JP2007228814A - Gene cassette for expressing foreign protein and method for producing foreign protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a genetic engineering material for insects, which does not need use of a recombinant baculovirus and facilitates purification of objective protein and simultaneously to provide a method for producing a foreign protein by utilizing the genetic engineering material. <P>SOLUTION: A gene cassette for expression obtained by fusing a DNA sequence at the 5' terminal part of sericin gene and a DNA sequence at the 3' terminal part with a foreign protein gene is transferred to a silk gland cells or the like so as to produce a large amount of foreign protein in silk gland cells, in silk glands, especially in middle silk glands, further in a sericin layer of silk, By the new method, the foreign protein production technique is established by producing a foreign protein using silk glands without using a recombinant baculovirus. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene introduction vector for insect cells, insect cells transfected with the vectors, insect tissues, and methods for producing foreign proteins using insects. Furthermore, the present invention relates to a silk thread containing a foreign protein produced by the recombinant silkworm obtained in the present invention.

  Production of foreign proteins using gene recombination techniques is used in various industries. As the host, Escherichia coli, yeast, animal cells, plant cells, insect cells and the like are mainly used. However, no host capable of efficiently producing any foreign protein has been developed, and it is necessary to construct a production system for each target protein, and further technological innovation is desired in the foreign protein production technology for each host. ing.

  In bacterial or yeast systems such as E. coli, there are problems with post-translational modifications, and proteins may not be synthesized in a fully functional form. In addition, animal cells can often synthesize proteins in a functional form, but are generally difficult to grow and low in production and are not economical.

  On the other hand, in the production of genetically engineered proteins using insects or insect cells, it is known that useful proteins with enzymes and physiological activities can be mass-produced relatively inexpensively and post-translational modification of proteins close to animals can be obtained. Yes. Specifically, a method for infecting insects or insect cells with a baculovirus incorporating a foreign protein gene enables mass production of the foreign protein at a relatively low cost, and bioactive proteins that have been commercialized as pharmaceuticals are also known. (See Patent Documents 1 and 2).

  However, the conventional technology for producing recombinant proteins using insects or insect cells uses recombinant viruses for the introduction of foreign genes, so that inactivation and containment are necessary for safety reasons. There is. In addition, in the method of inoculating the silkworm with the recombinant virus, the inoculation of the recombinant virus is complicated and the target foreign protein is produced in the body fluid, so that the target recombinant protein is contaminated in large quantities derived from the silkworm body fluid. It was necessary to purify from the protein. For this reason, there existed a subject that it was difficult to obtain a highly purified recombinant protein.

  On the other hand, in recent years, recombination of foreign genes into insect chromosomes has been attempted, and using the DNA of Autographa californica nuclear polyhedrosis virus (AcNPV), a jellyfish green fluorescent protein for silkworm fibroin L chain gene. A method for introducing and expressing a fusion gene to which a gene has been added on a silkworm chromosome by homologous recombination has been developed (see Non-Patent Document 1), and a silkworm introduced with a human collagen gene using the technique and a method for producing the same Is disclosed (see Patent Document 3). However, in the method for introducing a foreign protein gene into an insect chromosome using AcNPV as described above, since a recombinant baculovirus (AcNPV) is used, there remain problems of inactivation and containment of the recombinant virus.

  Recently, a method of stably introducing a foreign gene into a silkworm chromosome using piggyBac, a transposon derived from a lepidopteran insect, and expressing a protein encoded by the foreign gene was studied using a jellyfish green fluorescent protein as a model, It was also confirmed that the gene was stably transmitted to the offspring by crossing (see Non-Patent Document 2). However, since the amount of green fluorescent protein produced is not sufficient and is produced throughout the silkworm, advanced purification techniques must be used to recover the expressed recombinant green fluorescent protein in high purity. There was an economic problem because it was necessary.

  In addition, by introducing a gene cassette for expression in which the DNA sequence of the 5 ′ end portion and the DNA sequence of the 3 ′ end portion of the fibroin H chain gene are fused to a foreign protein gene into silk gland cells, a large amount of foreign protein is converted into silk thread. Production was possible in glandular cells, outside the silk glandular cells, and even in the fibroin layer of silk (see Patent Document 4). However, since foreign protein is produced in the fibroin layer, it was necessary to recover the foreign protein after solubilizing the fibroin layer after removing the sericin layer covering the fibroin layer.

  Recently, a method has been reported in which a sericin layer is dissolved to prepare a peptide solution, or powdered sericin is prepared using the peptide solution (see Patent Documents 5 and 6). However, these are for the use of silk protein (mainly sericin), and are not intended for the recovery / utilization of the recombinant protein present in the silk thread.

That is, in the foreign protein production technology using such insects as a host, it is necessary to inactivate and contain recombinant baculovirus, or it is difficult to purify the target protein from the fibroin layer of silk. There are several problems such as low protein expression.
JP 61-9288 JP 61-9297 JP 2001-161214 A Japanese Patent Laid-Open No. 2004-254681 Japanese Patent Laid-Open No. 10-29909 JP 2001-39999 Jeans and Development 1999, Vol. 13, pages 511-516 Nature Biotechnology 2000, Vol. 18, p81-84

  Recombinant protein production technology using insects has been energetically studied, but it is necessary to inactivate and contain recombinant baculoviruses that incorporate foreign protein genes. There are problems such as complexity. Further, in the production of foreign proteins in silkworms using recombinant baculoviruses, there has been a problem that it is difficult to extract and purify the target protein from body fluids containing a large amount of contaminants.

  The production of recombinant proteins that introduce foreign protein genes into the silkworm chromosome has been studied, but the production of the target foreign protein is low, and the target protein must be purified from the silkworm tissue or silk fibroin layer. There is also a problem that is difficult.

  In view of such circumstances, the present invention provides a genetic engineering material for insects that does not require the use of a recombinant baculovirus and can facilitate the purification of the target protein, and at the same time, utilizes the genetic engineering material. It is an object to provide a method for producing a foreign protein.

  As a result of intensive studies to produce a recombinant protein in a sericin layer that occupies 20 to 30% of the silk protein, the present inventors include a first intron of the sericin gene downstream of the promoter expressed in the silk gland 5 The amount of foreign protein produced in the sericin layer of silk by introducing a gene cassette in which the 5 'end of the foreign protein gene is linked to the 3' end of the 'terminal portion so that the amino acid frame is continuous. Found a dramatic improvement.

  That is, the present inventors introduce a gene cassette for expression in which a DNA sequence of the 5 ′ end portion of the sericin gene is fused to a foreign protein gene into a silk gland cell, etc. It was successfully produced in large quantities in the gland, especially the middle silk wire, and the sericin layer of silk. It was possible to establish a foreign protein production technology that facilitates purification by using a silk gland to produce foreign protein without using a recombinant baculovirus. In particular, when a foreign protein is produced in the silk sericin layer, sericin containing the foreign protein is obtained by solubilization with water, buffer solution, alkaline aqueous solution (preferably alkaline ionized water produced by electrolyzing water), etc. A solution can be easily obtained. From the sericin solution containing the foreign protein, the foreign protein can be easily recovered by using a normal purification technique with few other contaminating proteins.

  The present invention relates to genetic engineering materials that can be used for the production of foreign proteins in insects, such as the following gene cassettes and vectors, transformants, methods for producing foreign proteins using the transformants, and silk threads containing foreign proteins.

1) (1) a promoter expressed in the silk gland;
(2) A gene cassette for expression of a foreign protein, comprising a gene fused to the 5 ′ side of a foreign protein structural gene and linked to the downstream of (1) above.

  2) The gene cassette for expression of a foreign protein according to 1), wherein the 5 'end portion of the sericin gene contains a part of the first exon, the first intron, and the second exon of the sericin gene.

  3) The gene cassette for the expression of a foreign protein according to 2), wherein the portion of the sericin that combines the first exon and the second exon is a secretory signal gene region of the sericin gene.

  4) The gene cassette for expressing a foreign protein according to any one of 1) to 3) above, wherein (1) the silk gland promoter and / or (2) 5 ′ of the sericin gene is contained in the gene cassette for expressing the foreign protein. A gene cassette for the expression of a foreign protein characterized by having one or several base additions, deletions, or substitutions with other bases within a range where the terminal portions have substantially the same function.

