JPH05508768A - Immunoglobulin production by Trichoderma - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cross-references regarding applications related to immunoglobulin production by Trichoderma This application is filed under United States Application No. 071552757, filed July 16, 1990. This application is a continuation-in-part application dated March 19, 1990, This is a continuation in part of Application No. 07/496155, which was filed in 1987. In a continuation-in-part of U.S. Application No. 071044077, filed April 29, All such applications are fully cited and incorporated herein by reference.

field of invention The present invention relates to a heterologous protein produced by Trichoderma, and specifically to a heterologous protein produced by Trichoderma. It relates to immunoglobulins produced by. Herein, Trichodel A method for producing the immunoglobulin by subjecting a member of the genus genus to genetic engineering technology. is disclosed. These products are particularly useful in treating medical conditions.

Background of the invention 1. Molecular biology of Trichoderma Filamentous bacteria are widely used in biotechnology to produce various fermentation products. It is a lower eukaryote that is Fungi produce many industrially important enzymes, such as glucose. Coamylase, protease, lactase, pectinase and glucose oxidase secretes sidase.

Filamentous fungi have some advantages with respect to biotechnology. They are generally numerous. Producing large amounts of protein, large-scale cultivation is not complicated, and mycelium from the culture solution after fermentation separation is easy. For example, the high cellulose decomposition of certain species of the filamentous fungus Trichoderma Strains can secrete more than 40 grams of cellulase per liter of culture medium. (Durand, Hl et al., “Biochemistry and Genetics In "Cellulase Degradation", Academic Press, San Diego. , 135-151 (1988), Durand, H8 et al., “Enzyme and. Microvial Technology, 10:341-345 (1988)). difference In addition, Trichoderma produces excess glycodilation of proteins in the case of Saccharomyces yeast. It has been pointed out that it will not be standardized (EP215594, Pentik, M9 , “Construction and Characterization on Cellulite "Chick Easts", VTT Publication Series No. 39, VT T Technical Research Center, Nispoh, Finland (1987)).

The mesophilic organism Trichoderma reesei (formerly Trichoderma viride) ( Simmons, E. G., “Abstracts on Second International Le Mycolon Cal Combres J Junior High, Tampa, Florida, Age' Yurou, H , E. et al. (eds.), p. 618 (1977)) on the conversion of cellulosic biomass. manufactures the significant quantities of enzymes needed and is probably the only cellulase-producing organism of all. It seems to be the most widely studied.

Three types of enzyme activities are required to hydrolyze cellulose to glucose. It is said to attack normally substituted soluble substrates and has activity against crystalline cellulose. Randomly cleavable endoglucanase (1,4,-β-D-glucanase) Lucanohydrase, EC3,2,1,4), can degrade crystalline cellulose, but Cellobiohydrolase (1,4-β- D-glucan cellobiohydrolase, EC3,2,1,9) and cellobiose β-glucosidase that attacks cellooligosaccharides and produces glucose (β-D-glucoside glycohydrolase, EC3,2,1,21). these Synergism in some of the enzymes has already been demonstrated (Bergem, L., E. R. et al., "European Journal on Biochemistry", 97: 2l-30 (1973), Gong, C.S., et al. Story Series”, 181:261-287 (1979), Fergustam. , L., G. et al., rFEBs Letters, 119:97-100 (1980)) .

Sono's cellulases have already been purified and characterized, and three main types of fermentation It was shown that all the elements exist in various forms (Enari, T, M,, r micro In "Vial Enzymes and Biotechnology", Fogarty, W.

M. (ii), Applied Science Publishers, London and New York, pp. 183-223 (1983)). Two immunologically distinct species Cellobiohydrolases CBHI and CBHII are cultured from Trichoderma reesei. Detected from the nutrient medium (Fergustam, L., G. et al., rFEBS Letters) , 119:97-100 (1980), Gilbert, 1. G, etc., “Annual ・Reports on Fermentation Processes”, 6:323-358 ( 1983)). Five to eight types of electrophoretically distinct endoglucanases have been reported. and many of them exhibit various substrate specificities (Shoemaker, S., p. , etc., [Biochimica, Eng., Biophysics, Acta. 978), Shoemaker, S., P., et al. Acta J, 523:147-161 (1978), Bisset, F. Null On Chromatography J, 178:517-523 (1979), Farkas, V., A., et al. : 105-110 (1982)). Characterization of two types of extracellular β-glucosidases have been reported (Enari, T. M. et al., Proceedings on Second International Symposium on Bioconversion and ・Biochemical Engineering”, in Goth, T., (ed.), Indy. An Institute on Technology, New York, 1:87-9. 5 (1980)).

Through intensive strain development using direct mutagenesis and screening methods , we were able to produce several high-yielding Trichoderma reesei mutant strains. (Bailey, M. J., et al., Enzyme and Microvial Technol. Gee”, 3:153-157 (1981), Andreotti, R2 et al. Seedings on Second International Symposium on "Bioconversion and Biochemical Engineering", Go S, T, K.

(ed.), Indian Institute on Technology, Newpuri. -, 1:353-388 (1980), Farkas, ■9 et al. Ikrobei.

26:129-132 (1981), Montencourt, B.S., et al. , Adhannes in Chemistry Series J, 181, 289-30. 01979), Mandels, M. et al., Applied Microbiology J, 2 1:152-154 (1971), Gallo, B. J., et al. and Bioengineering Symposium J, 8:89-102 (197 9), Warziwarda, M. et al., Biotechnology Letters, 5:24. 3-246 (1983), Montencourt, B.S., et al. "Microbiology", 34 Ney 77-782 (1977), Scheer Ney S., Go et al., “Applied Microbiology and Viatechnology. -”, 20:46-53 (1984), Shoemaker, S., p., et al., “Tre. Nuns in the biology on fermentation "Rules and Chemicals" medium, Polender-1A, E, (ig), Fre Nam P L/S, pp. 89-109 (1981), Nehalainen, K. M. H., et al. , Enzyme and Microvial Technology, 3:59-60. (1980)). Fine particles produced by the best Trichoderma reesei mutants The amount of extravesicular protein exceeds 50% of total cellular protein. Main part of secreted protein contains cellulases, among which the CBHI component is the most abundant and secreted. It accounts for less than 60% of the total cellulase protein.

