TW201433572A - Method for manufacturing human epidermal cell growth factor - Google Patents

Abstract

Disclosed is a novel means making possible the manufacture of an epidermal growth factor (EGF) with good efficiency and lower cost than conventional techniques. As a result of thoroughgoing research aimed at developing a technique for producing human EGF using plants, the inventors of the present application succeeded in finding a modified EGF coding sequence and a signal peptide coding sequence that allow production of EGF with high efficiency using a transient expression system. Using these sequences allows EGF to be produced with good efficiency even when the transient expression system of a viral vector is used.

Description

Method for producing human epithelial cell proliferative factor

The present invention relates to a method for producing a human epithelial cell proliferation factor, and a nucleic acid construct suitable for use in the method.

The epithelial growth factor (EGF) system was originally discovered by Cohen et al. from the submandibular gland of a mouse (Non-Patent Document 1). Thereafter, it is purified from human urine as a substance having a gastric acid secretion inhibiting action (Non-Patent Document 2). Human EGF is synthesized in vivo and synthesized as a precursor protein formed by 1207 amino acids, and then has 3 peptides in the molecule by a processed peptide of about 6 kDa formed by 53 amino acids. Disulfide bond (Non-Patent Document 3). As the physiological activity of EGF, cell proliferation, gastric acid secretion inhibition, anti-ulcer effect, gastrointestinal mucosal protection, DNA synthesis promotion, corneal repair, wound healing, anti-inflammatory, analgesic, etc. are known. . It has also been attempted to apply EGF to a wound healing medicine, an anti-ulcer agent, and the like which are obtained by such an action, and is now utilized as one of ingredients of medicines and cosmetics.

In this way, while applying the application research of EGF, on the other hand, an efficient production method for EGF has also been studied. Various EGF production methods by genetic recombination have been reported. As an example of such reports, production by coliform bacteria (Non-Patent Document 4), production by yeast (Non-Patent Documents 5 to 7), and production by plants (Patent Document 1 and Non-Patent Document 8) )Wait.

In the case of using bacteria, it is necessary to closely monitor the subtle culture conditions, and Import expensive systems for control. In addition, there is also the possibility of potential risks such as the incorporation of heat-generating substances from bacteria, and there are problems in being accused. Further, EGF is considered to have six cysteine acids in the molecule, thereby forming three disulfide bonds to form a certain three-dimensional structure, thereby exerting biological activity. A protein which is a eukaryotic cell produced by recombinant DNA technology and which is a host of prokaryotic cells and which is produced by recombinant DNA technology is known to be incapable of constituting a stereoscopic structure of a protein, and is present in the form of insoluble particles in the body. Therefore, when producing a biologically active protein derived from eukaryotic cells by coliform, in many cases, the operation of converting into a natural protein must be performed, and the steps become complicated.

In contrast, a plant belonging to a eukaryotic organism has a function of forming a biologically active three-dimensional structure such as an enzyme for producing a disulfide bond and a chaperone in a cell. Patent Document 1 describes a method for stably reconstituting a gene of EGF in a cell gene of barley to produce barley. However, this method has problems in the management of genetically modified plants. On the other hand, Non-Patent Document 8 describes a method for stably recombining an EGF gene in a genome of a tobacco plant to produce EGF, and a method of using a viral vector, that is, a recombinant human being is described. An infectious virus of the EGF gene, which infects the virus to tobacco, by a method of producing EGF by transiently expressing human EGF. However, the method using a viral vector described in Non-Patent Document 8 has a problem in yield and cannot be regarded as a practical method.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] WO/2011/083500

[Non-patent literature]

[Non-Patent Document 1] S. Cohen, J. Biol. Chem., 237, 1555 (1962)

[Non-Patent Document 2] H. Gregory, Nature, 257, 325 (1975)

[Non-Patent Document 3] G. I. Bell, et al., Nucleic Acids Research 14(21), 8427 (1986)

[Non-Patent Document 4] F. kishimoto et al., Gene, 45, 311 (1986)

[Non-Patent Document 5] M. S. Urdea, et al. Proc. Natl. Acad, Sci. USA, 80 7461 (1983)

[Non-Patent Document 6] J. M. Clements, et al., Gene 106, 267 (1991)

[Non-Patent Document 7] Z. Hambali, Journal of International Studies, 10, 36 (2009)

[Non-Patent Document 8] Sonin Wirth, et al., Molecular Breeding 13:23-35, 2004

It is an object of the present invention to provide a novel means of manufacturing EGF that is less cost effective and efficient in accordance with conventional techniques.

The inventors of the present invention have diligently studied the development of human EGF production technology using plants, and have succeeded in discovering altered EGF coding sequences and message peptide coding sequences which can produce EGF efficiently by using a transient expression system. It has been found that by using such sequences, even if a transient expression system derived from a viral vector is used, EGF can be efficiently produced, and the present invention is completed.