  5) The gene cassette for expressing a foreign protein according to any one of 1) to 4) above, wherein (1) the silk gland promoter portion and / or (2) 5 of the sericin gene is contained in the gene cassette for expressing the foreign protein. 'A gene cassette for expression of a foreign protein characterized in that it has a gene that hybridizes with the part under stringent conditions instead of the terminal part and has a function substantially equivalent to that part.

  6) The promoter expressed in the silk gland is at least one promoter selected from a promoter of a sericin gene, a promoter of a fibroin L chain gene, and a promoter of a fibroin H chain gene 1) to 5) The gene cassette for expressing a foreign protein according to any one of the above.

  7) At least one poly A addition region selected from the poly A addition region of the sericin gene, the poly A addition region of the fibroin L chain gene, and the poly A addition region of the fibroin H chain gene downstream of the foreign protein expression gene cassette The gene cassette for expression of a foreign protein according to any one of 1) to 6), wherein

  8) An insect having a pair of piggyBac transposon inverted repeats on both sides of the foreign protein expression gene cassette according to any one of 1) to 6) above A gene cassette for gene transfer into the cell chromosome.

  9) An insect cell expression vector comprising the foreign protein expression gene cassette according to any one of 1) to 8) above.

  10) A gene introduction vector for insect cells comprising the gene cassette for gene introduction into the chromosome of insect cells described in 8) above.

  11) A recombinant silkworm in which the gene cassette for expressing a foreign protein according to any one of 1) to 8) is introduced into a chromosome and has the ability to produce a foreign protein in a silk gland or silk thread.

  12) A silk thread containing a foreign protein produced by the recombinant silkworm described in 11) above.

  13) A method for producing a foreign protein, which comprises introducing the gene transfer vector for insect cells according to 9) or 10) above into insect cells.

  14) The method for producing a foreign protein according to 12), wherein the insect cells are derived from lepidopterous insects.

  15) The method for producing a foreign protein according to 13), wherein the insect cells are derived from Bombyx mori.

  16) The method for producing a foreign protein according to 14), wherein the insect cell is a silk gland cell of Bombyx mori.

  17) Using the DNA transfer activity of the piggyBac transposase, a recombinant silkworm in which a gene cassette for expression of a foreign protein is incorporated into a chromosome is prepared, and the foreign protein is added to the silk gland or silk of the resulting recombinant silkworm. The method for producing a foreign protein according to 13), wherein the foreign protein is recovered from the silk gland or silk thread after the production of.

  18) A recombinant silkworm in which a gene cassette for expression of a foreign protein is integrated into a chromosome by microinjecting a gene transfer vector for insect cells and a DNA or RNA producing piggyBac transposase into a silkworm egg simultaneously. 17) The method for producing a foreign protein according to 17).

  19) A method for producing a foreign protein, characterized in that the useful protein is recovered from an aqueous solution in which the silk thread described in 12) is suspended in an aqueous solution and the sericin layer is dissolved.

  20) The method for producing a foreign protein according to 19), wherein the aqueous solution is at least one selected from water, a buffer solution, or an alkaline aqueous solution.

  21) The method for producing a foreign protein according to 20), wherein the alkaline aqueous solution is alkaline ionized water obtained by electrolyzing water and collecting an aqueous solution near the cathode.

  22) The method for producing a foreign protein according to any one of 19) to 21), wherein the aqueous solution described above contains a surfactant.

  23) The method for producing a foreign protein according to any one of 19) to 22), wherein the foreign protein is recovered from the aqueous solution in which the silk thread is dissolved by using column chromatography.

  24) The method for producing a foreign protein according to any one of 11) to 23), wherein the foreign protein is soluble in an aqueous solution.

  25) The method for producing a foreign protein according to any one of 11) to 24), wherein the foreign protein is either a cytokine or an antigen protein.

  26) The method for producing a foreign protein according to 25), wherein the cytokine is interferon and the antigenic protein is a viral antigenic protein.

  By introducing a gene cassette for expression in which the DNA sequence of the 5 'end of the sericin gene is fused to a foreign protein gene into a silk gland cell, a large amount of the foreign protein can be introduced into the silk gland cell, especially the silk gland, It has become possible to produce even a silk sericin layer. By this new method, we established a foreign protein production technology that can be easily purified by using the silk gland to produce foreign protein without using recombinant baculovirus.

  In the present invention, the “gene cassette for expressing a foreign protein” refers to a set of DNAs necessary for expressing a protein encoded by a foreign protein structural gene when introduced into an insect cell. The foreign protein expression cassette includes a foreign protein structural gene and a promoter that promotes the expression of the gene. Usually, it further contains a terminator and a poly A addition region, preferably a promoter, a foreign protein structural gene, a terminator, and a poly A addition region. Furthermore, a secretory signal gene linked to a foreign protein structural gene may be included between the promoter and the promoter. An arbitrary gene sequence may be linked to the poly A addition sequence. In addition, artificially designed and synthesized gene sequences can be linked.

  In addition, the “gene cassette for gene introduction” is a gene cassette for expression of a foreign protein having a pair of inverted piggyback transposon pairs on both sides, and piggyBac transposase. The DNA set introduced into the insect cell chromosome by the action of In the present invention, the term “insect cell expression vector” includes a gene cassette for expression of a foreign protein or a gene cassette for gene introduction, and a set of DNA necessary for expressing the foreign protein when introduced into an insect cell. Say. In the present invention, the “insect cell gene transfer vector” includes a gene cassette for expression of a foreign protein or a gene cassette for gene transfer, and has a pair of inverted piggyback transposon inverted sequences at both ends, and This refers to a DNA set that is introduced into insect cell chromosomes by the action of piggyback transposase.

  There is no particular limitation on the method for obtaining the DNA used in the present invention. A method to amplify and acquire necessary gene regions using PCR (polymerase chain reactiion) method based on known gene information, a method to screen homology from genomic libraries and cDNA libraries based on known gene information, etc. Is mentioned. In the present invention, these genes include genetic polymorphisms and mutants obtained by artificial mutation treatment using mutation agents. A genetic polymorphism is one in which the base sequence of a gene is partially changed due to a natural mutation on the gene.

  The promoter in the foreign protein expression cassette is not particularly limited, but a promoter having high activity for promoting the expression of the foreign protein gene is preferable. For example, the Drosophila heat shock protein gene promoter and the silkworm actin gene promoter (Nature Biotechnology 18, 81-84, 2000) described in JP-A-6-261770 and JP-A-62-285787 Examples include promoters of fibroin heavy chain genes (base numbers 255 to 574 of GenBank accession number V00094, base numbers 62118 to 62437 of GenBank accession number AF226688), promoters of fibroin light chain genes (Gene, 100: 151-158). : GenBank accession number M76430), promoters of sericin genes (base numbers 599 to 1656 of GenBank accession number AB007831) and other promoters having high transcription promoting activity in silkworm silk gland cells are suitable.

  The “foreign protein structural gene” is a gene that is not possessed by the host cell that is to express the gene, and is a gene that encodes a protein that is not originally produced by the host cell, and is not particularly limited. In view of industrial value, proteins produced by humans and mammals such as genes for growth hormones, cytokines, growth factors and cytoskeletal proteins can be mentioned. In addition, enzymes produced by microorganisms, plants or insects, genes of various proteins, and the like are also included in the scope of the present invention.

  In the gene cassette for foreign protein expression in the present invention, the 5 ′ end portion of the sericin gene is a DNA sequence having an action of enhancing the expression of the foreign protein by the promoter, and the first exon and the first intron of the sericin gene or the full length thereof. A DNA sequence comprising a portion and a portion of the second exon. In the present invention, it has been clarified that the 5'-terminal portion of the sericin gene promotes gene expression and transcription in the middle posterior silk gland in the recombinant individuals. It is well recognized that these regions that promote the transcriptional activity of genes retain their basic activity even if they are modified by various methods.

  Addition or deletion of one or two or more bases, whether artificially or naturally occurring, to the 5 ′ end of the sericin gene that promotes the transcriptional activity of the gene, regardless of whether it has substantially the same function Alternatively, DNA in which substitution with another base has occurred can also be used. "Addition or deletion of one or several bases or substitution with other bases" means that one or more bases are added or deleted or one or more bases compared to the base sequence This means that there is a mutation such that one of the bases is replaced with another base, but it has the same function as the basic non-mutated sequence.