The CBHI gene is derived from Shoemaker et al. (Shoemaker, Sl et al., “Bio/ Technology'', 1:691-696 (1983)) and Teeli et al. Li, T.

et al., Bio/Technology, 1:696-699 (1983)). The entire nucleotide sequence of the gene was published (Shoemaker, Sl. et al., Bio/Technology, 1:691-696 (1983)). Tricode The gene for the major endonuclease EG I was also cloned from Luma reesei. and characterized (Ventilla, Ml et al., Gene, 45:253-2 63 (1986), patent application EP 137280. Van Alspoor, J.N. V. et al., Bio/Technology, 5:6O-64 (1987)). other separation The cellulase gene identified was Cbh2 (Tieli et al., Gene, 51:43- 52 (1987), Patent No. 1 [Wo 85104672, Cheno, C.M., et al. Bio/Technology, 5:274-278 (1987)) and previously was called EGL 3 (EGL 2 (Saloheimo, Ml, etc., "Gene", 63:1l-21 (1988)).

The molecular biology of industrially important filamentous fungi is generally poorly known. This is one due to lack of reproductive reproduction cycles and/or genetic transformation systems. Ru. The transformation system is suitable for some filamentous fungi, such as N. crassa. Isu, Ml et al., [Bronding's on the National Academy of Sciences] Sciences on the United Stake on America J. 76:5259-5263 (1979)), Aspergillus nidulans (B. Lance, J.

etc., “Biochemical and Biophysical Research Communications” 112:284-289 (1983), Tilburn, Jl et al. 26:205-221 (1983), Inilton, Ml et al. dings on the national academy on sciences on ・The United Stake on America”, 81:1470-1474 (1984)) and Aspergillus niger (Kelly, J. M. et al., rEMB 4:475-479 (1985)), Aspergillus oryza E (Gomi, K1, etc., “Agricultural and Biological Chemistry”) Tory J, 51:2549-2555 (1987)) and Mattern, 1. E, et al., Molecular and General Genetics, 210:460. -461 (1987)) and Aspergillus millet 1 Babelka, R, M, et al. , Gene, 86:153-162 (1990)) has been developed. , these are generally complemented in the mutant host by each functional gene carried by the vector molecule. have their basis in sexuality. However, among these fungi, Aspergillus Only S. niger, Aspergillus awamori and Aspergillus oryzae are It is currently of industrial interest.

In the classification of fungi, the genus Aspergillus is classified into the class Ascomycetes and the class Euascomycetes. Included in the subclass Cethes. Euascomycetes is a plectomycete based on the fruiting body. It is divided into three groups: Cethes, Bilenomycetes, and Discomycetes.

The most important genera are Aspergillus and Penicillium (Toy, Y9, etc., 'Bureta' On Natural Science On Mast J, 11:185-189 ( 1968)). On the other hand, Trichoderma is classified as a member of the Deuteromycetes . Deuteromycota are sexually regenerative or sexually regenerative genera, such as the highly characteristic Aspera. It is a catch-all type of fungus that has no obvious affinity with Lugilus. to Lycoderma is a less well-defined incomplete state of the complete ascomycete Hypophlea Although it has been reported to have a reproductive stage with no Biotechnology Biogenesis”, 22:2429-2432 (1980 )), the genera Aspergillus and Trichoderma are clearly very different taxonomically It is considered a thing. Also, arg B from Aspergillus nidulans gene and the pYr4 gene from Neurospora crassa is a non-stringent It was shown that it does not hybridize with each Trichoderma gene under gentle conditions. This led to the idea that Trichoderma is evolutionarily distinct from other ascomycetes. It's proven.

Due to its remarkable ability to secrete proteins into the growth medium, Trichoderma lyse B. is an excellent candidate as a promising host for protein production. However, Genetic testing for Coderma reesei has so far focused almost exclusively on fungal cellulase production. The aim is to improve production characteristics and develop a Trichoderma strain with high cellulase productivity. The only techniques used were traditional mutagenesis and screening. .

i.e., the fungal traits first developed for Aspergillus and Ninirospora In transformation systems, transformation is performed using protoplasts. usually trait Integrate the convertible DNA into the host genome. Selection to identify stable transformants In order to provide a selective system, the vector system must be able to detect the corresponding mutations in the host genome. activities that supplement or are required for the growth of prototrophic strains in specific growth media; Usually it must have a functional gene (selectable marker) that supplies the enzyme.

Durand et al., “Biochemistry and Chemistry on Cellulose 'Essential Degradation', edited by Araberto et al., Academic Press, 1988. ．． On pages 135-151, Trichoderma forms based on phleomycin resistance. A transformation system was reported. However, the transformants remain free for long periods of time. Mycin was unstable if not linearly inoculated or kept on plates (Berges, T1 etc., “Abstracts, Tricell 89, International ・Symposium on Trichoderma Cellulases'', Vienna, 1989. (p. 21, also reporting a Trichoderma transformation system). But long The Berges system also produced unstable transformants.

Il, immunoglobulins Immunoglobulins consist of two identical light chains joined by a sulfur bridge and two identical Composed of heavyweights. Immunoglobulin polypeptide chains fold independently formed from continuous domains that can be The light chain is composed of two domains, A heavyweight is usually composed of 4-5 domains. Immunoglobulins protect against these It is present at the N-terminus of both the light and heavy chains that can recognize and specifically bind to This is because the variable domains are different.