That is, the present invention provides a nucleic acid construct comprising a base sequence encoding mature human EGF and for expressing mature human EGF in a plant cell, The above-described base sequence encoding the mature human EGF has a base sequence which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1. Furthermore, the present invention provides an expression vector comprising the above nucleic acid construct of the present invention. Furthermore, the present invention provides a recombinant virus comprising the above nucleic acid construct of the present invention. Furthermore, the present invention provides a method for producing human EGF comprising infecting a recombinant virus of the present invention into a plant, and recovering the mature human EGF protein molecule produced by the virus after the virus is proliferated. Furthermore, the present invention provides a method for producing human EGF, which comprises infecting a recombinant virus prepared by the above-described expression vector of the present invention into a plant body, and recovering the mature human EGF protein molecule produced by the virus after the virus is proliferated. . Furthermore, the present invention provides a polynucleotide which encodes a mature human EGF, and the nucleotide sequence of the polynucleotide has a nucleotide sequence which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1. Furthermore, the present invention provides a polynucleotide encoding a mature human EGF, wherein the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 1, or 1 to 15 in SEQ ID NO: 1. The base sequence of the base. Furthermore, the present invention provides a polynucleotide which encodes a tobacco extensin message peptide having a nucleotide sequence which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 4. Furthermore, the present invention provides a peptide encoding a tobacco extensin message peptide, wherein the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 4, or the base in SEQ ID NO: 4 is replaced by 1 to several bases. Base sequence. Furthermore, the present invention provides a polynucleotide which encodes an endoplasmic reticulum retention message, and the nucleotide sequence of the polynucleotide has an identity of 80% or more with the nucleotide sequence shown in SEQ ID NO: 7. Furthermore, the present invention provides a polynucleotide which encodes an endoplasmic reticulum retention message, wherein the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 7, or is substituted 1 to 2 in SEQ ID NO: 7. The base sequence of a base. Furthermore, the present invention provides a recombinant virus comprising the above polynucleotide of the present invention.

According to the present invention, there is provided a novel means of efficiently producing EGF using a transient performance system. Plants and humans belong to eukaryotes, and the cells of both have very similar protein expression mechanisms. In addition, the plant is an energy-efficient protein synthesis system based on independent vegetative organisms synthesized by light. In the prior art for producing EGF in bacteria, it is necessary to introduce an expensive system in order to closely monitor and control the fine culture conditions; compared with this technique, the method of the present invention is based on production cost, equipment investment, and production expansion. It is advantageous from the point of view. According to the present invention, since EGF can be provided at a lower cost than conventionally, it is advantageous to reduce the cost of products such as pharmaceuticals or cosmetics using EGF. Although a technique for producing EGF in plants has been known in the past, in order to achieve a certain production efficiency in the prior art, it is necessary to stably recombine the EGF gene in the genome of a plant. According to the method of the present invention, since it is not necessary to change the plant gene, it is advantageous in the management of the plant body as compared with the conventional technique using the plant.

Fig. 1 is a comparison diagram of a natural human gene sequence (NM_001963) encoding mature human EGF, and an altered gene sequence (SEQ ID NO: 1) used in the present invention.

Figure 2 is a comparison of the N. plumbaginifolia gene sequence (M34371) encoding the extensin message peptide with the altered gene sequence shown in SEQ ID NO: 4 used in the present invention.

Fig. 3 is a schematic view showing an example of a structure of a gene construct used for producing a recombinant TMV into which an EGF gene has been introduced.

Fig. 4 is a configuration diagram of a TMV expression carrier UB001 used in the embodiment.

Figure 5 is a diagram showing the EGF extracted and purified by recombinant TMV infected tobacco leaves. The results of silver staining were performed after SDS-PAGE. Band 1: molecular marker, with 2: standard, with 3: C endoplasmic reticulum message KDEL (5 times concentrated), with 4: no C endoplasmic reticulum message (5 times concentrated), with 5: C end Quality network message, with 6: no C-end endoplasmic reticulum message, with 7: molecular markers.

Figure 6 is a graph showing the results of bioactivity assay of EGF extracted and purified from recombinant TMV-infected tobacco leaves. A. The results of the proliferation promoting effect of human fibroblast PDL3 were measured using samples having different EGF concentrations. B. A graph showing the results shown in A as EGF concentrations.

Fig. 7 is a graph showing the results of measuring the biological activity (proliferation promoting action) of EGF extracted and purified by recombinant TMV-infected tobacco leaves. A. Human fibroblast PDL3. B. BALB/3T3 from mouse embryos.

Fig. 8 shows the results of measurement of biological activity (proliferation promoting action) of a commercially available human EGF protein standard. A. Human fibroblast PDL3. B. BALB/3T3 from mouse embryos.

The nucleotide sequence shown in SEQ ID NO: 1 can be optimized by using a transient expression system to express the mature human EGF formed by the 53 residue in a plant cell, and the codon is optimized to encode an amine. The sequence of the EGF protein molecule having the same base acid sequence as the native mature human EGF (SEQ ID NO: 3). The human EGF gene is registered in the NCBI database Gene 1950, and its sequence is, for example, registered in GenBank under accession No. NM_001963. SEQ ID NO: 2 shows the nucleotide sequence of a region encoding a mature EGF in the natural human EGF gene. If the natural base sequence is compared with the optimized modified sequence shown in SEQ ID NO: 1, as shown in FIG. 1, the base sequence of SEQ ID NO: 1 contains a change in amino acid-free acid. Complex base Replacement.