  The substantially equivalent function in this case represents a high transcription promoting activity in silkworm silk gland cells. Means for addition, deletion or substitution with one or several bases are known per se, such as random mutagenesis, site-directed mutagenesis, gene homologous recombination, or polymerase The chain amplification method (PCR) can be performed alone or in appropriate combination. For example, a method of replacing cytosine bases with uracil bases by chemical treatment with sodium bisulfite, a method of reducing the accuracy of nucleotide incorporation during DNA synthesis by performing PCR in a reaction solution containing manganese Various commercially available kits for introducing specific mutations can also be used.

  For example, edited by Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, edited by Masami Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE. Chapter [PCR Technology, Principle and Application of DNA Amplification] It can be carried out according to the method described in the book of Stockton Press, 1989, etc., or by modifying those methods. In this case, the substantially equivalent function represents a high transcription activity or transcription promotion activity in silkworm silk gland cells.

  Specifically, in the silkworm posterior silk gland or the middle silk gland on the 2nd day of the 5th day, the transcription amount of the foreign protein gene in the recombinant silkworm having the mutated sequence is the silkworm actin gene, fibroin H chain gene or fibroin L chain. It shows that it is confirmed by Northern analysis that it is within the range of 0.1 to 1 times as much as the transcription amount of the gene or sericin gene. Preferably, it is within the range of 0.1 to 1 times the amount of transcription of the fibroin heavy chain gene.

  For comparison of transcription amount, the molecular imager FXPro manufactured by BIORAD can be used to quantitatively detect and compare signals detected and detected by Gene Images Random-Prime Labeling and Detection System manufactured by Amersham Pharmacia. . At this time, the probe amount, hybridization conditions, and detection conditions are determined in advance so that the detection sensitivity of the foreign protein gene detection probe and the silkworm actin gene, fibroin H chain gene, fibroin L chain gene, or sericin gene detection probe are comparable. To do. Using the conditions, the foreign protein gene detection probe and the chicoactin gene or fibroin H chain gene or fibroin L chain gene or sericin gene detection probe to be compared are mixed and hybridized for detection and comparison.

  Similarly, a DNA in which the homologous sequence at the 5 'end of the sericin gene hybridizes under stringent conditions as long as it has substantially the same function. Single-stranded DNA hybridizes with the partner strand depending on the degree of homology. At this time, by setting the stringency of the hybridization conditions more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the salt concentration (formamide, etc.) concentration and temperature conditions in the hybridization and washing steps. Details are described in detail in US Pat. No. 6,100,037. The DNA identified by the hybridization screening under such stringent conditions is 70% or more, preferably 80% or more, more preferably 90% or more at the level of homology.

  The amount of exogenous protein produced can be improved by fusing the 3 ′ side of the second exon with the 5 ′ side of the foreign protein structural gene so that the amino acid frame is continuous. If the length is too long, an extra amino acid residue is added to the N-terminal side of the target foreign protein, and the structure and activity of the target foreign protein may be lost. It is necessary to.

  In many cases, a favorable result can be obtained by setting the second exon portion immediately after the secretion signal gene of the sericin gene or up to several amino acid residues.

  For comparison of protein production, signals from foreign proteins detected by ECL Western Blotting Detection System manufactured by Amersham Pharmacia can be detected and compared by using Molecular Imager FXPro manufactured by BIORAD.

  The poly A region is not particularly limited, but a poly A region of a protein gene that is expressed in a large amount in the silk gland such as fibroin H chain, fibroin L chain, sericin can be preferably used.

  The vector in the present invention means a vector having a circular DNA structure or a linear DNA structure. In particular, a vector that can replicate in E. coli and has a circular DNA structure can be preferably used. For the purpose of facilitating selection of transformants, a marker gene such as an antibiotic resistance gene or a jellyfish-derived fluorescent green protein gene can be incorporated into this vector.

  The insect cells used in the present invention are not particularly limited, but are preferably lepidopterous insects, more preferably cells derived from Bombyx mori, more preferably silkworm silk gland cells or Bombyx mori eggs. It is a contained cell. Among the silk gland cells, the middle silk gland cells of the silkworm 5th instar larvae which are active in the synthesis of sericin protein and are easy to handle are suitable.

  There is no particular limitation on the method for introducing a gene cassette and vector for expression of a foreign protein into insect cells. As an introduction method into insect cultured cells, calcium phosphate method, electroporation method, liposome method, gene gun method, microinjection method, etc. can be used, but in introduction into silkworm silk gland cells, For example, a gene can be easily introduced into a middle silk gland tissue taken out from the body of a silkworm fifth-instar larva by using a gene gun.

  Gene transfer to the middle silk gland using a gene gun, for example, by using a particle gun (model number: PDS-1000 / He) of Bio-Rad, gold particles coated with a vector containing a gene cassette for expression of a foreign protein, This can be achieved by spraying the middle silk gland fixed to an agar plate or the like with a He gas pressure of 1,100 to 1,800 psi.

  When a gene is introduced into cells contained in Bombyx mori eggs, a microinjection method is preferred. Here, when microinjection is performed on an egg, it is not necessary to perform microinjection directly on cells in the egg, and it is possible to introduce a gene simply by microinjection into the egg.

  The vector having the “gene cassette for gene transfer” of the present invention is microinjected into the eggs of Bombyx mori, so that the recombinant silkworm in which the “gene cassette for expressing foreign protein” of the present invention is introduced into the chromosome can be obtained. It is possible to obtain. For example, according to the method of Tamura et al. (Nature Biotechnology 18, 81-84, 2000), a vector having a “gene transfer gene cassette” and a plasmid in which a PiggyBac transposase gene is placed under the control of a silkwormctin promoter At the same time, microinjection is performed on silkworm eggs, and hatched larvae are bred, and the resulting adult worms (G0) are crossed within a group to obtain next-generation (G1) silkworm larvae.

  Recombinant silkworms usually appear at a frequency of 1-2% in this G1 generation. The selection of recombinant silkworms can be performed by PCR using DNA extracted from a part of the tissue of G1 generation silkworms and using primers designed based on foreign protein genes. Alternatively, if a gene encoding a green fluorescent protein (for example, GFP and its analog) linked downstream of a promoter that can be expressed in silkworm cells is introduced into a “gene cassette for gene introduction” in advance, the G1 generation By selecting an individual that emits green fluorescence under ultraviolet light for silkworms, for example, 1st instar larvae, it is possible to easily select recombinant silkworms.

  Examples of promoters that can be expressed in silkworm cells, linking genes encoding green fluorescent proteins, such as the Drosophila heat shock protein gene promoter described in JP-A-6-261770 and JP-A-62-285787. And the promoter of the silkworm actin gene (Nature Biotechnology 18, 81-84, 2000) and the 3xP3 promoter known to be expressed in the Drosophila optic nerve. Preferably, the promoter of the silkworm actin gene (Nature Biotechnology 18, 81-84, 2000) or the 3xP3 promoter is used.

  In addition, as a promoter that can be expressed in silkworm cells, a promoter having high transcription promoting activity in silkworm silk gland cells, for example, a fibroin H chain gene promoter (base numbers 255 to 574 of GenBank accession number V00094, GenBank accession number AF226688 Base number 62118-62437), fibroin L chain gene promoter (Gene, 100: 151-158: GenBank accession number M76430), sericin gene promoter (base numbers 599-1656 of GenBank accession number AB007831) Is also possible.

  However, when linked to a gene encoding green fluorescent protein to a promoter having high transcription promoting activity in silkworm silk gland cells according to the present invention, a large amount of green fluorescent protein is produced in silkworm silk gland cells. In addition, in the silk thread, a green fluorescent protein is produced in addition to the target foreign protein. In this case, when the target foreign protein is purified and recovered from the silk wire and silk thread, the green fluorescent protein is difficult to purify and recover the target foreign protein as a contaminating protein. In addition, there is a possibility that the green fluorescent protein may give unwanted properties such as a decrease in the strength and elongation to the physical properties of the silk thread.

  In addition, for the purpose of obtaining a recombinant silkworm in which a `` gene cassette for expression of a foreign protein '' is introduced into a chromosome, when microinjecting a vector having a `` gene cassette for gene introduction '' into an egg of Bombyx mori, Recombinant silkworms can also be obtained in the same manner as described above by simultaneously microinjecting PiggyBac transposase proteins.