Antibody molecules consist of protein hydrates in two parts, the so-called Fc and Fab fragments. Can be decomposed. Fab fragments contain light and light molecules involved in antigen identification and binding. Contains sites from both the heavy and heavy variable domains.

The domains of Fc fragments play several roles in the activation of immune responses in cells. Play the wari. Biochemical tests show that Fc fragments are not required for antigen binding. It shows.

Genetic engineering technology has made it possible to produce only the Fab antigen-binding site of antibodies. (Bellator, M9 et al., Science, 240:1041-1042 (1988 ), Cabin, S2, Gene, 85:552-557 (1989)). Mr. By combining independently foldable domains of antibodies in various ways, It has also become possible to produce chimeric antibodies (Morrison, S. L., EP173 494, Cabin, S8, EP125023). For example, human antibody domain Altering the antibody spectrum by combining with variable mouse antibody domains became possible. In addition, genetic engineering technology has enabled the antigen-binding site to become a unique polypeptide. It is also possible to produce single-chain antibodies formed by tide chains.

Monoclonal antibodies are produced in hybridoma and myeloma tissue culture; Human or mouse hybridoma cell lines are often used. These methods monoclonal antibodies from human hybridoma cell lines are quite expensive. The body yield is relatively low (compared to 100 μg/ml for mouse vibrid-7). (1 μg for human hybridomas).

Furthermore, most human monoclonal antibodies obtained in cell culture are of the 1gM type. belongs to If it is desired to obtain human monoclonal antibodies of the IgG type, Using cell sorting techniques, IgG or other antibodies can be extracted from large numbers of 1 gM type antibodies. It was necessary to isolate the few cells that had switched to producing antibodies. child In this case, the cost increases significantly and the yield of human 1gG type monoclonal antibody is 1. It is even lower than that of the gM type.

The antibody gene is transferred to a host organism, such as the bacterium Escherichia coli (vector, Mo, etc.). Science J, 240:1041-1042 (1988)) and yeast Satsukaro Mysis cerevisiae (Horovitz, A., H. et al., “Proceedings ON THE NATIONAL ACADEMY ON SCIENCES ON THE UNAI Ted Steve on America, 85:8678-8682 (1988) ) efforts have been made to develop better methods. These are F It has been used in the production of ab sites. Escherichia coli also produces single chain antibodies. used for construction.

However, perhaps in most cases the organism's ability to secrete proteins is inherently poor. The yield of active product is still low due to the That is, there is still a large amount of immunity globulins and monoclonal antibodies especially required for industrial and medical applications. There is a need for an economical method of manufacturing.

Summary of the invention Violet of immunoglobulin and especially monoclonal antibody applications in industry and medicine We acknowledge that the production costs of antibodies in the quantities required for these applications are high. Knowing that, the inventors have developed a large number of recombinantly produced immunoglobulins. Research on the use of recombinant DNA technology in designing hosts useful as scale sources. I've been researching it.

As a result of these tests, large amounts of foreign proteins and especially immunoglobulins, phosphopeptides and their production of immunoglobulins and immunoglobulin peptides. A sono host expressing the biologically active moiety was developed.

The present invention relates to the expression of heterologous proteins and particularly to immunoglobulins produced by recombinant techniques. immunoglobulins, immunoglobulin peptides and their immunoglobulins and immunoglobulins Trichoderma transformation set for expression of biologically active portions of bulin peptides Replacement DNA cloning vectors and vector systems are described. Main departure Transformation of Trichoderma with bright vectors or vector systems results in transformation. When microorganisms grown in a suitable culture medium are grown, high levels of the desired foreign protein can be obtained. is expressed and secreted.

According to its first aspect, the invention provides: a) Encodes an expressible protein of interest or a biologically active fragment thereof at least one gene, and b) A selection mask that allows the selection of Trichoderma hosts transformed by the vector. Provides a vector containing.

According to its second aspect, the invention provides vectors for one or more plasmids. system, a) a protein product of interest or a biologically active fragment thereof; both one gene, and b) a small number of cells that allow selection of Trichoderma hosts transformed with the above vectors; at least one selectable marker that encodes a protein product of interest; or a plasmid that is not the same plasmid that provides more genes ( vector containing a selectable marker given to the Trichoderma host as described above (there may be more than one marker). Provides a tar system.

In preferred embodiments of the first and second aspects of the invention, the protein product of interest or Genes encoding biologically active fragments of immunoglobulins or their immunoglobulin This gene encodes an epidemiologically active fragment.

In another embodiment of the first and second aspects of the invention, the protein product of interest or The gene encoding its biologically active fragment is a signal or leader sequence. It is in close proximity to the column at its 5° end and is functionally linked to it during the transcription-decoding phase. , said signal or leader sequence is said to be used in said target cell from said Trichoderma host cell. The amino acid sequence is capable of directing the secretion of the protein, and the amino acid sequence is capable of directing the secretion of the protein of interest or its biological activity. can be cleaved from the sexual fragment.

In another embodiment of the first and second aspects of the invention, the protein product of interest or The gene encoding its biologically active fragment is -, signal/leader sequences, terminators, and combinations thereof. in close proximity to, and in association with, one or more DNA regulatory agents selected from the group (and, if necessary, in the accompanying transcription-decoding phase); The DNA sequence is operably linked to the gene to express the desired protein product. and/or control secretion.

In another embodiment of the first and second aspects of the invention, the protein product of interest or its A DNA fragment operably linked to a gene encoding a biologically active fragment. The nodal sequence is a DNA sequence homologous to the Trichoderma genome sequence.

