, and OM-FGF2 were expressed in DF-1 cells. Anti-FGF2 antibody (Cell Signaling #20102) was used. Signals not observed in the negative control were observed in the DF-1 cell lysate, and these signals were identified as FGF2 proteins fused with ovalbumin, lysozyme, and ovomucoid based on their mobilities. Meanwhile, signals were observed in the culture medium of each cell line only when OM-FGF2 was expressed. This indicates that the FGF2 protein fused with ovomucoid was efficiently secreted when expressed in chicken cells, but was hardly secreted when fused with ovalbumin or lysozyme.

[0042] Figure 3 shows the results of Western blotting using anti-FGF2 antibody on samples of medium expressing OM-FGF2 and FGF2 in DF-1 cells. The mobility signals of FGF2 fused with ovomucoid and FGF2 were confirmed (arrows). Comparison of protein amounts based on chemiluminescence intensity revealed that the amount of FGF2 fused with ovomucoid in the medium was approximately 34-fold higher than that of FGF2 alone, indicating highly efficient secretion. Furthermore, comparison with a commercially available recombinant human mature FGF2 (Peprotech, whose amino acid sequence is identical to that of bovine mature FGF2) blot performed simultaneously indicated that the concentration of FGF2 alone in the medium was estimated to be 0.02 ng / μL, and the concentration of FGF2 fused with ovomucoid was estimated to be 0.68 ng / μL.

[0043] Figure 4 shows the results of Western blotting using anti-FGF2 antibodies on samples of medium expressing OM1D-FGF2 and FGF2 in DF-1 cells. The mobility signals of FGF2 fused to the first domain of the mature ovomucoid protein and FGF2 were confirmed (arrows). Comparison of protein amounts based on chemiluminescence intensity revealed that the amount of FGF2 fused to the first domain of the mature ovomucoid protein in the medium was approximately 22-fold higher than that of FGF2 alone, indicating highly efficient secretion. In other words, even if the fusion protein does not contain the full-length ovomucoid, it is possible to induce highly efficient secretion in chicken cells by simply including a portion of the ovomucoid protein in the fusion protein. Furthermore, comparison with commercially available recombinant human mature FGF2 blots performed simultaneously indicated that the concentration of FGF2 alone in the medium was estimated to be 0.02 ng / μL, and the concentration of FGF2 fused to the first domain of the mature ovomucoid protein was estimated to be 0.43 ng / μL.

[0044] (3) Constructs with Linkers Inserted into FGF2 Fusion Proteins The ovomucoid or partial ovomucoid sequence to be fused to the target protein may be directly linked, or a preferred amino acid sequence may be inserted depending on the purpose. As schematically shown in Figure 5, three types of constructs were prepared in which a GS linker (amino acid sequence GGGGS (SEQ ID NO: 61)) was inserted between the first domain of the ovomucoid mature protein of OM1D-FGF2 and FGF2. The number of inserted GS linkers was 1, 3, and 7, and they are encoded by the DNAs of SEQ ID NOs: 13, 14, and 15, respectively. The overall sequences of the respective constructs are OM1D-GS1-FGF2 (SEQ ID NO: 16), OM1D-GS3-FGF2 (SEQ ID NO: 17), and OM1D-GS7-FGF2 (SEQ ID NO: 18). Like OM-FGF2 and FGF2, these were inserted into the pCAGGS vector and subjected to gene expression.

[0045] (4) Assay of the specific activity of secreted FGF2 fusion proteins. OM1D-FGF2, OM1D-GS1-FGF2, OM1D-GS3-FGF2, OM1D-GS7-FGF2, and FGF2 alone were expressed in DF-1 cells as described above, and the FGF2 protein secreted into the medium was analyzed by Western blotting using an anti-FGF2 antibody (Figure 6). The signals for fusion FGF2 and FGF2 were confirmed from their respective mobilities, and the relative expression levels (relative concentrations) of each protein could be determined from the chemiluminescence intensity. It was found that all fusion proteins containing the GS linker were secreted into the medium more efficiently than FGF2 expressed alone.