  The PiggyBac transposon is a DNA transposable element having an inverted sequence of 13 base pairs at both ends and an ORF of about 2.1 k base pairs inside. The piggyback transposon used in the present invention is not particularly limited. For example, those derived from Trichoplusia ni cell line TN-368, Autographa californica NPV (AcNPV), and Galleria mellonea NPV (GmMNPV) can be used. Preferably, using a plasmid pHA3PIG and pPIGA3GFP (Nature biotechnology 18,81-84,2000) having a part of PiggyBac derived from Trichoplusia ni cell line TN-368, the piggyback transposase having the gene and DNA transfer activity Can be prepared.

  The transgenic silkworm used in the present invention is a silkworm in which a foreign protein gene has been introduced into the silkworm chromosome, and the foreign protein gene labeled with the conventional method after treating the silkworm chromosomal DNA with a restriction enzyme according to a conventional method. This is a silkworm that gives a positive signal when Southern blotting is performed using as a probe. The gene locus on the chromosome into which the foreign protein gene is introduced is not particularly limited as long as it does not inhibit the development, differentiation, and growth of silkworm. Recombinant silkworms have the ability to produce foreign proteins in their silk gland cells, silk gland lumen, and silk. Recombinant silkworms can grow normally and can be mated, and can stably carry the introduced foreign protein gene and transmit it to offspring.

  Therefore, it is possible to easily scale up the amount of foreign protein produced by subculturing recombinant silkworms and increasing the number of heads. In the mating, it is possible to improve the production amount of the foreign protein by mating with a wild type silkworm. In this case, it is necessary to subculture while appropriately selecting silkworms into which the target foreign protein gene has been introduced. In this case, using the DNA of cells obtained from any tissue, the presence and structure of marker genes and foreign protein genes used for selection of recombinant silkworms can be easily analyzed by PCR, Southern blotting, etc. It is possible to discriminate offspring that have inherited the gene of the recombinant silkworm.

  Insect cells and silkworm silk glands introduced with the gene cassette for expression of the foreign protein of the present invention can be produced in culture supernatants and cells by culturing them in a culture solution suitable for each culture. Is possible. For example, BmN cells, which are silkworm ovary-derived cells into which the gene cassette for expression of the present invention has been introduced, are cultured in TC-100 medium (manufactured by Pharmingen) at 27 ° C., so that they can be cultured in 3 to 4 days. To produce the desired foreign protein. Further, the silkworm posterior silk gland, for example, is aseptically extracted from 5th instar larvae and then cultured at 25 ° C. in an insect grace medium to produce a foreign protein. When protein production is performed in the silk gland, it is preferable to maintain a high dissolved oxygen concentration in the medium, and a factor that inhibits low-molecular protein synthesis that accumulates in the medium is, for example, an ultrafiltration membrane. It is preferable to cultivate while removing the protein, etc., and protein synthesis for a long time is possible.

  The recombinant silkworm obtained in the present invention can be bred in the same manner as a normal silkworm, and foreign proteins can be produced by bred under normal conditions. Depending on the target foreign protein, it is also possible to improve the production amount of the foreign protein, particularly by optimizing the culture temperature, humidity, feeding conditions, etc. at the age of 5 years.

  A recombinant silkworm introduced with a foreign protein gene fused with the 5 ′ end of the sericin gene of the present invention can produce a large amount of the desired foreign protein in its cage. The target foreign protein can be easily purified and recovered from the obtained koji. Moreover, depending on the function of the produced foreign protein, the obtained silk thread containing the foreign protein can be used in various industrial applications as it is or in a partially processed form.

  Silkworm silk is formed by a fibroin layer and a sericin layer covering the surface. Since the sericin layer is relatively highly soluble, a foreign protein can be obtained from the silk gland or silk of the recombinant silkworm obtained by the present invention by a simple extraction operation. Although there is no restriction | limiting in particular about the solvent used in order to extract a foreign protein from a silk gland or a silk thread, In many cases, water or aqueous solution is preferable. The aqueous solution used for extraction can contain a suitable solute to facilitate the extraction of foreign proteins. For example, a buffer solution can be used. As types, acetic acid, citric acid, boric acid, tartaric acid, phosphate buffer and the like can be used. The pH is not particularly limited, and any pH can be used as long as it does not deactivate the function of the target foreign protein. An alkaline aqueous solution having a pH of 8 to 13 is preferable. Sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, aqueous ammonia, sodium hydrogen carbonate, magnesium hydroxide, barium hydroxide, copper hydroxide, iron hydroxide and the like can be used for the alkaline aqueous solution. Of these, alkaline ionized water having a pH of 8 to 11 is preferable. Alkaline ion water is an alkaline aqueous solution prepared by electrolyzing water and collecting the liquid near the cathode.

  Moreover, it is also possible to use the above-mentioned aqueous solution containing a surfactant capable of weakening the surface tension. Surfactants are roughly classified into ionic surfactants and nonionic surfactants. The ionic surfactant is further classified into an anionic (anionic) surfactant, a cationic (cationic) surfactant, and an amphoteric surfactant. Anionic (anionic) surfactants include sodium alkyl sulfate, stearyldimethylbenzylammonium chloride, soap (higher fatty acid salt of alkali metal), sodium polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl ether phosphate, poly Examples include oxyethylene alkylphenyl ether phosphoric acid, coconut oil fatty acid methyl taurine sodium, lauryl sulfate triethanolamine, sodium lauryl sulfate, lauroyl sarcosine sodium, lauroyl methyl β-alanine sodium solution, and lauroyl methyl taurine sodium. Cationic (cationic) surfactants include ethyl lanolin sulfate fatty acid aminopropylethyldimethylammonium chloride, alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, distearyldimethylammonium chloride, stearyltrimethylammonium chloride, benzalkonium chloride, Examples include benzethonium chloride. Amphoteric surfactants include alkyldiaminoethylglycine hydrochloride solution, 2-alkyl-N-carboxymethyl-N-hydrokishi ethylimidazolinium betaine, lauryldimethylaminoacetic acid betaine, and the like. Nonionic surfactants include dimethicone copolyol, sucrose fatty acid ester, polyoxyethylene sorbitol tetraoleate, bemulene, polymethylpolysiloxane copolymer, polyoxyethylene octylphenyl ether, polyoxyethylene hydrogenated castor oil, Polyoxyethylene stearyl ether, polyoxyethylene stearamide, polyoxyethylene cetyl ether, polyoxyethylene polyoxypropylene glycol, polyglycerin fatty acid ester, polyoxyethylene sorbitan monooleate, ethylene glycol monoautearate, monostearic acid Propylene glycol, polyoxyethylene glyceryl monostearate, polyoxyethylene sorbitan monostearate, poly monopalmitate Carboxymethyl sorbitan, polyethylene glycol monolaurate, polyoxyethylene sorbitan monolaurate, and the like monolaurate polyoxyethylene sorbit. Any type of surfactant can be used, but it is preferred to use an anionic surfactant that is an ionic surfactant.

  The amount of the surfactant used is preferably about 0.1% (w / vol) to 5% (w / vol) with respect to the aqueous solution.

  There is no particular limitation on the method for isolating and purifying the extracted foreign protein, and a normal protein purification method can be used. For example, it can be purified and isolated by combining desalting and concentration by ultrafiltration, gel filtration, dialysis, salting out, and the like. In addition, it can be purified and isolated by column chromatography using a silica gel carrier, an ion exchange carrier, a gel filtration carrier, a chelating carrier, a dye carrier, etc., with the function of the intended useful protein as an index. . Among these, an ion exchange carrier is preferably used. For example, the canine parvovirus VP2 antigen can be recovered in a soluble fraction obtained by homogenizing a silk gland or silk thread of a silkworm introduced with a canine parvovirus VP2 gene by adding alkaline ionized water. Furthermore, the purity of the canine parvovirus VP2 antigen can be increased by adsorbing the obtained extract on, for example, a blue sepharose carrier, washing, and eluting with a buffer containing salts.