In another embodiment of the first and second aspects of the invention, the protein product of interest or is a phase operably linked to the gene encoding the biologically active fragment. The same DNA regulatory sequence contains genes expressed in glucose, cbh1 gene, cb Selected from DNA regulatory regions of h2 gene, egll gene, and eg12 gene be done.

In another embodiment of the first and second aspects of the invention, transformed by said vector The selectable marker that allows selection of transformed Trichoderma hosts is phleomycin resistance, asdS, trpC and argB selectivity.

In its third aspect, the present invention provides a method for transforming Trichoderma; In this method, suitable Trichoderma strains are transformed with the vector or vector system of the present invention. be converted.

In its fourth aspect, the present invention provides a method for producing a heterologous protein using Trichoderma. and the method provides for the production of Trichoderma by the vector or vector system of the present invention. The transformed strain was cultured in an appropriate medium, and expressed and secreted from the medium. Collect the product.

In a preferred embodiment of the fourth aspect, the heterologous protein is an immunoglobulin, an immunoglobulin, Globulin light chain, immunoglobulin heavy chain, and the above-mentioned immunoglobulins, immunoglobulins selected from the group consisting of immunologically active fragments of immunoglobulin light chains and immunoglobulin heavyweights. selected.

In its fifth aspect, the present invention provides immunoglobulins and immunoglobulins in Trichoderma. Robulin heavy chain, immunoglobulin light chain or said immunoglobulin or immunoglobulin Provides a method for producing biologically active fragments of bulin heavy or immunoglobulin light chains. The method involves transforming Trichoderma with the vector or vector system of the present invention. The transformed strain is cultured in an appropriate medium, and the immunoglobulins expressed and secreted from the medium are collected. Collect the Robulin product.

In another embodiment of the fourth and fifth aspects of the invention, said immunoglobulin globulin heavy, immunoglobulin light chain or said immunoglobulin or immunoglycemic agent Robulin heavy or biologically active fragments of immunoglobulin light chains can be globulin, immunoglobulin heavy, immunoglobulin light chain or said immunoglobulin biologically active fragments of immunoglobulin heavy chains or immunoglobulin light chains. re-associate in vitro with patients.

In its sixth aspect, the present invention uses the vectors and vector systems of the present invention. To provide a Trichoderma strain stably transformed with a gene of interest.

In its seventh aspect, the invention provides a fusion protein.

In a third aspect of the present invention, the heterologous protein, particularly immunoglobulin, immunoglobulin, immunoglobulin heavy chain, immunoglobulin light chain or the above immunoglobulin, immunoglobulin Provides compositions enriched in biologically active fragments of phosphorus heavy chains and immunoglobulin light chains do.

Brief description of the drawing Construction drawings are not drawn to scale.

FIG. 1 shows the principle of inactivation of chromosomal cellulase genes.

Figure 2 shows insertional mutagenesis of the Trichoderma reesei chromosomal CBHI locus. The construction of the plasmid pMS4 used is shown. Inactivation of the EeoR1 site induces Mark the position of the emitted frame soft mutation with a ★.

Figure 3 shows the construction of plasmid p285°proC.

Figure 4 shows the construction of plasmids pMT648 and pMT813.

Figure 5 shows the construction of plasmid pMT837.

Figure 6 shows the restriction map of plasmid pR27.

Figure 7 shows the restriction map of plasmid pAMH100.

Figure 8 shows the construction of plasmid pAMH105.

Figure 9 shows the results using synthetic oligonucleotides OAMHI, OAMH2 and OAMH3. Plasmids pAMH1103, pAMH1106 by loop mutagenesis and construction of pAMHllol.

Figure 10 shows linkers NOR202, N0R203, OAMHI, OAMH2 and and OAMH3.

Figure 11 shows the restriction map of plasmids pAMH102 and pAMH104.

Figure 12 shows the restriction map of plasmid pAMH1103 and the construction of pAMH103. shows.

Figure 13 shows the restriction map of plasmid pAMH1106 and the construction of pAMH106. shows.

Figure 14 shows the restriction map of plasmid pAMH1101 and construction of pAMHlol. shows.

Figure 15 shows the construction of plasmid pAHM110.

Figure 16 shows the platform used for the expression of EGI under the function of the CBHI promoter. Figure 2 shows the construction of sumid pAMH111.

Figure 17 shows Western profiling of chymosin expression in Trichoderma reesei. Show cut. Lane 1: Purified prochymosin control, traces of pseudochymosin and Contains chymosin. Lane 2; culture supernatant from growth of pAMH102-containing strain.

Lane 3; control supernatant from plasmid-free strain. Lane 4; containing pAMH102 Mycelium from the strain.

FIG. 18 is a diagram of plasmid pLT1 with subclones of light chain cDNA.

Figure 19 shows the subclone of IgG3 heavy chain CDNA (■H, 01N, partial CH□). FIG. 2 is a diagram of plasmid pLT2 containing

Figure 20 shows the subclones of IgG1 heavy chain cDNA (V ■ and Cut). FIG.

Figure 21 shows plasmid pEN10 containing a subclone of the chromosome cbh1 gene. It is a figure of 1.

Figure 22 shows bacterial expression of plasmids pEN304 and pEN305, light chain cDNA. An outline of vector construction is shown.

Figure 23 shows intermediate vectors for constructing plasmid pEN207 and plasmid pEN209. An outline of the construction of the

Figure 24 shows plasmid pEN209, expression vector for cbh 1-heavy chain Fab fusion. An outline of the construction of the

Figure 25 shows the construction of plasmid pAJ202, an expression vector for heavy chain Fab cDNA. The outline is shown below.

Figure 26 shows a schematic of the construction of plasmid pUJ10.

Figure 27 shows the construction of plasmids pEN401 and pEN402.