[0046] Next, FGF2 activity in the medium was examined by cell proliferation assay. NIH3T3 cells were seeded in a 96-well plate at 1,000 cells / well / 100 μl of medium using the above-mentioned DMEM medium with a final FCS concentration of 0.1%. DF-1 cells were expressing OM1D-FGF2, OM1D-GS1-FGF2, OM1D-GS3-FGF2, OM1D-GS7-FGF2, and FGF2 alone. Serial dilutions of the medium were made from 1:1 to 25:1, and 1 microliter of each was added to the NIH3T3 cell medium and cultured for 2 days. Cell counts were then counted using a proliferation assay kit (Dojindo Laboratories Cell Counting Kit-8) according to the attached protocol, and the activity of the growth factors contained in each DF-1 medium was determined. This cell proliferation activity was divided by the relative amount of FGF2 contained in the medium, and the FGF2 activity ratios of each medium were compared (Figure 7). Compared to expression of FGF2 alone, OM1D-FGF2 without a GS linker exhibited only about 20% of the activity, whereas insertion of the GS linker resulted in a specific proliferation activity that was almost equal to or greater than that of expression of FGF2 alone.

[0047] Example 2: Implementation in a Chicken Egg Bioreactor We found that fusing a gene with a full-length or partial ovomucoid sequence enabled highly efficient secretion of poorly secreted proteins from chicken cells. Next, we engineered chickens to incorporate this expression system, and expressed the foreign protein in the oviduct cells of the chickens under the control of the ovalbumin promoter, resulting in highly efficient secretion, thereby enabling the expression of large amounts of the poorly secreted foreign protein in egg white. A schematic diagram of the donor construct constructed for chicken genetic engineering (gene knock-in) is shown in Figure 8. OM1D-GS3-FGF2 or FGF2 was ligated to the 3' side of the 2.8 kb upstream of the ovalbumin translation initiation site (SEQ ID NO: 19), and further 3' to that, the SV40 promoter, neomycin resistance gene (SEQ ID NO: 20), and the 3.0 kb gene sequence from ovalbumin exon 2 onward (SEQ ID NO: 21) were placed. The entire sequences were OM1D-GS3-FGF2 donor (SEQ ID NO: 22) and FGF2 donor (SEQ ID NO: 23), and these were inserted into the plasmid pBlue Script II (SK+) (Stratagene, USA, now Agilent Technologies) to create pBS-OM1D-GS3-FGF2 donor and pBS-FGF2 donor.

[0048] (1) Genome editing using chicken male primordial germ cells and production of knock-in chickens Knock-in to the ovalbumin locus was performed using a chicken male primordial germ cell line in accordance with International Application Publication WO2017 / 111144A1, targeting the ovalbumin gene using the CRISPR method. OligoDNAs represented by SEQ ID NO: 26 and SEQ ID NO: 27 were synthesized targeting SEQ ID NO: 25 (OVATg1) in the sequence (SEQ ID NO: 24) containing the translation start point of exon 2 of the ovalbumin gene shown in Figure 9a, and the 5'-ends were phosphorylated using T4 Polynucleotide Kinase. The mixture was then heated to 98 ° C and annealed by slowly cooling to room temperature. This DNA fragment was inserted into the BbsI cleavage site of the plasmid px330-Puror (px330-Puror-OVATg1), which had been prepared by inserting the puromycin resistance gene unit of SEQ ID NO: 28 into the NotI site of the plasmid px330 (AddGENE, USA).