  The foreign protein thus produced is not particularly limited, but a protein that is soluble in an aqueous solution is desirable. For example, cytokines are preferred. Cytokines perform cell proliferation, differentiation, and functional expression, and can be classified into various types according to structure and action mechanism. Typical examples include interferon. Also preferred are antigenic proteins of the vaccine. Antigen proteins of vaccines are those that have antigenicity to animals and induce antibody production and cellular immunity when administered into animals, as well as viruses, microorganisms, parasites, tumors, It is a protein derived from other animals and can be expected to prevent or cure diseases caused by the protein. Amino acid composition, molecular weight, and the presence or absence of sugar chains are not a problem, and any protein or peptide can be used. The protein produced in this way can be used for pharmaceutical use, various measurements, and diagnostic use, as in the case of cytokine and vaccine antigen proteins produced by other conventional production methods. In this case, you may use as a mixture which added various additives. Silkworm tissues or silkworms expressing cytokine or vaccine antigen proteins can be used as medical or clothing fibers as they are or after being processed. Moreover, the recombinant silkworm tissue or silk thread expressing the enzymes can be used for the enzymatic reaction as it is.

  Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.

Example 1. Preparation of Bombyx mori genomic DNA
The silkworms at 5 days of age were dissected and the middle silk gland tissue was removed. After washing with 1 × SSC, 200 μl of DNA extraction buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl) was added. Proteinase K (final 200 μg / ml) was added and the tissue was thoroughly ground with a grinder. Further, 350 μl of DNA extraction buffer and 60 μl of 10% SDS were added and mixed, and then incubated at 50 ° C. for 2 hours. Tris-HCl saturated phenol pH 8.0 500 μl was added and mixed for 10 minutes, followed by centrifugation at 10,000 rpm for 5 minutes at 4 ° C. to recover the supernatant.

  An equal amount of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added to the supernatant, mixed, and then centrifuged. Phenol / chloroform / isoamyl alcohol was added again, and the supernatant was recovered after centrifugation. After adding an equal amount of chloroform / isoamyl alcohol (24: 1) and mixing, chloroform / isoamyl alcohol was added again to the centrifuged supernatant, and the supernatant was recovered after centrifugation. 1/10 volume of 3M sodium acetate (pH 5.2) was added to the resulting supernatant and mixed, and 2.5 times the amount of cold ethanol was added and left at -80 ° C for 30 minutes, then 15,000 rpm for 10 minutes at 4 ° C. The mixture was centrifuged to precipitate genomic DNA. The DNA precipitate was washed with 70% ethanol and then air-dried. A genomic DNA solution was prepared by dissolving and diluting with RNase-containing sterile water to 100 μg / ml.

Example 2 Preparation of Gene The gene used was obtained by preparing a primer at both ends using a known sequence and performing PCR using an appropriate DNA source as a template. A restriction enzyme cleavage site was added to the end of the primer for later gene manipulation.

  The sericin promoter (base number 599 to 1656th of GeneBank registration number AB007831: P region) is composed of Bombyx mori genomic DNA as a template and two primers, primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2). Obtained by PCR used.

  The sericin promoter, sericin gene first exon, first intron, and second exon region (hereinafter referred to as SP region) are two types, primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), using Bombyx mori genomic DNA as a template. It acquired by PCR using the primer of.

  The feline interferon-ω gene (base numbers 9 to 593 of GeneBank registration number S62636: IC region hereinafter) is primer 5 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 5) using baculovirus rBNV100 encoding the feline interferon-ω gene as a template. It was obtained by PCR using the two types of primers of 6). rBNV100 is obtained by, for example, cutting out the feline interferon-ω gene from a plasmid extracted from E. Coli (pFeIFN1) (Microtechnical Research Institute No. 1633), and then cloning a silkworm cloning vector (T. Horiuchi et al., Agric. ., 51, 1573-1580, 1987) and a silkworm polynuclear disease virus DNA can be produced by co-transfecting silkworm established cells.

  There are two types of sericin poly A signal region (GeneBank accession number AF226688, nucleotide numbers 79099-79995: the SA region): Bombyx mori genomic DNA as a template, primer 7 (SEQ ID NO: 7) and primer 8 (SEQ ID NO: 8) It acquired by PCR using the primer of.

  These reaction solutions were electrophoresed on a 1% agarose gel, and DNA fragments of about 0.3 kbp in the P region, about 3.5 kbp. In the SP region, about 580 bp in the IC region, and about 0.9 bp in the SA region were used in a conventional method. Extracted and prepared according to These DNA fragments were phosphorylated with a polynucleotide kinase (Takara Shuzo Co., Ltd.), then cleaved with HincII and then dephosphorylated with a DNA ligation kit Ver. 2 of Takara Shuzo Co., Ltd. at 16 ° C. The reaction was performed overnight and ligated. Using these, E. coli was transformed according to a conventional method, and it was confirmed by PCR that the obtained colony was inserted into the obtained transformant under the same conditions as described above. The inserted plasmid was prepared by a conventional method. By sequencing these plasmids, it was confirmed that the fragments obtained were the base sequences of the respective genes.

Example 3 Production of Plasmid for Gene Introduction A plasmid containing a gene structure in which an expression cassette for a feline interferon-ω gene was inserted between a pair of inverted repeats of transposon piggyBac was used as a gene introduction plasmid.

  That is, the transposase-encoding region was removed from plasmid p3E1.2 disclosed in US Pat. No. 6,218,185, its Bgl II site and Hpa I site were smoothed, and an expression cassette for the feline interferon-ω gene was inserted.

  The gene expression cassette in this example consists of a sericin promoter / cat interferon-ω / sericin poly A signal region (P / IC / SA) or a sericin promoter / sericin gene first exon / first intron / second exon region. -Cat interferon-ω-Sericin poly A signal region (SP, IC, SA).

  A specific method is shown below.

  The P / IC / SA construct was prepared by the following method. The plasmid having feline interferon-ω (IC region) prepared in Example 2 was digested with Sal I and Hind III, and about 1 kbp. Fragment (P region) excised from the plasmid with sericin promoter by Sal I and Hind III. ) Was inserted. Further, this was cleaved with BamH I, and about 0.9 kbp. Fragment (SA region) cut out with BamH I from a plasmid having a sericin poly A signal region was inserted therein. This plasmid having P, IC, and SA was digested with Asc I, and the excised about 2.5 kbp fragment was blunted with Takara Shuzo T4 DNA Polymerase, and p3E1.2 was digested with Bgl II and Hpa I. After removing the gene fragment of 750 bp, the gene fragment that had been smoothed and dephosphorylated was ligated to produce a gene introduction construct containing the P • IC • SA gene cassette.

  The SP / IC / SA construct was prepared by the following method. The plasmid having the cat interferon-ω (IC region) prepared in Example 2 was cleaved with Sal I and Hind III, and the plasmid containing the sericin promoter / sericin gene first exon / first intron / second exon region An approximately 3.5 kbp fragment (SP region) excised with Sal I and Hind III was inserted.

Further, this was cleaved with BamH I, and about 0.9 kbp. Fragment (SA region) cut out with BamH I from a plasmid having a sericin poly A signal region was inserted therein. This SP / IC / SA-containing plasmid was digested with Asc I, and the excised approximately 5 kbp fragment was blunted with Takara Shuzo T4 DNA Polymerase, and p3E1.2 was digested with Bgl II and Hpa I to obtain 750 bp. After the gene fragment was removed, the gene fragment was smoothed and dephosphorylated and ligated to produce a gene introduction construct containing the SP / IC / SA gene cassette.
The P / IC / SA gene introduction construct and the SP / IC / SA gene introduction construct were purified using the QIAGEN Plasmid Maxi Kit according to the attached protocol.

Example 4 Preparation of piggyback transposase protein (1) Gene cloning and expression vector preparation For piggyback transposase gene cloning, an oligo was used with reference to the nucleotide sequence of the piggyback transposase gene disclosed in US Pat. No. 6,218,185. Nucleotide primers were synthesized. A restriction enzyme cleavage site was added to the end of the primer for later gene manipulation.

  For cloning the piggyback transposase protein gene, two types of primers, primer 9 (SEQ ID NO: 9) and primer 10 (SEQ ID NO: 10), were used, and the plasmid p3E1.2 disclosed in US Pat. No. 6,218,185 was used as a template. Obtained by PCR.