The nucleotide sequence shown in SEQ ID NO: 4 can be suitably used because it expresses mature human EGF efficiently in a plant cell using a transient expression system, and is a sequence encoding a message peptide for intracellular delivery. In the nucleotide sequence shown in SEQ ID NO: 4, a message peptide consisting of the same amino acid sequence as the extensin message peptide (SEQ ID NO: 6) possessed by Tobacco N. plumbaginifolia is used, in order to use a transient expression system. The EGF is produced to optimize the codon. The extensin gene sequence of N. plumbaginifolia is registered in GenBank according to accession No. M34371. The signal peptide coding region seen in this registration sequence is shown in SEQ ID NO: 5. If the natural base sequence is compared with the optimized altered sequence shown in SEQ ID NO: 4, as shown in FIG. 2, the base sequence of SEQ ID NO: 4 contains an unrelated amino acid. Altered base substitutions.

The nucleotide sequence shown in SEQ ID NO: 7 can be suitably used because it expresses mature human EGF efficiently in a plant cell using a transient expression system, and is a sequence encoding a message peptide for local presence in cells. Specifically, in the nucleotide sequence shown in SEQ ID NO: 7, the endoplasmic reticulum retention message KDEL (SEQ ID NO: 8) is encoded. The base sequence shown in SEQ ID NO: 7 is also optimized for use in conjunction with the above-described optimized human EGF optimized alteration sequence.

It is known that 18 amino acid groups other than Met and Trp are encoded as 2 to 6 synonymous codons, but the frequency of synonymous codon usage varies depending on the biological species (Grantham, R. (1980) Trends Biochem. Sci. 5, 327 -331.; Grantham, R., Gautier, C. and Gouy, M. (t980) Nucl. Acids Res. 8, 1893-1912.; Grantham, R., Gautier, C., Gouy, M., Mercier, R. and Pave, A. (1980) Nucl. Acids Res. 8, r49-r62.; Grantham, R., Gautier, C., Gouyt, M., Jacobzone, M. and Mercier, R. (1981) Nucd .Acids Res.9, r43-r74.; Aota, S.-I., Gojobori, T., Ishibashi, F., Maruvama, T. and Ikemura, T. (1988) Nucl. Acids Res. 16, r315-r402.). Murray et al. also reported the frequency of codon usage in plants (Murray et al. (1989) Nucl. Acids Res. 17, 477-498), with reference to this report, which can be used to enhance the desired amines introduced into plant cells. The manner in which the codons of the base sequence of the acid sequence are expressed in plant cells is altered.

Among the synonymous codons of the 18 amino acids, specific examples of the codons frequently used in plants (especially dicotyledons) include those shown in Table 1. These codons can be preferably used for codons of plants.

The nucleic acid construct of the present invention is suitable for use in a nucleic acid construct that transiently expresses mature human EGF in a plant cell, which encodes a mature human EGF The base sequence of the amino acid sequence. However, the nucleic acid construct can produce EGF in plants even when it is expressed by the Agrobacterium tumefaciens recombination into the plant genome, so the use of the nucleic acid construct of the present invention is not limited to transientity. which performed.

The nucleotide sequence shown in the above SEQ ID NO: 1 is a nucleotide sequence encoding the amino acid sequence of the mature human EGF, and is the most suitable base sequence in the present invention.

Further, not only the nucleotide sequence identical to the SEQ ID NO: 1, but also a sequence in which a few bases are different, in particular, a sequence in which a codon having a high frequency of use in a plant as exemplified in Table 1 above is used. , can be used as well as the base sequence of SEQ ID NO: 1. Here, "a few bases are different" may be, for example, 1 to 15 bases, 1 to 12, 1 to several, 1 to 5, 1 to 3, and 1 to 2 bases. Or one replacement. In the case of expressing the sequence identity (%) according to SEQ ID NO: 1, the base sequence can be changed in the same manner as in SEQ ID NO: 1, and the identity with SEQ ID NO: 1 is 90% or more, 92% or more, 95% or more. , or 98% or more.

Here, the "identity" of the sequence number is such that the two sequences are aligned as many as possible so that the bases of the two base sequences to be compared are as many as possible, and the total number of bases is divided by the number of identical bases. The value obtained is expressed as a percentage. In the case of the above pairing, a gap may be appropriately inserted in one or both of the two sequences to be compared as needed. The alignment of such sequences can be carried out using well-known procedures such as BLAST, FASTA, CLUSTAL W. In the case of inserting a gap, the total number of bases described above is calculated by using one gap as one base. The total number of bases thus calculated is different between the two sequences to be compared, and the identity (%) is calculated by dividing the total number of bases of the longer sequence by the number of identical bases. However, when the sequence to be compared exhibits a state of being linked to another arbitrary sequence (for example, recombination to the state of the expression vector), only the corresponding region is taken out and the sequence is aligned, and the identity is calculated. example For example, when the base sequence encoding the mature human EGF is compared, only the region corresponding to the region encoding the mature human EGF is taken out and the sequence is aligned.

The nucleic acid construct of the present invention may further comprise a base sequence encoding a message peptide which can be used for intracellular delivery or localization of an EGF protein molecule produced in a plant somatic cell. The message peptide is usually a peptide formed by the number of amino acids of about 3 to 60, and is a structure indicating the transport and localization of proteins synthesized in the cytoplasm.

As the message peptide which can be utilized for intracellular delivery, for example, a message peptide of extensin can be mentioned. The base sequence encoding the extensin message peptide may be contained on the upstream side of the base sequence encoding EGF.