[0049] The above gene (plasmid) was introduced into a chicken male primordial germ cell line (prepared according to Non-Patent Document 3) collected from the blood of a White Leghorn male embryo. ５ ~5 x 10 ５ Male primordial germ cell lines were washed with PBS, suspended in OPTI-MEM, and transfected with 0.8 μg of px330-Puror-OVATg1 and 0.8 μg of pBS-OM1D-GS3-FGF2 donor or 0.8 μg of pBS-FGF2 donor using 3 μl of Lipofectamine 2000. More specifically, Lipofectamine 2000 and the plasmid were mixed in 80 μl of OPTI-MEM, mixed with the male primordial germ cell line, and then allowed to stand at room temperature for approximately 5 minutes. 500 μl of antibiotic-free medium was then added, and the mixture was allowed to stand at 37°C for approximately 1 to 4 hours before being plated on feeder cells. From 3 days after transfection, neomycin (G418 disulfate, Nacalai Tesque, Japan) was added to a final concentration of 0.5 mg / ml. The medium was replaced as appropriate, and cells growing in the presence of neomycin at a final concentration of 0.5 mg / ml were collected and genomic DNA was prepared. Genomic PCR was used to confirm that the donor construct had been knocked into the ovalbumin locus. PCR was performed on the 5' region using primers for the exogenous gene in the donor construct and the 5' region of ovalbumin not contained in the donor construct, as follows. PCR was performed using an antisense primer for the ovotransferrin signal peptide shown in SEQ ID NO:29 (P2 in Figure 9a) and a sense primer for the region approximately 3.0 kb 5' of the ovalbumin translation start site shown in SEQ ID NO:30 (P1 in Figure 9a). As shown in the left panel of Figure 9b, when the genome derived from primordial germ cells (knock-in PGCs) into which the donor construct was introduced together with px330-Puror-OVATg1 and drug-selected was used as a template, an amplification product was observed at a position of approximately 3.0 kb, which is expected when the donor construct is inserted, whereas no amplification product was observed when the genome derived from control primordial germ cells that had not been subjected to gene introduction (control PGCs) was used as a template (UT).

[0050] Similarly, the 3' region was confirmed by genomic PCR using primers for the exogenous gene of the donor construct and the 3' region of ovalbumin, which is not included in the donor construct. PCR was performed using a sense primer for the drug resistance gene unit shown in SEQ ID NO: 31 (Fig. 9a, P3) and an antisense primer for a region approximately 3.4 kb 3' from the ovalbumin translation start site shown in SEQ ID NO: 32 (Fig. 9a, P4). As shown in the right panel of Fig. 9b, when the genome derived from primordial germ cells (knock-in PGCs) that had been drug-selected after introducing the donor construct together with px330-Neor-OVATg1 was used as a template, an amplification product was observed at approximately 3.4 kb, as would be expected if the donor construct had been inserted. However, when the genome derived from control primordial germ cells (control PGCs) that had not been introduced with the gene was used as a template, no amplification product was observed (UT). From the above, it is believed that cells in which a donor construct containing an exogenous gene portion has been knocked into the ovalbumin locus are present in the drug-selected cell population.

[0051] The primordial germ cells containing the OM1D-GS3-FGF2 donor and FGF2 donor constructs were transferred into the blood of 2.5-day-old White Leghorn embryos (recipient embryos) by microinjection. Prior to transfer, the fertilized eggs were irradiated with 5 or 6 Gy of ionizing radiation before incubation to reduce the number of primordial germ cells endogenous to the recipient embryos.

[0052] After 2.5 days of incubation, a window approximately 2 cm in diameter was opened at the tip of the egg to expose the embryo, and approximately 1,000 to 5,000 drug-selected cells (suspended in 1 to 2 μl of PBS) were transplanted using a microglass needle into the blood of a recipient embryo at Hamburger Hamilton stage 13 to 15. After sealing the window with cellophane tape, the embryos were cultured at 38.5°C and 60 to 80% humidity until hatching (chimeric chicks (G0)). Six OM1D-GS3-FGF2 donor knock-in male chimeras and seven FGF2 donor knock-in male chimeras were obtained. These produced knock-in sperm after sexual maturation, allowing for the generation of knock-in chickens. Knockin female chickens express OM1D-GS3-FGF2 and FGF2 under the control of the ovalbumin promoter in oviduct cells, and the secreted foreign proteins accumulate in the lumen of the oviduct and are contained in egg white; therefore, eggs laid by OM1D-GS3-FGF2 knockin chickens contain more foreign proteins than eggs laid by FGF2 knockin chickens.