PCR was performed using a 0.2 ml microcentrifuge tube, 10 ng of template DNA, 50 pmol of each primer, 10 μl of the attached 10 × PCR buffer, 1 mM MgCl ₂ , 0.2 mM dNTPs, and 2 units of KODplus were added to each reagent. The total volume was 100 μl. Use a BioRad thermal cycler for 30 cycles under conditions of DNA denaturation at 94 ° C for 30 seconds, primer annealing at 60 ° C for 30 seconds, and DNA primer extension at 72 ° C for 3 minutes. It was. This reaction solution was electrophoresed on a 1% agarose gel, and a DNA fragment of about 1.8 kbp was extracted and prepared according to a conventional method.

  This DNA fragment was phosphorylated with a polynucleotide kinase (Takara Shuzo Co., Ltd.), cleaved with HincII, and then dephosphorylated with the pUC19 vector using Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 2 at 16 ° C. overnight. Reactions were performed and ligated. Using this, Escherichia coli was transformed according to a conventional method, and it was confirmed by PCR that the obtained colony was inserted into the obtained transformant under the same conditions as described above. The inserted plasmid was prepared by a conventional method. By sequencing this plasmid, it was confirmed that the obtained fragment was the base sequence of the gene.

  This plasmid was digested with NcoI and BamHI, and the resulting 1.8 kbp NcoI / BamHI fragment was always inserted into the NcoI / BamHI gap of pTV118N (Takara Shuzo Co., Ltd.) previously digested with NcoI and BamHI. The resulting plasmid was inserted by a ligation reaction according to the method, and the resulting plasmid was named pTV-piggy. By culturing Escherichia coli into which this plasmid has been introduced, a recombinant piggyback transposase protein having a molecular weight of about 70 kDa in which 10 histidine residues have been added to the N-terminal amino acid sequence of the piggyback transposase protein can be produced.

(2) Production of recombinant piggyback transposase protein E. coli BL21 strain was transformed to ampicillin resistance with pTV-piggy, and the resulting transformants were named BL21-piggy strain, respectively.

  Next, recombinant piggyback transposase protein was produced in BL21-piggy strain. First, BL21-piggy strain was sterilized LB medium (Sambrook, J. et al., 2001, Molecular Cloning 3rd. Edition, Cold Spring Harbor Lab. Press) containing 50 μg / ml ampicillin sodium (LB-amp medium) 5 ml. One platinum ear was inoculated and pre-cultured by shaking at 37 ° C. for 24 hours.

  Inoculate the entire volume of this preculture in 50 ml of LB-amp medium, incubate for 3 hours at 37 ° C, amplitude 30 cm, and 180 rpm, then add 1 mM IPTG (isopropyl-1-thio-β-D-galactoside). The culture was further continued for 4 hours. As a control experiment, the same culture was performed using a transformant obtained by transforming BL21 with pTV118N as a control. The bacterial cells thus obtained were collected and resuspended in 5 ml of TBS buffer (Takara Shuzo), and then a cell disruption solution was prepared by ultrasonic disruption and centrifugation. This cell lysate was fractionated by SDS-PAGE and subjected to Western blotting with Penta-His Antibody antibody (QIAGEN). As a result, a recombinant piggyback transposase with a molecular weight of about 70 kDa was obtained from the cell lysate derived from BL21-piggy strain. Protein was detected.

(3) Purification of recombinant piggyback transposase protein Since this recombinant protein has 10 histidine residues in the N-terminal amino acid sequence, it was purified using interaction with nickel ions.

  First, a column system packed with 10 ml of a chelating Sepharose Fast Flow carrier (Amersham Biosciences) was constructed. After flowing 50 ml of 50 mM nickel sulfate aqueous solution and 50 ml of TBS buffer in this order, 50 ml cell disruption solution derived from 500 ml culture solution of BL21-piggy strain obtained by the same method as (2) was passed. . Thereafter, 100 ml of TBS buffer containing 5 mM imidazole and 100 ml of TBS buffer containing 50 mM imidazole were run in this order. An additional 50 ml of TBS buffer containing 600 mM imidazole was run. Western blotting was performed on each buffer solution applied to the column in the same manner as in (2). As a result, about 70 kDa protein was detected in a TBS buffer solution containing 600 mM imidazole. In addition, when each buffer solution passed through the column was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue, a single band of about 70 kDa was detected from the TBS buffer containing 600 mM imidazole. It was confirmed to be a back transposase protein. After removing imidazole by dialysis, protein concentration was quantified using Pierce BCA reagent with bovine serum albumin as a standard protein.

Example 5. Preparation of transgenic silkworm 0.5 mM phosphate buffer (pH 7.0) / 5 mM KCl solution containing 200 μg / ml each of the plasmid for gene transfer described in Example 3 and the piggyback transposase protein described in Example 4 And 3-20 nl were microinjected into 500 silkworm eggs within 4 hours after laying eggs.

  The larvae hatched from the silkworm eggs are reared, and the next generation (G1) obtained by multiplying the obtained adult worms (G0) within the group is collected with the injection needle (21G) at the age of four, and PCR is performed. To screen for gene transfer. PCR was performed with Premix Taq manufactured by Takara Shuzo Co., Ltd. using the primers of SEQ ID NO: 5 and SEQ ID NO: 6. In other words, 20 μL of Premix Taq diluted to a final concentration of 2 × and each primer adjusted to 0.5 μM was dispensed, 0.5 to 2 μL of body fluid was added, DNA denaturation conditions were 94 ° C., 30 seconds, primer annealing The reaction was carried out for 30 cycles using a BioCycle thermal cycler under conditions of 55 ° C. for 30 seconds and DNA primer extension conditions of 72 ° C. for 1 minute. These reaction solutions were electrophoresed on a 1% agarose gel, and those in which a DNA fragment of about 580 bp was confirmed were judged as gene transfer positive. As a result, a transgenic silkworm having a feline interferon ω gene expression cassette introduced into the chromosome was obtained.

Example 6: Measurement of recombinant protein in silk by Western analysis The secretion of a foreign protein, namely cat interferon ω, into silk was examined.
Weigh 10 mg each of non-transformed silkworms, transformed silkworms (P / IC / SA gene-introduced transformed silkworms, SP / IC / SA gene-introduced silkworm), and complete the silk thread (fibroin layer, sericin layer) 4 ml of dissolved 60% lithium thiocyanate (LiSCN) was added and stirred, and then allowed to stand overnight at room temperature to dissolve the soot. The sample was diluted 10 times with 8M urea, 2% SDS, 5% 2-mercaptoethanol, and the cat interferon was detected using the ECL PlusTM Western blotting Kit (Amersham Pharmacia) according to the attached protocol. It was. The signal intensity was measured using a molecular imager (manufactured by BioRad), and the result was compared with the signal intensity of feline interferon with a known concentration to determine the protein content. In addition, sodium phosphate buffer can be added to the cocoons of non-transformed silkworms and transformed silkworms (P, IC, SA gene-introduced transgenic silkworms, SP, IC, SA gene-introduced silkworms) to allow only the sericin layer. The cat interferon was detected by Western blotting in the same manner as a sample.

  As a result, no signal was detected from the silkworm of the non-transformed silkworm and the silkworm transformed with the P • IC • SA gene, whereas the signal was detected from the silkworm of the silkworm transformed with the SP • IC • SA gene. It was confirmed that the interferon protein was secreted into the silk thread. The content was about 0.8 to 2.0% for SP, IC and SA transformed silkworms. This was 0.4 to 2 mg in terms of weight per silkworm. The result is shown in FIG. Further, when only the sericin layer was solubilized and fractionated into a soluble fraction and an insoluble fraction, feline interferon protein was confirmed in the soluble fraction. The result is shown in FIG.

Example 7 Expression analysis of recombinant protein in middle silk gland tissue by Western analysis Middle silk gland of non-transformed silkworm, transformed silkworm (P, IC, SA gene-introduced silkworm, SP, IC, SA gene-introduced silkworm) The tissue was collected, and the expression of cat interferon ω in the tissue was examined by Western analysis. Homogenize the silkworm middle silk gland in 100 mM sodium phosphate buffer (pH 7.0), collect the supernatant after centrifugation, and use the ECL PlusTM Western blotting Kit (manufactured by Amersham Pharmacia) as a sample according to the attached protocol. Interferon was detected. That is, the blotted membrane was blocked overnight at 4 ° C. in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS).