The nucleotide sequence shown in the above SEQ ID NO: 4 is a base sequence encoding the extensin message peptide, and is the most suitable base sequence in the present invention.

In addition, it is not only the sequence identical to SEQ ID NO: 4, but is a few bases different (for example, one to several, one to five, one to three, one to two, or one base). The sequence of the basal substitution, especially the sequence using a codon having a high frequency of use in plants as exemplified in Table 1 above, can be suitably used similarly to the nucleotide sequence of SEQ ID NO: 4. In the case of sequence identity (%), the base sequence can be changed in the same manner as in SEQ ID NO: 4, and the identity with SEQ ID NO: 4 is 90% or more, 92% or more, 95% or more, or 98% or more. .

As the message peptide which can be utilized for localization, for example, an endoplasmic reticulum retention message formed by a sequence of KDEL (SEQ ID NO: 8) can be mentioned. Since the endoplasmic reticulum retention message KDEL is used for addition to the C-terminal side of the EGF protein, the base sequence encoding the endoplasmic reticulum retention message can be included in the downstream side of the base sequence encoding EGF.

The nucleotide sequence shown in SEQ ID NO: 7 is encoded as endoplasmic reticulum The base sequence of the message is the most suitable base sequence for use in the present invention.

In addition, not only the sequence identical to SEQ ID NO: 7, but a sequence in which a few bases are different (for example, 1 to 2 or 1 base is substituted), in particular, most of them are exemplified as shown in Table 1 above. A sequence using a frequently used codon in a plant can be used as well as the nucleotide sequence of SEQ ID NO: 7. When the sequence identity (%) is expressed, the base sequence can be changed in the same manner as in SEQ ID NO: 7, and the identity with SEQ ID NO: 7 is 80% or more, or 90% or more.

The nucleotide sequence shown in SEQ ID NO: 11 is ligated to the upstream side of the nucleotide sequence (SEQ ID NO: 1) optimized for the codon encoding the mature EGF sequence, and the codon encoding the stretch protein peptide sequence is ligated. The optimized base sequence (SEQ ID NO: 4) is ligated to the downstream side of the base sequence (SEQ ID NO: 7) encoding the codon for the endoplasmic reticulum retention message KDEL. This base sequence is a particularly excellent example of the base sequence contained in the nucleic acid construct of the present invention. Foreign proteins that incorporate the extensin message peptide and endoplasmic reticulum retention information, if expressed in plant cells, are thought to accumulate in the cytoplasm of the cell, especially the endoplasmic reticulum.

Not only the sequence identical to the serial number 11, but for example, 1 to 25, 1 to 20, 1 to 12, 1 to several, 1 to 5, 1 to 3, and 1 to 2 Or a sequence having a different base, particularly a sequence using a codon having a high frequency of use in a plant as exemplified in Table 1 above, can be suitably used similarly to the nucleotide sequence of SEQ ID NO: 11. When the sequence identity (%) is expressed, the base sequence can be changed in the same manner as in SEQ ID NO: 11, and the identity with SEQ ID NO: 11 is 90% or more, 92% or more, 95% or more, or 98% or more.

The nucleic acid construct may be DNA or RNA, or may be a nucleic acid derivative such as PNA (Peptide Nucleic Acid) or LNA (Locked Nucleic Acid), but is typically DNA or RNA. A nucleic acid construct for constructing a viral expression vector, typically a DNA construct. Further, in the case of the "recombinant virus containing a nucleic acid construct", since the virus contains the nucleic acid construct in the genome, if the virus is an RNA virus, the nucleic acid construct is RNA, and if the virus is a DNA virus, The nucleic acid construct is then DNA.

The nucleic acid construct containing the above-described altered base sequence can be synthesized by chemical synthesis by a conventional method. Alternatively, a cDNA having a naturally occurring sequence can be synthesized by a well-known genetic engineering method, and the cDNA can be used as a template, and a suitable primer can be used to appropriately introduce a mutation. Alternatively, the nucleic acid construct to be synthesized is divided into a plurality of short regions and chemically synthesized into a plurality of DNA fragments, and the DNA constructs are synthesized by a known fusion PCR method. RNA constructs can be modulated by a transcription reaction from a DNA construct.

The term "transient expression" refers to a state in which a foreign gene is expressed in a host cell without being recombined into the genome of the host cell. Such transient performance can be carried out using a viral vector. A plant viral vector for expressing a desired foreign gene in a plant somatic cell is known as various substances (for example, refer to "protein nucleic acid enzyme", vol. 45, p. 607-613, etc.), and a commercial product is also present. Various things. Among the plant viral vectors, the most actively studied and widely used is the tobacco mosaic virus (TMV) vector, and the TMV vector can also be suitably used in the present invention.

When the nucleic acid construct of the present invention is transiently expressed in a plant using a viral vector, the nucleic acid construct of the present invention containing a region encoding human EGF is synthesized by a transcription reaction to synthesize a recombinant viral RNA, and an appropriate RNA is required. A synthetase promoter (for example, a phage synthetase promoter such as a T7 promoter), a viral replicase cDNA, a mobile protein cDNA, and a coat protein cDNA are ligated and used as a viral expression vector in the form of recombinant plastid DNA. If it is a tobacco mosaic virus vector such as TMV In the case, the nucleic acid construct is ligated to the viral factor cDNA in the order of [RNA synthetase promoter]-[replicase cDNA]-[mobile protein cDNA]-[nucleic acid construct of the present invention]-[capsid protein cDNA] Yes (Figure 3). A transcription reaction is carried out using a TMV expression vector containing such a structure as a template, whereby a recombinant TMV RNA which recombines a nucleic acid construct encoding a mature human EGF protein can be prepared.