[0053] Example 3: Increasing the efficiency of secretion using DF1 cells with puromycin N-acetyltransferase as a model It was demonstrated using puromycin N-acetyltransferase (PAC) from Streptomyces actinomycetes that not only FGF2 but also foreign proteins that do not enter the endoplasmic reticulum-Golgi pathway can be efficiently secreted extracellularly by fusing them with ovomucoid and expressing them in chicken cells.

[0054] A schematic diagram of the expression construct of PAC used in the experiment is shown in Figure 10. DNA (SEQ ID NO: 1) encoding the ovotransferrin signal peptide and the four amino acids following it (amino acids 1-23 of the immature ovotransferrin protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 59)) was used as the common signal peptide. To the 3' side of this sequence were linked DNA encoding chicken ovalbumin mature protein (SEQ ID NO: 2), DNA encoding chicken lysozyme mature protein (SEQ ID NO: 3), DNA encoding chicken ovomucoid mature protein (SEQ ID NO: 4), and DNA encoding an amino acid sequence corresponding to the first domain of chicken ovomucoid mature protein (amino acids 1 to 69, counting from the N-terminus) (SEQ ID NO: 5). Further linked to the 3' side of this was DNA encoding PAC (SEQ ID NO: 33), and further linked to the 3' side of this was DNA encoding a Flag epitope tag (amino acid sequence: GDYKDDDDK (SEQ ID NO: 63)) containing a stop codon at its terminal (SEQ ID NO: 34). The respective overall sequences are OA-PAC (SEQ ID NO: 35), LZ-PAC (SEQ ID NO: 36), and OM-PAC (SEQ ID NO: 37). Additionally, DNA encoding only the ovotransferrin signal peptide and the four amino acids following it, PAC, and Flag tag (PAC, SEQ ID NO: 38), DNA encoding only the ovotransferrin signal peptide, PAC, and Flag tag (4aa(-)PAC, SEQ ID NO: 39), and DNA encoding only PAC and Flag tag (SP(-)PAC, SEQ ID NO: 40) were used as controls. Each gene construct was inserted into the pCAGGS vector and subjected to gene expression. As with FGF2, these genes were introduced into chicken DF-1 cells using Lipofectamine 2000, and the foreign proteins contained in the medium and cell lysates three days later were analyzed by Western blotting.

[0055] Figure 11 shows the results of Western blotting of the culture supernatant (Sup, left, Figure 11) and cell lysate (WCL, right, Figure 11) obtained when SP(-)PAC, 4aa(-)PAC, PAC, OA-PAC, LZ-PAC, and OM-PAC were expressed in DF-1 cells. Anti-Flag antibody (Sigma Aldrich M2) was used. Western blotting of the culture supernatant demonstrated that, like FGF2, PAC can be secreted efficiently by fusing it with full-length ovomucoid. However, no secretion was observed when PAC fused with ovalbumin or lysozyme or when PAC was expressed alone (regardless of the presence or absence of a signal peptide or the amino acid sequence following the signal peptide). Western blotting of the cell lysate demonstrated clear expression of PAC and PAC fusion proteins, although the expression levels varied.

[0056] Example 4 Improving the efficiency of secretion using DF1 cells as a model for CRE recombinase Construct of CRE recombinase expression gene It was revealed that, in addition to FGF2 and PAC, CRE recombinase (CRE) derived from bacteriophage P1, which has a nuclear localization signal and is present in the cytoplasm or nucleus in mammalian cells but does not normally enter the endoplasmic reticulum-Golgi apparatus pathway, can be fused with ovomucoid and expressed in chicken cells, allowing for efficient extracellular secretion.