  The membrane was washed twice with TPBS (0.1% Tween20 · PBS) and treated with an anti-cat interferon antibody diluted 1000 times with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes. Thereafter, the resultant was diluted 10,000 times with TPBS, and then treated with an HRP-labeled anti-rabbit IgG antibody for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes, and then a detection reagent (solution A + solution B) of ECL Plus ™ Western blotting Detection System (Amersham Pharmacia) was added. Luminescence was exposed and developed in HyperfilmTM ECLTM. For comparison of signals, a molecular imager FXPro manufactured by BIORAD was used.

  As a result, no signal was detected in the middle silk gland tissue of the non-transformed silkworm and the transformed silkworm introduced with the P / IC / SA construct, whereas the silk gland tissue of the transformed silkworm with the SP / IC / SA construct introduced A signal was detected. Based on the results of this experiment, regions other than the sericin promoter, ie, the first exon, first intron, and second exon region, play an important role in dramatically improving protein synthesis or gene expression in the middle silk gland cells of the silkworm. It was reconfirmed that

Example 8. Preparation of gene for antigen protein (canine parvovirus VP2) The gene used was obtained by preparing primers at both ends using a known sequence and performing PCR using an appropriate DNA source as a template. did. A restriction enzyme cleavage site was added to the end of the primer for later gene manipulation.

  The canine parvovirus VP2 gene (base numbers 1 to 1755 of GeneBank accession number AB128923: VP2 region) is primer 11 (SEQ ID NO: 11) and primer 12 (SEQ ID NO: 12) using Canine parvovirus encoding canine parvovirus VP2 gene as a template. ) Was obtained by PCR using two types of primers.

  The reaction solution was electrophoresed on a 1% agarose gel, and a DNA fragment of about 1700 bp was extracted from the VP2 region according to a conventional method. This DNA fragment was phosphorylated with a polynucleotide kinase (Takara Shuzo Co., Ltd.), then cleaved with HincII, and then dephosphorylated with the pUC19 vector using Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 2 at 16 ° C. overnight. Reactions were performed and ligated. Using this, E. coli was transformed according to a conventional method, and it was confirmed by PCR that the obtained colony was inserted into the resulting transformant under the same conditions as described above. The inserted plasmid was prepared by a conventional method. By sequencing this plasmid, it was confirmed that the obtained fragment was the base sequence of each gene.

Example 9 Preparation of Plasmid for Gene Introduction A plasmid containing a gene structure in which an expression cassette for canine parvovirus VP2 gene was inserted between a pair of inverted repeats of transposon piggyBac was used as a gene introduction plasmid.
That is, the transposase-encoding region was removed from plasmid p3E1.2 disclosed in US Pat. No. 6,218,185, its Bgl II site and Hpa I site were smoothed, and an expression cassette for canine parvovirus VP2 gene was inserted.

The gene expression cassette in this example consists of a sericin promoter / canine parvovirus VP2 / sericin poly A signal region (P / VP2 / SA), or a sericin promoter / sericin gene first exon / first intron / second exon region. It is a canine parvovirus VP2 / sericin poly A signal region (SP / VP2 / SA).
A specific method is shown below.

  The P · VP2 · SA construct was produced by the following method. The plasmid having the canine parvovirus VP2 (VP2 region) prepared in Example 8 was cleaved with Sal I and Hind III, and about 1 kbp. Fragment (P region) excised from the plasmid with sericin promoter by Sal I and Hind III. ) Was inserted. Further, this was cleaved with BamH I, and about 0.9 kbp. Fragment (SA region) cut out with BamH I from a plasmid having a sericin poly A signal region was inserted therein. This plasmid having P · VP2 · SA was digested with Asc I, and the excised approximately 2.5 kbp fragment was blunted with Takara Shuzo T4 DNA Polymerase, and p3E1.2 was digested with Bgl II and Hpa I. After removing the gene fragment of 750 bp, the resulting product was ligated with the smoothed and dephosphorylated product to prepare a gene introduction construct containing the P • VP2 • SA gene cassette.

  The SP, VP2, and SA constructs were produced by the following method. The plasmid having the canine parvovirus VP2 (VP2 region) prepared in Example 7 was cleaved with Sal I and Hind III, and the plasmid containing the sericin promoter, sericin gene first exon, first intron, and second exon region. An approximately 3.5 kbp fragment (SP region) excised with Sal I and Hind III was inserted.

  Further, this was cleaved with BamH I, and about 0.9 kbp. Fragment (SA region) cut out with BamH I from a plasmid having a sericin poly A signal region was inserted therein. This plasmid having SP, VP2, and SA was cleaved with Asc I, and the excised approximately 7 kbp fragment was blunted with Takara Shuzo T4 DNA Polymerase, and p3E1.2 was cleaved with Bgl II and Hpa I to obtain 750 bp. After the gene fragment was removed, the gene fragment was smoothed and dephosphorylated and ligated to prepare a gene introduction construct containing the SP / VP2 / SA gene cassette.

  The P / VP2 / SA gene introduction construct and the SP / VP2 / SA gene introduction construct were purified using QIAGEN Plasmid Maxi Kit according to the attached protocol.

Example 10. Production of transgenic silkworm 0.5 mM phosphate buffer (pH 7.0), 5 mM KCl containing 200 μg / ml each of the plasmid for gene transfer described in Example 9 and the piggyback transposase protein described in Example 4. A solution was prepared, and 3 to 20 nl was microinjected into 500 silkworm eggs within 4 hours after laying eggs.

  The larvae hatched from the silkworm eggs are reared, and the next generation (G1) obtained by multiplying the obtained adult worms (G0) within the group is collected with the injection needle (21G) at the age of four, and PCR is performed. To screen for gene transfer. PCR was performed with Takara Shuzo Co., Ltd. Premix Taq using the primers of SEQ ID NO: 11 and SEQ ID NO: 12. In other words, 20 μL of Premix Taq diluted to a final concentration of 2 × and each primer adjusted to 0.5 μM were dispensed, 0.5 to 2 μL of body fluid was added, DNA denaturation conditions were 94 ° C., 30 seconds, primer annealing The reaction was carried out for 30 cycles using a thermal cycler manufactured by BioRad under the conditions of 55 ° C. for 30 seconds and DNA primer extension conditions of 72 ° C. for 2 minutes. These reaction solutions were electrophoresed on a 1% agarose gel, and those in which a DNA fragment of about 1700 bp was confirmed were judged to be gene transfer positive. As a result, transgenic silkworms in which the expression cassette of canine parvovirus VP2 gene was introduced into the chromosome were obtained.

Example 11 Measurement of Recombinant Protein in Silk by Western Analysis The secretion of a foreign protein, ie, an antigen protein (canine parvovirus VP2), into silk was examined.
Weigh 10 mg each of non-transformed silkworms, transformed silkworms (P / VP2 / SA gene-introduced transformed silkworms, SP / VP2 / SA gene-introduced silkworms), and complete the silk thread (fibroin layer, sericin layer) 4 ml of dissolved 60% lithium thiocyanate (LiSCN) was added and stirred, and then allowed to stand overnight at room temperature to dissolve the soot. This was diluted with 10M with 8M urea, 2% SDS, 5% 2-mercaptoethanol as a sample, and using ECL Plus ™ Western blotting Kit (Amersham Pharmacia), antigen protein (canine parvovirus VP2) according to the attached protocol ) Was detected. The signal intensity was measured using a molecular imager (manufactured by BioRad), and the result was compared with the signal intensity of an antigen protein (canine parvovirus VP2) with a known concentration to determine the protein content. In addition, PBS is added to the cocoons of non-transformed silkworms and transformed silkworms (P · VP2 · SA gene-introduced transformed silkworms, SP · VP2 · SA gene-introduced silkworms) to solubilize only the sericin layer, Using this as a sample, canine parvovirus VP2 was similarly detected by Western blotting.

  As a result, no signal was detected from the cocoons of non-transformed silkworms and P · VP2 · SA gene-introduced silkworms, whereas signals were detected from the cocoons of SP · VP2 · SA gene-introduced silkworms. It was confirmed that the protein (canine parvovirus VP2) was secreted into the silk thread. The content was about 0.1 to 1.2% for SP, VP2, and SA transformed silkworms. This was 0.1 to 1.2 mg in terms of weight per silkworm. The result is shown in FIG. Further, when only the sericin layer was solubilized and fractionated into a soluble fraction and an insoluble fraction, an antigen protein (canine parvovirus VP2) was confirmed in the soluble fraction. The result is shown in FIG.