Figure 28 shows the construction of plasmid pMPH31.

Figure 29 shows the structural gene of a single heavy chain antibody with cbh f-ring as a linker. An outline of the elements of plasmid pML5 is shown.

Detailed description of the invention 1. Deposit information The following deposits of biological material shall be made in this matter by the Central Bureau of Fall. Schimmelkurtles (P-O Box 273, Austilstrad 113 It was held in 740 A.D. Baarn, Netherlands). Receiving number CB5 As 252.90 (dated June 25, 1990) light chain productivity Trichoderma li Sei strain (pEN304/RUT-C-30), receiving number CBS 287.9 1 (dated June 3, 1991) CBHI-IgGI Fab fusion antibody productivity Trichodel? -Reesei strain (pEN304/pEN209/RUT-C-30) and as acceptance number CBS 288.91 (dated June 3, 1991) Ig GI Fab antibody-producing Trichoderma reesei strain (pEN304/pAJ20 2/RUT-C-30). Also, Masheroder Bake 1Bay Day-33 00 Fuselaunschweik (Germany) Teutche Samlung Fono Miku Roorganismus Land Zerkrullen Gesellschaft Mitsut Beshi Müllenkter Haffrang (German Collection in Micro Auger) Deposit was made with Nisms and Cell Cultures). Receiving number D As SM6584 (dated June 21, 1991) Plus in Escherichia Cori Mido pAJ202, received as number DSM6585 (June 21, 1991) Attachment) Plasmid pEN209 in Escherichia coli, accession number DSM65 86 (dated June 21, 1991) Plasmid pE in Escherichia coli N304, received as serial number DSM6587 (dated June 21, 1991). Plasmid pEN401 in E. coli and accession number DSM6588 (dated June 21, 1991) as plasmid pEN4 in Escherichia coli. 02゜ 2. Definition In the following description, recombinant DNA (rDNA) used in science and immunology The word tsukuhi is widely used. of the claim, including the scope given to those words. In order to provide a clear and consistent understanding of this specification, including its scope, the following definitions are provided: give righteousness.

gene. A DNA sequence that contains a template for RNA polymerase. RN encoding protein A is called metsenger RNA (+II RNA) and is an RNA port in eukaryotes. Transcribed by limerase II.

“Complementary DNAJ or rcDNAJ genes are synthesized by reverse transcription of mRNA. contains a recombinant gene from which intervening sequences (introns) have been removed.

Proteins homologous to the Trichoderma host of the present invention are non-transforming proteins of the same species as the host species. This refers to cases in which Trichoderma spp. naturally produces a certain amount of natural protein. Main departure A gene homologous to the bright Trichoderma host is defined as a non-transformed trichoderma homologous to the host species. Refers to genes found in the Luma genome.

Proteins that are foreign to the Trichoderma host of the present invention are non-transparent proteins of the same species as the host species. This refers to the case in which Trichoderma does not produce natural proteins at all in its natural state. Main departure Genes that are foreign to the Ming Trichoderma host are This gene is not found in the Koderma genome.

cloning vehicle. suitable for transferring genes of interest to host cells. Plasmid or phage DNA or other DNA sequences that provide a nucleic acid environment (e.g. linear DNA). The cloning vehicle of the present invention is suitable for prokaryotes and eukaryotes. Can be designed to replicate autonomously in a biological host. In Trichoderma, Loning vehicles generally do not autonomously replicate, but instead simply act as objects of interest. It simply provides a vehicle to carry a certain gene to the Trichoderma host, resulting in This is followed by insertion into the Trichoderma genome. In addition, cloning bee The DNA sequence of the vehicle can be determined without loss of essential biological function of the vehicle. The DNA can be cleaved in a manner that allows the DNA to be spliced into one or a few endonuclei upon which DNA can be replicated and cloned ase recognition site. Furthermore, cloning vehicles are capable of cloning May contain markers suitable for use in identifying cells transformed by the vehicle .

The word "vector" is also used in relation to "cloning vehicles". be.

Alternatively, those markers are independent of those supplying the gene of interest. can be included in various cloning vehicles.

Expression vehicle. Similar to cloning vehicles, but for transformation into the desired host. After conversion, a vehicle or vehicle capable of expressing the gene of interest cloned into it or vector. In preferred embodiments, the expression vehicles are transformed into the desired host. After transformation, the cloned gene of interest is expressed at a high rate. .

In a preferred embodiment, the gene of interest that is desired to be expressed is cloned. It is provided to the bacterial host as part of an expression vehicle and integrated into the bacterial chromosome. Ru. Additionally, sequences derived from cloning or expression vehicles may be can be integrated together with the gene of interest during the reading process.

The expression vehicle may also contain the gene of interest, e.g. or a light chain sequence, or a desired biologically active portion thereof, or a portion thereof; , which, when reassembled with another peptide, yields a biologically active protein product. plasmids or other vectors. The above genes of interest are usually , a suitable overhang designed such that the sequence with a suitable overhang can be easily inserted into the Contains restriction sites. The above-mentioned vehicle is a protein of interest in a suitable Trichoderma host. It can be used as an intermediate for quality expression.

immunoglobulin. The term "immunoglobulin" refers to immunological properties (e.g., antigen binding). e.g., a multi-subunit protein containing two heavy chains and two light chains. It is intended to include target antibodies. As used in this specification, the term also refers to the protein containing the amino acid sequence of the individual light chain or heavy chain of such an antibody; A light or heavy compound is modified (e.g., by reassociation with another immunoglobulin) shall be capable of demonstrating their inherent immunological activity.