[0057] A schematic diagram of the CRE expression construct used in the experiment is shown in Figure 12. DNA (SEQ ID NO: 1) encoding the ovotransferrin signal peptide and the four amino acids following it (amino acids 1-23 of the ovotransferrin immature protein, sequence MKLILCTVLSLGIAAVCFAAPPK (SEQ ID NO: 59)) was used as the common signal peptide. To the 3' side of this sequence were linked DNA encoding chicken ovalbumin mature protein (SEQ ID NO: 2), DNA encoding chicken lysozyme mature protein (SEQ ID NO: 3), DNA encoding chicken ovomucoid mature protein (SEQ ID NO: 4), and DNA encoding an amino acid sequence corresponding to the first domain of chicken ovomucoid mature protein (amino acids 1 to 69 counting from the N-terminus) (SEQ ID NO: 5), and then DNA encoding CRE (SEQ ID NO: 41) was linked to the 3' side of each of these. The respective full sequences are OA-CRE (SEQ ID NO: 42), LZ-CRE (SEQ ID NO: 43), OM-CRE (SEQ ID NO: 44), and OM1D-CRE (SEQ ID NO: 45). DNA encoding only CRE (SEQ ID NO: 41) was used as a control. Each gene construct was inserted into a pCAGGS vector and subjected to gene expression. pEGFP-N1 (SEQ ID NO: 12) was also transfected as a negative control. As with FGF2, these genes were transfected into chicken DF-1 cells using Lipofectamine 2000, and the foreign proteins contained in the medium after 3 days were analyzed by Western blotting.

[0058] Figure 13 shows the results of Western blotting when CRE, OA-CRE, LZ-CRE, OM-CRE, and OM1D-CRE were expressed in DF-1 cells. Anti-CRE antibody (Cell Signaling #15036) was used. Similar to FGF2 and PAC, CRE can be secreted highly efficiently by fusing it to the full-length ovomucoid or a partial region of ovomucoid. Furthermore, CRE secretion into the medium was observed even when fused to ovalbumin. Compared to expression of CRE alone, fusion with ovomucoid resulted in approximately 23-fold secretion, fusion with the first domain of ovomucoid resulted in 15-fold secretion, and fusion with ovalbumin resulted in approximately 21-fold secretion.

[0059] Example 5 Secretion of a Target Protein Using a Signal Peptide Other than the Ovotransferrin Signal Peptide Although the ovotransferrin signal peptide was used as the signal peptide in Examples 1-5, this example demonstrates that a target protein can also be expressed and secreted when a signal peptide other than the ovotransferrin signal peptide is used. The lysozyme signal peptide (amino acids 1-18 of the immature lysozyme protein, sequence MRSLLILVLCFLPLAALG (SEQ ID NO: 62)) was used as the signal peptide, and DNA encoding this (SEQ ID NO: 46) was used. To the 3' side of this sequence, DNA encoding the four amino acids following the ovotransferrin signal peptide (APPK (SEQ ID NO: 60)) and DNA encoding chicken ovalbumin mature protein (SEQ ID NO: 2) were linked, and to the 3' side of this, DNA encoding PAC (SEQ ID NO: 37) was linked, and to the 3' side of this, DNA encoding a Flag epitope tag (amino acid sequence: GDYKDDDDK (SEQ ID NO: 63)) containing a stop codon at its terminus (SEQ ID NO: 34) was linked. The entire sequence is referred to as LZsp-OM-PAC, and the DNA sequence is shown in SEQ ID NO: 47. Furthermore, LZsp-PAC (SEQ ID NO: 48), which was prepared by removing the DNA encoding the mature chicken ovalbumin protein (SEQ ID NO: 2) from LZsp-OM-PAC, served as a control. Each gene construct was inserted into the pCAGGS vector and subjected to gene expression. These genes were introduced into chicken DF-1 cells using Lipofectamine 2000, and the foreign proteins contained in the medium and cell lysates three days later were analyzed by Western blotting.

[0060] Figure 14 shows the results of Western blotting when LZsp-OM-PAC and LZsp-PAC were expressed in DF-1 cells (AI881 and AI888, respectively). The antibody used was an anti-Flag antibody (Sigma Aldrich M2). Western blotting showed that the PAC gene product was secreted with high efficiency from LZsp-OM-PAC-transfected cells, but secretion of the PAC gene product was barely detectable from LZsp-PAC-transfected cells that did not contain ovomucoid.