Example 12 Extraction of Recombinant Protein from Silk Thread A foreign protein, ie, an antigen protein (canine parvovirus VP2) was extracted and collected from silk by the following method. An aqueous solution shown below was used as a solvent that can dissolve silk thread (sericin layer) and recover foreign protein.

Aqueous solution that completely dissolves silk thread (fibroin layer, sericin layer): 60% LiSCN aqueous solution Aqueous solution that dissolves only sericin layer: phosphoric acid aqueous solution (buffer solution), 1% SDS aqueous solution, alkaline ionized water 1 g of the transformed silkworm (SP / VP2 / SA gene-introduced transgenic silkworm) was weighed and dissolved while stirring at room temperature for 12 hours. A part of the lysate was taken, and antigen protein (canine parvovirus VP2) was detected using ECL Plus ™ Western blotting Kit (Amersham Pharmacia) according to the attached protocol. The signal intensity was measured using a molecular imager (BioRad). In addition, silkworms (fibroin layer, sericin layer) are completely solubilized in the silkworms of non-transformed silkworms, and canine parvovirus VP2 is similarly detected by Western blotting using this as a sample. It was. The results are shown in FIG.

  From the results shown in FIG. 5 and Table 1, it was found that those dissolved in alkaline ionized water most dissolved the sericin layer and the extraction of foreign proteins was high.

  In the evaluation method, the signal intensity of a sample obtained by solubilizing silkworms (fibroin layer, sericin layer) of a silkworm (SP · VP2 · SA gene-introduced transformed silkworm) with a 60% LiSCN solution that completely dissolves silkworms (++, The signal intensity of the other samples was shown as relative, with the signal intensity of the sample obtained by solubilizing the silkworm of non-transformed silkworm with 60% LiSCN aqueous solution completely dissolving silk thread (fibroin layer, sericin layer) as-.

  Therefore, the extract obtained with alkaline ionized water was concentrated using an ultrafiltration membrane, and the buffer was exchanged with 20 mM phosphate buffer. The solution was adsorbed on a blue sepharose carrier, washed with 20 mM phosphate buffer, and eluted with 20 mM phosphate buffer containing sodium chloride, whereby canine parvovirus VP2 antigen could be obtained with high purity. FIG. 6 shows the result of silver staining analysis.

  By introducing a gene cassette for expression in which the DNA sequence of the 5 'end of the sericin gene is fused to a foreign protein gene into a silk gland cell, a large amount of the foreign protein can be introduced into the silk gland cell, especially the silk gland, It has become possible to produce even a silk sericin layer. By this new method, we established a foreign protein production technology that can be easily purified by using the silk gland to produce foreign protein without using recombinant baculovirus.

FIG. 1 is a view obtained by analyzing the production of a recombinant protein on silk by Western analysis, and is a drawing-substituting photograph showing electrophoresis. FIG. 2 is a view obtained by analyzing a sample in which only a sericin layer of silk thread is solubilized by Western analysis, and is a drawing-substituting photograph showing electrophoresis. FIG. 3 is a view obtained by analyzing the production of a recombinant protein on silk by Western analysis, and is a drawing-substituting photograph showing electrophoresis. FIG. 4 is a diagram in which a sample in which only the sericin layer of silk thread is solubilized is analyzed by Western analysis, and is a drawing-substituting photograph showing electrophoresis. FIG. 5 is a drawing obtained by solubilizing the sericin layer of silk thread and analyzing the extraction of the recombinant protein by Western analysis, and is a drawing-substituting photograph showing electrophoresis. FIG. 6 is a drawing substituting a photograph showing electrophoresis by purifying an aqueous solution obtained by solubilizing a silk sericin layer and extracting a recombinant protein by column chromatography and silver staining.

Claims (26)

(1) a promoter expressed in the silk gland;
(2) A gene cassette for expression of a foreign protein, comprising a gene fused to the 5 ′ side of a foreign protein structural gene and linked to the downstream of (1) above. The gene cassette for expression of a foreign protein according to claim 1, wherein the 5 'end portion of the sericin gene includes a part of the first exon, the first intron, and the second exon of the sericin gene. The gene cassette for expression of a foreign protein according to claim 2, wherein the portion of the sericin that combines the first exon and the second exon is a secretory signal gene region of the sericin gene. The gene cassette for the expression of a foreign protein according to any one of claims 1 to 3, wherein (1) the silk gland promoter and / or (2) the 5 'end portion of the sericin gene contained in the gene cassette for the expression of the foreign protein A gene cassette for expression of a foreign protein characterized by having one or several base additions, deletions or substitutions with other bases within a range having substantially equivalent functions. The gene cassette for expressing a foreign protein according to any one of claims 1 to 4, wherein (1) the silk gland promoter portion and / or (2) the 5 'end portion of the sericin gene contained in the gene cassette for expressing the foreign protein Alternatively, a gene cassette for expression of a foreign protein comprising a gene that hybridizes with the portion under stringent conditions and has a function substantially equivalent to the portion. 6. The promoter expressed in the silk gland is at least one promoter selected from a promoter of a sericin gene, a promoter of a fibroin H chain gene, and a promoter of a fibroin L chain gene. A gene cassette for expression of a foreign protein according to claim 1. At least one poly A addition region selected from the poly A addition region of the sericin gene, the poly A addition region of the fibroin H chain gene, and the poly A addition region of the fibroin L chain gene exists downstream of the foreign protein expression gene cassette. The gene cassette for expression of a foreign protein according to any one of claims 1 to 6. A chromosome of an insect cell, characterized in that a pair of piggyBac transposon inverted repeats are present on both sides of the foreign protein expression gene cassette according to any one of claims 1 to 7. Gene cassette for gene transfer into An insect cell expression vector comprising the foreign protein expression gene cassette according to any one of claims 1 to 8. A gene transfer vector for insect cells comprising the gene cassette for gene transfer into the chromosome of insect cells according to claim 8. A recombinant silkworm, wherein the gene cassette for expression of a foreign protein according to any one of claims 1 to 8 is introduced into a chromosome and has the ability to produce a foreign protein in a silk gland or silk thread. The silk thread containing the foreign protein which the recombinant silkworm of Claim 11 produces. A method for producing a foreign protein, which comprises introducing the gene introduction vector for insect cells according to claim 9 or 10 into insect cells. The method for producing a foreign protein according to claim 12, wherein the insect cell is derived from a lepidopterous insect. The method for producing a foreign protein according to claim 13, wherein the insect cells are derived from Bombyx mori. 15. The method for producing a foreign protein according to claim 14, wherein the insect cells are silk gland cells of Bombyx mori. Using the DNA transfer activity of piggyback (posyBac) transposase, create a recombinant silkworm that incorporates a gene cassette for the expression of a foreign protein into the chromosome, and produce the foreign protein in the silk gland or silk of the resulting recombinant silkworm The method for producing a foreign protein according to claim 13, wherein the foreign protein is recovered from the silk gland or silk thread after the treatment. Gene transfer vector for insect cells and DNA or RNA producing piggyback (posyBac) transposase are simultaneously microinjected into silkworm eggs to produce recombinant silkworms that incorporate a gene cassette for expression of foreign proteins into the chromosome. The method for producing a foreign protein according to claim 17, wherein A method for producing a foreign protein, wherein the silk protein according to claim 12 is suspended in an aqueous solution, and useful proteins are recovered from the aqueous solution in which the sericin layer is dissolved. The method for producing a foreign protein according to claim 19, wherein the aqueous solution is one or more selected from water, a buffer solution, and an alkaline aqueous solution. 21. The method for producing a foreign protein according to claim 20, wherein the alkaline aqueous solution is alkaline ionized water obtained by electrolyzing water and collecting an aqueous solution near the cathode. The method for producing a foreign protein according to any one of claims 19 to 21, wherein the aqueous solution described above contains a surfactant. The method for producing a foreign protein according to any one of claims 19 to 22, wherein the foreign protein is recovered from the aqueous solution in which the silk thread is dissolved by using column chromatography. The method for producing a foreign protein according to any one of claims 11 to 23, wherein the foreign protein is soluble in an aqueous solution. The method for producing a foreign protein according to any one of claims 11 to 24, wherein the foreign protein is either a cytokine or an antigen protein. The method for producing a foreign protein according to claim 25, wherein the cytokine is interferon and the antigen protein is a viral antigen protein.

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