Specific examples of the transient expression system according to TMV which can be suitably used in the present invention include BSG1037 and BSG1057 described in Japanese Patent No. 4750285, and GENEWARE (registered trademark) of Kentucky BioProcessing Co., Ltd., which is commercially available. This is a system that expresses the systemic infectious recombinant TMV plastid, including the RNA synthetase promoter, TMV replication enzyme (RNA-dependent RNA synthetase) cDNA, mobile protein cDNA, and coat protein cDNA. The desired foreign gene is inserted between the mobile protein cDNA and the coat protein cDNA, and is synthesized as a template to the RNA having the end cap at the 5' end, thereby obtaining a whole body which can express the desired foreign gene in the plant cell. Infectious TMV viral RNA. The synthesis of RNA by end cap addition can be easily carried out using a commercially available kit (for example, Life Technologies' mMESSAGE mMACHINE (registered trademark) kit, etc.).

Further, in the present invention, the "replicase cDNA", the "mobile protein", and the "shell protein" are included in the case of having the same sequence as those of the natural plant virus. Optimized for sexual performance, with a sequence including human changes. For example, in the TMV expression vector BSG1057 described in Japanese Patent No. 4750285, the replication of the replicase cDNA and the mobile protein cDNA are changed to produce a replicating enzyme and a mobile protein having a sequence slightly different from the native TMV, and such a sequence changer is also It is included in the scope of the invention.

Recombination of the whole body of the nucleic acid encoding the mature human EGF protein The stained recombinant TMV RNA can be inoculated directly into the plant according to the state of the naked virus genome, or can be mixed with the purified coat protein to form a shell, which is made into a virion state and then inoculated into the plant. The term "recombinant virus" includes both the naked virus genome and the virions that form the shell. The inoculation of the virus is carried out by mechanically injuring the plant leaves with fine particles such as carbon powder or diatomaceous earth, and bringing them into contact with the inoculum.

In plants infected with recombinant virus, the virus proliferates and produces a large amount of EGF in plant cells. After 10 to 15 days of cultivation after virus inoculation, the leaves are harvested and the EGF protein molecules are extracted and recovered. Extraction and purification of EGF protein molecules by plant tissues infected with recombinant virus can basically use general protein purification methods (CD Mount et al., Archives of Biochemistry and Biophysics, 240 33 (1985), UHGregory and IRWillshire, Hoppe-Sayler's Z. Physiol. Chem., 356 1765 (1975)). Specifically, it can be carried out as follows.

The infected plant tissue is added to a buffer containing a proteolytic enzyme inhibitor, homogenized by a mixer, and the protein is extracted. After centrifuging the solution, the supernatant is recovered, and the impure protein in the supernatant is removed by ultrafiltration, and further concentrated by ultrafiltration through a membrane to obtain a crude product. The ultrafiltration filter can be carried out, for example, by filtration through a filter such as Miracloth (CALBIOCHEM) or glass fiber filter paper, and the filtrate is subjected to cross-flow filtration by a film having a molecular weight of about 10 to 30 kDa.

The obtained crude product was further purified one time or more using a hydrophobic interaction chromatography carrier. The carrier is not particularly limited, and may be a known carrier generally used for hydrophobic interaction chromatography such as a gel or a resin. As the hydrophobic interaction chromatography carrier, a group having a phenyl group as a functional group such as a phenyl sepharose (Sepharose) carrier is suitably used. Adding a final concentration of 1 to 3 M ammonium sulfate to the crude product to increase the salt concentration, and then The hydrophobic interaction chromatography carrier is contacted to adsorb the target protein to the carrier. Next, the adsorbed protein is eluted by a buffer such as sodium phosphate or the like having a concentration of about mM (the pH is used in the vicinity of neutral).

Further, if necessary, the eluate is adsorbed by a strong anion exchange chromatography carrier, and is adjusted to be alkaline (pH 9 to 11) sodium chloride-containing glycine buffer to be eluted, thereby recovering, An EGF protein that is refined in plants. The obtained EGF protein may be subjected to further treatment such as desalting treatment as needed.

The refined EGF can be characterized by a well-known chemical analysis method by mass analysis. Further, the EGF purity can also be determined by a known method such as HPLC analysis (L.M. Matrisian et al., Analytical Biochemistry, 125, 339 (1982)). Furthermore, the ability to promote cell proliferation, such as one of the important physiological activities of EGF, can be performed by a well-known method (G. Carpenter and J. Zendegui, Analytical Biochemistry, 153, 279 (1985)), and binding ability to EGF receptor. Determination (RLLadda, et al, Analytical Biochemistry, 93, 286 (1979)).

The human EGF produced by the method of the present invention has good physiological activity and can be used for wound healing drugs, anti-ulcer agents, cosmetics, and the like as well as conventional materials.