[0061] Example 6 Restriction of ovomucoid region that promotes highly efficient secretion In Examples 1 and 4, it was shown that the secretion efficiency of a foreign protein can be significantly improved not only by using the full-length chicken ovomucoid, but also by using an amino acid sequence corresponding to the first domain of the mature chicken ovomucoid protein (amino acids 1 to 69 counting from the N-terminus of the mature ovomucoid protein). To verify the region of ovomucoid that has the effect of improving secretion efficiency, the 1st to 69th amino acids counting from the N-terminus of the mature ovomucoid protein (sequence AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLLCAYSIEFGTNISKEHDGECKETVPMN (amino acid sequence: SEQ ID NO: 64, DNA sequence encoding it: SEQ ID NO: 5)) or the 1st to 31st amino acids (sequence AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (amino acid sequence: SEQ ID NO: 65, DNA sequence encoding it: SEQ ID NO: 5)) were analyzed. Constructs were prepared by inserting DNA encoding the chicken ovomucoid mature protein (SEQ ID NO: 4) of OM-PAC (SEQ ID NO: 37) in place of the DNA encoding the chicken ovomucoid mature protein (SEQ ID NO: 4) of Example 3. The overall sequences are OM1D-PAC (SEQ ID NO: 52), OM1D(1-31)-PAC (SEQ ID NO: 53), OM1D(1-22)-PAC (SEQ ID NO: 54), and OM1D(1-15)PAC (SEQ ID NO: 55). Each gene construct was inserted into a pCAGGS vector and subjected to gene expression. These genes were introduced into chicken DF-1 cells using Lipofectamine 2000, and after 3 days, the foreign proteins contained in the medium and the cell lysate were analyzed by Western blotting.

[0062] 15 shows the results of Western blotting when OM-PAC, OM1D-PAC, OM1D(1-31)-PAC, OM1D(1-22)-PAC, and OM1D(1-15)PAC were expressed in DF-1 cells. Anti-Flag antibody (Sigma Aldrich M2) was used. Western blotting demonstrated that the PAC gene product was secreted into the culture supernatant not only from the full-length ovomucoid but also from forms containing a partial region of the ovomucoid. Thus, the effects of the present invention can be achieved by including the first domain of ovomucoid or a portion thereof, and this region can be a sequence containing the 1st to 15th amino acids, the 1st to 21st amino acids, the 1st to 31st amino acids, or the 1st to 69th amino acids counting from the N-terminus of the mature ovomucoid protein.

[0063] Example 7: Secretion of a target protein is promoted by fusing ovomucoid with the target protein when the target protein is expressed in oviduct cells under the control of an oviduct-specific promoter. Expression and secretion of a target protein using DF-1 cells or promoters other than a general-purpose promoter was demonstrated by promoting secretion of a target protein fused with ovomucoid under the control of an oviduct-specific promoter using poultry oviduct-derived cells. A DNA sequence 2.8 kb upstream of the ovalbumin translation initiation site (SEQ ID NO: 19) was inserted into the 5' side of OM-PAC (SEQ ID NO: 37) (OVAp-OM-PAC, SEQ ID NO: 56), and a DNA sequence 2.8 kb upstream of the ovalbumin translation initiation site (SEQ ID NO: 19) was inserted into the 5' side of PAC of SEQ ID NO: 38 (OVAp-PAC, SEQ ID NO: 57) were prepared, and these constructs were inserted into pBS and used for gene expression. Poultry oviduct cells obtained by treating poultry oviduct tissue with a solution containing collagenase, trypsin, and pronase were used as transfected cells. These genes were introduced into fowl oviduct cells using Lipofectamine 2000, and foreign proteins contained in the medium and cell lysates were immunoprecipitated and then analyzed by Western blotting.

[0064] Figure 16 shows the results of Western blotting when OVAp-OM-PAC and OVAp-PAC were expressed in poultry oviduct cells. The antibody used was an anti-Flag antibody (Millipore). As a result of Western blotting, secretion of the PAC gene product was observed from OVAp-OM-PAC-introduced cells, but secretion of the PAC gene product was hardly observed from OVAp-OM-PAC-introduced cells that did not contain ovomucoid. This shows that the effects of the present invention can also be observed in poultry oviduct cells under the control of an oviduct-specific promoter.