[Immunoglobulin gene] as used herein refers to the above-mentioned immunoglobulin gene. or refers to a DNA sequence encoding a light chain.

immutable or mutable. The terms "invariant" and "variable" refer to the variable and an immunoglobulin that is a heavy or light chain that encodes properties and characteristics possessed by constant regions Used according to function when indicating a region of the phosphorus chain. There is a functionally active area and, if available, there is a full encrypted region for mutable or immutable regions. There's no need to.

domain. The term "domain" may or may not function independently. used to describe the independently folded parts of a protein. natural protein A general definition of domain boundaries in Argos, R9, “Journal In Molecular Biology, 211:943-958 (1990). be discovered.

Functional derivatives. A "functional derivative" of a protein is a small peptide of that protein. the peptide has the desired biological activity (functional or structural) or has a known They can exhibit the above-mentioned activity by being modified in a known manner. For example, immunoglobulin Functional derivatives of light chains have been found in Fab fragments, with appropriate functional induction of the light chain. It retains its inherent activity until it is reassembled with the body into immunoactive Fab fragments. It may be a fragment of the heavy chain not shown. As used herein, a A protein is called another if it contains additional chemical moieties that are not normally part of the protein. It is said to be a "chemical derivative" of the protein. Those parts are dissolved in protein. properties, absorbability, biological half-life, etc. On the other hand, the above part is related to the toxicity of the protein. This can reduce or weaken the undesirable side effects of proteins. Them The part that can mediate the action is Seeds” (1980). The method for binding the above portion to protein is , is well known in the industry.

Functional derivatives of proteins are created by post-translational modifications due to the need for modification to perform specific functions. It may or may not include modifications, such as covalently bound carbohydrates. "function The term "derivative" refers to a "fragment," "variant," "analog" or shall include "chemical derivatives."

fragment. A protein "fragment" is a longer protein amino acid sequence. It refers to peptides that have some, but not all, portions.

As used herein, a protein is a function found in natural immunoglobulins. or by being modified (e.g. by another immunoglobulin flag) If the protein can exhibit said function (by re-association with protein), said protein It is said to be the active fragment.

"Mutant". A protein “variant” is a naturally occurring protein or its fragment. Substantially similar in structure and biological activity to agents, but which have been manipulated. refers to proteins containing different amino acid sequences. In other words, two proteins If the white matter has similar activity, the primary structure of one protein will be the sequence found in the other. The term is used herein even if it does not have the same amino acid sequence as As such, they are considered mutants.

IIl, Antibody Structure and Identification In the following description, various methods known to those skilled in the field of immunology are cited. There is. Standard references describing general principles of immunology include Caddy, D. “Antipodes, A Practical Approach”, Volume 1, IRL Print Res, Washington, DC (1988)), Frayn, J. (Immunology: The ・Science in Self-Non-Self Discrimination”, John ・Willey & Sons, New York (1982)), Kennett, R1, etc. (“Monoclonal Antipodes, Hybridomania Two Days”) Mention in Biological Analysis, Blenheim Press, New York (1980)), Campbell, A.F. “The Monoclonal Antibody” Laboratory Techniques in Biochemistry ・And Molecular Iology” Volume 13, (edited by Hardon, R., etc.) , Elsepia, Amsterdam (1984)) and Eisen, H.N., f. "Microbiology" 3rd edition, Davis, BD3, etc., Haber & Row , Philadelphia (1980)).

If an antibody can bind a molecule to the antibody by it reacting specifically with the molecule , is said to be "capable of binding" to a molecule. The term "epitope" refers to refers to the portion of an antigen that can be recognized and bound. The antigen is one or more antigens. May have pitopes. "Antigen" is an antigen that an animal can bind to an epitope of that antigen. can be induced to produce the body. The above-mentioned specific reaction occurs when the antigen responds to its corresponding Antibodies react with numerous other antibodies that react in a highly selective manner and can be evoked by other antigens. indicates that there is no reaction.

As used herein, "immunoglobulin", "antibody J (Ab)" or "monoglobulin" The term clonal antibody J (Mab) refers to the intact molecule and its fragments, their peptides, and biologically active fragments of said intact molecules and peptides. fragments, such as Fab and F(ab')2 flags capable of binding antigens shall include ment.

Fab and F(ab')2 fragments are Fc fragments of intact antibodies. is removed from the circulation more quickly and is less susceptible to non-specific antibodies than intact antibodies. Woven bonding properties may be poor (Wahl et al., Journal in Nuclear Medicine). ”, 24:316-325 (1983)).

Because immunoglobulins usually contain two pairs of chains linked together by disulfide bridges , as used herein the term "immunoglobulin" refers to the chains whose structure is characterized by their unique properties. even though it may require reassociation with a second immunoglobulin chain to exhibit biological activity. shall include individual immunoglobulin chains (“immunoglobulin peptides”) .

The immunoglobulins of the present invention can be of any type, such as IgM, IgG, IgAS IgD and IgE and any of the various subtypes of the individual types can belong to The light chain can be kappa or lambda. The heavyweights are μ( Mu), γ (gamma), δ (delta), α (alpha) or ε (nibusilon) ). In animals, different groups of genes encode light and heavy chains. those Bioactive parts of the chain or domains from various bells can be genetically engineered upon request. can be recombined.

Light and heavy are divided into domains with structural and functional homology.

Both light (■, ) and heavy (VH) variable regions are involved in immunoglobulin recognition and determine specificity and specificity. The light (C0) and heavy (CH) constant region domains are Provides essential biological properties, such as antibody chain association, secretion, transplacental mobility, and complement fixation. do.

A complex series of events induces immunoglobulin gene expression in B cells.

The V (variable) region gene sequences that confer antigen specificity and binding are V□, D (multiple). ) and JR (“J” for joining), or another called VL and JL. located in each germline gene segment. These gene segments are DN are joined by A rearrangement to form a complete V region expressed in the heavy and light chains, respectively. Ru. Then the rearranged junctions (VL-J, and VH-D-JH) V segments Each encodes a light and heavy complete variable region or antigen binding domain.