The present invention also provides a polynucleotide which enhances the efficiency of expression in plant cells and encodes mature human EGF. The polynucleotide has 90% or more, preferably 92% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1. Alternatively, the nucleotide sequence of the polynucleotide may be the nucleotide sequence of SEQ ID NO: 1, or may be 1 to 15, preferably 1 to 12, more preferably 1 to several in SEQ ID NO: 1. Further preferably, one to five, more preferably one to three, particularly preferably one to two, and even more preferably one base substituted base sequence. The base sequence of the polynucleotide is preferably the nucleotide sequence shown in SEQ ID NO: 1.

The present invention also provides a polynucleotide which enhances the efficiency of expression in a plant cell and encodes a tobacco extensin message peptide. The polynucleotide has 90% or more, preferably 92% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4. Alternatively, the nucleotide sequence of the polynucleotide may be the nucleotide sequence shown in SEQ ID NO: 4, or may be one to several, more preferably one to five, and even more preferably one to three in SEQ ID NO: One or more, preferably more than one base substituted base sequence. The base sequence of the polynucleotide is preferably the nucleotide sequence shown in SEQ ID NO: 4.

The present invention also provides a polynucleotide which enhances the efficiency of expression in plant cells and encodes an endoplasmic reticulum retention message. The polynucleotide has 80% or more, preferably 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 7. Alternatively, the nucleotide sequence of the polynucleotide may be the nucleotide sequence shown in SEQ ID NO: 7, or may be a base sequence in which 1 to 2 and more preferably 1 base in SEQ ID NO: 7. The base sequence of the polynucleotide is preferably the nucleotide sequence shown in SEQ ID NO: 7.

Any of the above-described polynucleotides which enhance the efficiency of expression in plant cells may be DNA or RNA, or may be a nucleic acid derivative such as PNA (Peptide Nucleic Acid) or LNA (Locked Nucleic Acid). It is not particularly limited, but is typically DNA.

The virus in which the above-described polynucleotide encoding mature human EGF or the like is recombined is as described above and can be prepared by a usual method. The recombinant virus is preferably a plant virus, such as a recombinant tobacco mosaic virus. The virus comprising the polynucleotide encoding the mature human EGF, as described above, preferably further comprises the polynucleotide encoding the tobacco extensin message peptide and the polynucleotide encoding the endoplasmic reticulum retention message.

(Example)

Hereinafter, the present invention will be more specifically described based on examples. However, the invention is not limited to the following examples.

1. Modulation of recombinant TMV expression vector

Containing DNA encoding the tobacco extensin message peptide, the 53-residue mature human EGF, and the endoplasmic reticulum retention message KDEL (SEQ ID NO: 10), and the DNA formed by the altered base sequence The embedded DNA (SEQ ID NO: 9) was commissioned by GeneArt (now Life Technologies) and produced by chemical synthesis.

On the other hand, the plastid UB001 (Fig. 4) produced by the well-known plastid BSG1057 (Japanese Patent No. 4750285) which can express the recombinant tobacco mosaic virus will be changed, and cut and ligated by PacI and XhoI, and agarose electrophoresed by agar The glue is extracted and refined.

The above-described embedded DNA was ligated to the linearized UB001 by T4 DNA ligase (Invitrogen) (37 ° C, 20 minutes). The obtained plastid DNA was introduced into Escherichia coli NEB5-α (New England BioLabs) by heat shock, and single colony proliferation was carried out by screening with ampicillin-containing medium, whereby the coliform cells were subjected to plastid DNA using a mini prep kit (Qiagen). The recombinant TMV-expressing plastids in which DNA is inserted between the MP region and the CP region of the tobacco mosaic virus of UB001 are obtained by extraction and purification. The plastids were cut by PacI and XhoI to confirm the embedded size.

2. Modulation of recombinant TMV particles

The transcription reaction was carried out using the Ambion mMessage mMachine kit (Life Technologies) using the recombinant TMV expressing plastid DNA obtained above as a template. The composition of the reaction liquid was set as follows.

The reaction solution was incubated at 37 ° C for 1 to 3 hours to prepare recombinant TMV RNA which can express the mature human EGF protein which is fused with the extensin message peptide and the endoplasmic reticulum retention message. 0.5 μL of the reaction mixture was subjected to electrophoresis on 1% agarose gel to confirm the transcription product.

19.5 μL of the reaction mixture was mixed with 1.58 mL of nuclease-free water, 0.2 mL of 1 M sodium phosphate pH 7.0, and 0.2 mL of purified TMV coat protein (10 mg/mL, purified by TMV-infected tobacco leaves). The shell formation reaction was carried out by culturing at room temperature for 2 hours to one night. Thereby, a recombinant TMV particle which can express the protein (SEQ ID NO: 10) in which the mature human EGF fuses the extensin message peptide and the endoplasmic reticulum retention message is obtained.

3. Inoculation of the original modulation

The inoculum was prepared by using the reaction solution after the reaction was formed in the shell. The buffer for modulating the vaccination (GENEWARE (registered trademark) Inoculation Buffer (GIB)) was prepared as follows.

250 mL of 18.2 MΩ water, 2.25 g of glycine, 3.14 g of K ₂ HPO ₄ , and 3.00 g of Na ₄ P ₂ O ₇ were placed in a beaker. 10H ₂ O, stirred with a stirrer for 15 minutes or more. The dissolved solution was transferred to a measuring cylinder of 500 mL capacity and adjusted to 300 mL with 18.2 MΩ water. This was transferred to a vessel which can be autoclaved, and 3.00 g of bentonite and 3.00 g of diatomaceous earth were added, and the bentonite and the diatomaceous earth were sufficiently wetted by rotary stirring, and then autoclaved (121 ° C, 20). minute). After cooling, it was stored at 4 ° C (steady at 4 ° C for 1 year).