[0065] According to the present invention, proteins, particularly poorly secreted proteins, can be secreted from poultry cells with high efficiency. The present invention can be used in the fields of medicine, food, livestock, etc.

Claims

1. A method for promoting secretion of a target protein, comprising expressing in poultry cells a construct encoding a fusion protein of ovomucoid or a portion thereof with a target protein, and secreting the fusion protein from the poultry cells, wherein the construct comprises a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a portion thereof, and a polynucleotide encoding the target protein, and the polynucleotide encoding the signal peptide, the polynucleotide encoding ovomucoid or a portion thereof, and the polynucleotide encoding the target protein are arranged in the construct from the 5' end to the 3' end.

2. The method of claim 1, wherein the portion of ovomucoid comprises the first domain of ovomucoid or a portion thereof.

3. The method of claim 1 or 2, wherein the portion of ovomucoid comprises an amino acid sequence selected from the group consisting of the 1st to 69th amino acids, the 1st to 31st amino acids, the 1st to 22nd amino acids, and the 1st to 15th amino acids counting from the N-terminus of mature ovomucoid.

4. The method of any one of claims 1 to 3, wherein the portion of ovomucoid comprises AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMN (SEQ ID NO: 64), AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (SEQ ID NO: 65), AEVDCSRFPNATDKEGKDVLVC (SEQ ID NO: 66), or AEVDCSRFPNATDKE (SEQ ID NO: 67).

5. The method according to any one of claims 1 to 4, wherein the target protein is a poorly secreted protein.

6. The method according to any one of claims 1 to 5, wherein a polynucleotide encoding a linker is positioned between the polynucleotide encoding ovomucoid or a part thereof and the polynucleotide encoding the target protein.

7. A construct comprising a polynucleotide encoding a signal peptide, a polynucleotide encoding ovomucoid or a portion thereof, and a polynucleotide encoding a target protein, wherein the polynucleotide encoding the signal peptide, the polynucleotide encoding ovomucoid or a portion thereof, and the polynucleotide encoding the target protein are arranged from the 5' end to the 3' end.

8. The construct of claim 7, wherein the portion of ovomucoid comprises the first domain of ovomucoid or a portion thereof.

9. The construct of claim 7 or 8, wherein the portion of ovomucoid comprises an amino acid sequence selected from the group consisting of amino acids 1 to 69, amino acids 1 to 31, amino acids 1 to 22, and amino acids 1 to 15, counting from the N-terminus of mature ovomucoid.

10. The construct of any one of claims 7 to 9, wherein the portion of ovomucoid comprises AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMN (SEQ ID NO: 64), AEVDCSRFPNATDKEGKDVLVCNKDLRPICG (SEQ ID NO: 65), AEVDCSRFPNATDKEGKDVLVC (SEQ ID NO: 66), or AEVDCSRFPNATDKE (SEQ ID NO: 67).

11. The construct according to any one of claims 7 to 10, wherein the target protein is a poorly secreted protein.

12. A construct according to any one of claims 7 to 11, wherein a polynucleotide encoding a linker is positioned between the polynucleotide encoding ovomucoid or a portion thereof and the polynucleotide encoding the target protein.

13. A poultry cell comprising a construct according to any one of claims 7 to 12.

14. A vector comprising the construct of any one of claims 7 to 12.

15. A poultry cell containing the vector of claim 14.

16. A method for producing a target protein, comprising culturing the cells according to claim 15.

17. A poultry in which the construct according to any one of claims 7 to 12 has been knocked in.

18. A method for producing poultry according to claim 17, comprising knocking in the construct according to any one of claims 7 to 12 by placing it in-frame into an avian oviduct-specific locus.

19. Eggs produced by the poultry of claim 17.

20. A method for producing a target protein, comprising collecting the target protein from the eggs according to claim 19.

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