Monoclonal antibody hybrid desired to be cloned and expressed according to the present invention The mercury source is manufactured by any of a variety of methods. monoclonal Antibodies can be produced using hybridoma technology (Cola et al., Nature , 256:495 (1975). N. Immunology 6:511 (1976), Colla et al. Journal in Immunology”, 6:292 (1976), Hamerling et al. "Monoclonal Antipodes and T-Cell Hybridomas" ”, El Sepia, New York, pp. 563-681 (1981)). in general, The method involves immunizing an animal with the protein of interest. Extract the spleen cells of the animal , fused with an appropriate myeloma cell line. According to the present invention, a suitable myeloma cell line All can be used as long as they are available. However, the American Myeloma parent cell line available from the Lycan Type Culture Collection. It is preferable to use SP20. Hybridoma cells generated after fusion are selectively maintained in HAT medium and then, for example, in Wands, J. R., et al. 80, 225-232 (1981). (which is incorporated herein by reference), is cloned by limiting dilution. next , by assaying the hybridoma cells obtained through the above selections. Clones are identified that secrete antibodies capable of binding the protein.

By applying the above method, an additional antibody that recognizes the epitope of the target protein can be produced. Additional cell lines are obtained.

A wide range of donor hybridomas are suitable for the production of recombinant immunoglobulin constructs of the invention. It can be used immediately for construction. For example, the catalog FATCC Cell Lines & Hybridomas”, American Type Culture Collection (123 0 Everclone Drive, Rockville, MD, 20852 Unite Steve Stave in America), and ECACC Catalog, 2nd Edition (PH LSCAMR, Porton Down, Salisbury, Wills, UK) reference.

Hybridomas that secrete monoclonal antibodies that react with various antigens are These are listed here, are available from collections, and can be used in the present invention. Special Of interest is the ability to isolate antibodies that react with viral, bacterial, and tumor antigens. It is a secreting hybridoma.

If polyclonal antibodies are desired, the protein of interest or its antigenic fragment is By administering cells expressing a protein to an animal, a protein that can specifically bind to the protein can be detected. Production of serum containing reclonal antibodies can be induced. Another method is to simply isolate the protein. or chemically synthesize peptide fragments and purify them by HPLC. This renders it substantially free of contaminants. Then operate the product. A polyclonal antiserum with high specific activity can be produced by introducing the antiserum into a product.

The polyclonal antibody is a target monoclonal antibody produced according to the method of the present invention. It may be particularly useful for identifying clones that secrete the body.

IV, Trichoderma host The host strain can be prototrophic or trophic depending on the selection marker used in the transformation method. It may be a auxotrophic Trichoderma strain. Complement the corresponding mutagenesis of the host genome When using a selectable marker, construct an auxotrophic Trichoderma mutant strain. There must be. The auxotrophic Trichoderma mutant strain is a known mutant strain. It can be manufactured by a manufacturing method.

Nutrient requirements that require uracil, tryptophan or arginine to grow The sexual Trichoderma mutant strain was described by Nevalainen (Nevalainen, Hl, Technical Research Center in Finland, Publication 26 (1985)) filtration and concentration techniques. requires arginine A series of minimal auxotrophs supplemented with different intermediates in arginine biosynthesis were obtained. By using the plate, a mutant strain lacking the argB gene was identified.

Mutant strains lacking the trpC gene have PRA isomers in their mycelium. may be characterized by a lack of InCP synthetase activity (Creighton, T.E. , Biochemical Journal, 120:699-707 (1970)) . The trpC characteristics of mutant strains that exhibit no enzymatic activity are, for example, Aspergillus. ・Confirmed by transformation and complementation with trpC-plasmid of P. nidulans (Inilton, Ml et al., Proceedings in the National ・Academy in Sciences in the United Steve I America, 81:1470-1474 (1984)).

■7 Design of recombinant constructs The genetic engineering process for the host of the present invention may encode a protein of interest. This is facilitated through cloning of gene sequences and expression of said gene sequences.

As used herein, the term "gene sequence" refers to a nucleic acid molecule (preferably D NA). Gene sequences that can encode the target protein can be obtained from a variety of sources. Derived from the source. These sources include genomic DNA, cDNA, synthetic DNA and and combinations thereof, derived from sources that naturally encode the protein of interest. is preferable.

Produce immunoglobulins and especially monoclonal antibodies with defined specificity Hybridoma cell lines are used to isolate RNA or genomic DNA. Next, a gene capable of expressing the immunoglobulin according to the present invention is cloned. This is particularly useful when The mRNA or genomic DNA may be any of the following methods known in the art: techniques, such as Maniatis, T1, etc., ``Molecular Cloning (A Laboratory Cold Spring Harbor Laboratory, No. 1 2nd edition (1989).

A wide range of donor hybridomas are available for the production of mRNA or genomic DNA. can be done. For example, the catalog FATCC Cell Lines and Hybridomas American Type Culture Collection, 12301 Park Row 20852 United Stave Drive, Rockville, MD ・In America), and ECACC Catalog, 2nd Edition (PHLS CAMR , Porton Down, Salisbury, Wills, UK). Various hive that secretes immunoglobulins and especially monoclonal antibodies that react with antigens The redomas listed therein are available from the collection and can be used in the present invention. It is Noh. Of particular interest are reactions with viral, bacterial and tumor antigens. It is a hybridoma that secretes a responsive antibody.

The recombinant protein product according to the present invention is a tricogene modified by recombinant DNA technology. Produced by M. derma. By modifying the Trichoderma host of the present invention, Native or chimeric immunoglobulins are expressed. What is “natural” immunoglobulin? The coding sequence was originally formed as a result of genome rearrangement in animal cells. means an immunoglobulin. “Chimeric” immunoglobulin is its code sequence was originally formed as a result of in vitro recombinant DNA techniques. immunoglobulin.