Forming the shell into a stock solution of the reaction solution or appropriately diluted with nuclease-free water The liquid is mixed with an equal amount of GIB and used as an inoculum. To 2 leaves of each leaf of Nicotiana benthamiana, 30 μL of the inoculum was dropped on each leaf, and the leaves were wounded with a gloved finger to infect the leaves, thereby infecting the recombinant TMV to tobacco.

4. Recycling from tobacco leaf EGF (1)

After 12 days of inoculation, tobacco leaves were harvested. The 500 g of the harvested leaves were placed on ice for about 45 minutes before extraction. The extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 0.1% Triton X-100) was stored at 4 ° C until the extraction operation. 500 mL of the extraction buffer was added to 500 g of the leaves, and the extract was crushed and extracted by a juicer, placed in a glass bottle and stored in ice liquid. This solution was centrifuged at 10,000 g for 10 minutes as a filter material thereafter. After centrifugation, the supernatant was filtered through Miracloth and 30 kDa PES Kvick flow UF (0.1 m ² ) to remove most of the protein in the filtrate to concentrate EGF (30 KDa filtrate). Further, this 30 KDa filtrate was further concentrated to ² mL of Hydrosart Sartorius® (0.1 m ² ) to 90 mL (2 KDa filtrate). Further, each filtrate residue was washed with 7 times the extraction buffer (1,600 mL), and the washing liquid (30 KDa residue and 2 KDa residue) was collected.

For the samples of tobacco leaf breaking liquid, centrifugal supernatant, 30KDa filtrate, 30KDa residue, 2KDa filtrate and 2KDa residue, the EGF test was carried out by Western blotting method. As a result, in addition to the crushed liquid and the centrifugation supernatant, about 5.5 kDa of EGF was detected in the 30 kDa filtrate sample and the 2 kDa filter residue sample. EGF produced by recombinant TMV in tobacco leaf cells was translated according to the state of the addition of the message peptide, but since the message peptide was detached by the enzyme reaction in the plant cell, the result was determined to be the above size. .

5. Recycling from tobacco leaf EGF (2)

After 12 days of inoculation, tobacco leaves were harvested. The harvested 1500 g of leaves were tied to ice for about 45 minutes before extraction. The extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 0.1% Triton X-100) was stored at 4 ° C until the extraction operation. 1500 mL of the extraction buffer was added to 1500 g of the leaves, and the extract was crushed and extracted by a juicer, and placed in a glass bottle and stored in ice liquid. This solution was centrifuged at 10,000 g for 10 minutes as a filter material thereafter. After centrifugation, the supernatant was filtered through Miracloth and 30 kDa PES Kvick flow UF (0.1 m ² ) to remove most of the protein in the filtrate to concentrate EGF. To the 30 KDa filtrate, ammonium sulfate was added to 1.5 M to be adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). Further, ammonium sulfate was added to the 30 KDa filtrate to 2.5 M to be adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). This solution was passed through a NuviaQ column to adsorb EGF, and eluted by adding 500 mM sodium chloride in glycine buffer (pH 10.0). Using this as a sample, SDS-PAGE was implemented. Purification of silver staining and determination of biological activity.

The measurement of biological activity was carried out as follows. Human fibroblast PDL3 was placed in 5000 wells in each well of a 96-well microplate. The sample was diluted from 20 pg/mL to 2000 pg/mL in 11 stages, and 0.5 mL of each diluted sample was added to each column (6 holes) of the tray, and a total of 11 rows of wells. A column of 0.5 mL of the culture solution for PDL3 culture was separately added as a negative control group. The microplate was cultured for 4 days in a carbon dioxide gas cell culture incubator. After the culture, the culture solution was removed, and 1 mL of the tetrazolium-based germ cell staining solution was added for staining for 3 hours, and the absorbance at 490 nm was measured using a microplate analyzer.

Further, biological activity was measured in the same manner as BALB/3T3 from BALB/c mouse embryos except PDL3. As a reference data, the biological activity was measured in the same manner as the above recombinant protein sample, using a commercially available human EGF protein standard (Promega).

The silver staining results are shown in Fig. 5. It was confirmed that the EGF in the purified tobacco leaves can be recovered by high purity by the above method.

The bioactivity measurement results of EGF are shown in Fig. 6 and Fig. 7. Both PDL3 cells (Fig. 6, Fig. 7A) and BALB/3T3 cells (Fig. 7B) were combined with the added concentration of EGF purified from tobacco leaves to promote cell proliferation, which was not inferior to the commercially available human EGF protein (Fig. Results of 8A, B). Thereby, it was confirmed that EGF purified and purified from tobacco leaves by the above method has biological activity.

<110> Youyou Biological Co., Ltd.

<120> Manufacturing method of human EGF

<130> PF495-PCT

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<170> PatentIn version 3.1

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<212> DNA

<213> Artificial sequence

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<223> Modified mature human EGF sequence

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<213> Homo sapiens

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<223> Altered tobacco extensin message peptide sequence

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<213> Tobacco (Nicotiana plumbaginifolia)

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<213> Tobacco (Nicotiana plumbaginifolia)

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<223> Changed endoplasmic reticulum message sequence

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<223> Insertion sequence, SP-EGF-KDEL

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Claims (35)

A nucleic acid construct comprising a base sequence encoding mature human EGF for expressing mature human EGF in a plant cell, and the above-described base sequence encoding the mature human EGF has the sequence number 1 The base sequence is a base sequence having 90% or more identity. The nucleic acid construct according to the first aspect of the invention, wherein the base sequence encoding the mature human EGF has a base sequence which is 95% or more identical to the nucleotide sequence shown in SEQ ID NO: 1. The nucleic acid construct of claim 1, wherein the base sequence encoding the mature human EGF is SEQ ID NO: 1 or the SEQ ID NO: 1 is substituted by 1 to 15 bases. Base sequence. The nucleic acid construct of claim 1, wherein the base sequence of the mature human EGF is encoded by the nucleotide sequence shown in SEQ ID NO: 1. The nucleic acid construct according to any one of claims 1 to 4, further comprising a base sequence encoding a message peptide for intracellular delivery upstream of the above-mentioned base sequence encoding the mature human EGF . The nucleic acid construct according to claim 5, wherein the base sequence encoding the message peptide has a base sequence which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 4. The nucleic acid construct of claim 5, wherein the base sequence encoding the message peptide has a base sequence which is 95% or more identical to the nucleotide sequence shown in SEQ ID NO: 4. The nucleic acid construct of claim 5, wherein the base sequence encoding the message peptide is the nucleotide sequence shown in SEQ ID NO: 4, or one of the sequence numbers 4 is substituted. A base sequence of several bases. The nucleic acid construct of claim 5, wherein the base sequence encoding the message peptide is the nucleotide sequence shown in SEQ ID NO: 4. The nucleic acid construct according to any one of claims 1 to 9, which further comprises a base sequence encoding an endoplasmic reticulum retention message. The nucleic acid construct according to claim 10, wherein the nucleotide sequence encoding the endoplasmic reticulum retention information has a base sequence which is 80% or more identical to the nucleotide sequence shown in SEQ ID NO: 7. The nucleic acid construct of claim 10, wherein the base sequence encoding the endoplasmic reticulum retention message is the nucleotide sequence shown in SEQ ID NO: 7, or 1 to 2 bases are substituted in SEQ ID NO: 7. Base sequence. The nucleic acid construct of claim 10, wherein the base sequence encoding the endoplasmic reticulum retention message is the nucleotide sequence shown in SEQ ID NO: 7. The nucleic acid construct of any one of claims 1 to 13 which is a DNA construct. The nucleic acid construct of claim 14, which is linked to an RNA synthetase promoter, a viral replicase cDNA, a viral mobile protein cDNA, and a viral coat protein cDNA. The nucleic acid construct of claim 15, wherein the viral replicase, mobile protein and coat protein are derived from a tobacco mosaic virus. A performance vector comprising the nucleic acid construct of any one of claims 1 to 16. For example, the expression vector of claim 17 is a viral expression vector. A recombinant virus comprising the nucleic acid construction of any one of claims 1 to 13 Structure. A recombinant virus as claimed in claim 19, which is a recombinant virus prepared by the expression vector of claim 18 of the patent application. A method for producing human EGF, which comprises infecting a recombinant virus of claim 19 or 20 into a plant body, and recovering the mature human EGF protein molecule produced by the virus after the virus is proliferated. A method for producing human EGF, which comprises infecting a recombinant virus prepared by the expression vector of claim 18 of the patent application into a plant body, and recovering the mature human EGF protein molecule produced by the virus after the virus is proliferated. For example, the method of claim 21 or 22 uses tobacco as a plant body. A polynucleotide encoding a mature human EGF having a base sequence having 90% or more identity with a nucleotide sequence represented by SEQ ID NO: 1. The polynucleotide of claim 24, wherein the base sequence of the polynucleotide is the nucleotide sequence of SEQ ID NO: 1. A polynucleotide in which a mature human EGF is encoded, the base sequence of the polynucleotide is a nucleotide sequence represented by SEQ ID NO: 1, or a base substituted by 1 to 15 bases in SEQ ID NO: 1. Base sequence. A polynucleotide encoding a tobacco extensin message peptide, the base sequence of which has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 4. The polynucleotide of claim 27, wherein the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 4. A polynucleotide in which a tobacco extensin message peptide is encoded, the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 4, or 1 to several bases are substituted in SEQ ID NO: 4. Base sequence. A polynucleotide which encodes an endoplasmic reticulum retention message, the base sequence of which has an identity of 80% or more with the nucleotide sequence shown in SEQ ID NO: 7. The polynucleotide of claim 30, wherein the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 7. A polynucleotide in which the endoplasmic reticulum retention message is encoded, the base sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO: 7, or 1 to 2 bases are substituted in SEQ ID NO: 7. Base sequence. A recombinant virus comprising the polynucleotide of any one of claims 24 to 26. A recombinant virus according to claim 33, which is a plant virus. The recombinant virus according to claim 33 or 34, which further comprises the polynucleotide of any one of claims 27 to 29, and the polynucleotide of any one of claims 30 to 32 .