Modifications to the Trichoderma hosts of the present invention allow for one or more immune Antibiotic globulins and especially immunoglobulin chains can be produced in large quantities. With this invention Then, the expression of the target gene is carried out while it is operably linked to the promoter. By inserting the gene encoding the immunoglobulin as described above, It is possible to enrich the Trichoderma host with respect to the objective immunoglobulins. good In preferred embodiments, the promoter is a strong homologous promoter. very liked In a new embodiment, the gene of interest is inserted into the cbh1 locus or already A cassette containing the gene of interest operably linked to the homologous Cbhl promoter. is inserted into the cbh1 locus (U.S. Application No. 07152430 No. 8, this is cited as part of the explanation). In addition to cbh 1, promoter as well as other transcriptional, translational and secretory control signals useful in the expression vectors of the invention. Other homologous genes that can provide glucose include, for example, genes expressed in glucose and cellulase genes cbh2, egll, eg12, formerly called eg 13 (These are protein cellobiohydrolase 11 cellobiohydrolase II, encoding endoglucanase I and endoglucanase II) or this (Knowles, Jl et al., "Tibtech", 5:25) 5-2601987)). Trichoderma derived from genes related to homologous proteins. An example of a promoter that has been used is the Aspergillus glucoamylase promoter. , and Aspergillus amds, gpd, argB promoter (pente M9 et al., Gene, 61:155-164 (1987)) and trp C promoter. A synthetic promoter is a combination of two or more different promoters. can be constructed to include regulatory regions derived from natural promoters.

As a result of eliminating the activity of either cellulase gene, glucose is removed from cellulose. A host partially or completely deficient in the ability to decompose is obtained.

The cellulase-deficient strain is, for example, a plasmid containing only a portion of the homologous coding region. (insertional activation). This is an EP 244234. Alternatively, cellulase-deficient strains can also be A gene replacement strategy in which homologous recombination is achieved using 3′ and 3′ adjacent DNA. It can be manufactured by A marker gene or target protein product is inserted between adjacent regions. You can place the gene that encodes it. Lacking different cellulase enzymes (Trichodel The M. reesei strain has e.g. antibodies against different cellulases and especially monochrome Characterize for the presence or absence of specific cellulases using natural antibodies. (U.S. Application No. 07) No. 1524308).

Integration of the target gene with homologous recombination can be carried out using circular DNA, which is integrated into Trichoderma chromosomal DNA in a collinear manner. However, the line The use of shaped DNA particularly facilitates directed integration into homologous loci. emergency In a preferred example, the target gene is integrated into the Trichoderma cbh 1 gene. Directed to child seat.

Can be used to provide a source of homologous transcriptional and translational control sequences to a host of the invention Clones of cellulase enzymes are known in the art. For example, natural cellobiotic The gene related to the dolorase CBHI sequence was published by Shoemaker et al. , S9 et al., Bio/Technology, 1:691-696 (1983)) and (Thiri, T9 et al., “Bio/Technology”, 1:696-6 99 (1983)), and the entire nucleotide sequence of the gene is known (Shoemaker, Sl et al., Bio/Technology, 1:69 1-696 (1983)). , from Trichoderma reesei, the same (main species) The gene for EG I was cloned and characterized. (Pentilla, M5 et al., Gene, 45:253-263 (1986), special Patent application EP137280. Pan Alspur, J.N., V., et al., “Bio/Technology 5:6O-64 (1987)). Other isolated cellulase genes cbh 2 (Thieli et al., Gene, 51:43-52 (1987), Patent application WO35104672, Cheno, C.M., et al., “Bio/Technology ', 5:274-278 (1987)) and eg, formerly called eg13. 12 (Saloheimo, M9 et al., Gene J, 63:1l-201988)) .

According to this invention, proteins of interest, such as immunoglobulins of interest and especially immunoglobulins, can be A gene construct encoding the bulin chain can be introduced into the Trichoderma genome.

In addition, expression can be enhanced by using a strong promoter, such as cbh1. If desired, additional or modified regulatory regions, such as enhancer sequences, can be achieved. can be used. Preferably, said 111m sequence is homologous to Trichoderma. tone The nodal region and especially the promoter is an orthogonal region containing only the sequence components required for expression. and/or may be modified to retain regions responsible for high expression levels. workman The enhancer sequence is an independent DNA element, but one that is of interest. As a component operably bound to a gene, e.g. a gene of interest. as a colinear or NA sequence (e.g., 5' or 3' non-translated) can be introduced at the same time as the gene of interest (in a sequence or intron). Ru.

In a highly preferred embodiment according to the invention, the gene producing the protein of interest is specifically Introducing the gene locus and/or multiple copies of the gene into Trichoderma as described above. By introducing the protein into the genome of the target protein, an increased amount of the target protein can be obtained.

In another embodiment, the gene encoding the protein of interest also includes the gene encoding the protein of interest. Method of operably linking offspring to promoter and terminator sequences cloning, which allows the insertion of the gene of interest into an expression cassette. This method uses a vector that provides the site for integration into the Trichoderma genome. It can be done. Preferably, the cbhl gene promoter and terminator are used. be done. Additionally, the cassette contains a promoter and terminator that can be removed. Stuffer fragments may be provided between the sequences. For example, in patent application EP244234 Common expression vectors as described may be used, such as pAMHllo.

pAMHllo is derived from pUc19 (Yanishuperon et al., “gene”; 33:103-119 (1985)). The stuffer fragment in pAMH110 is , 5acII and NdeI. Coat the target protein After blunting the ends of the double-stranded DNA sequence to be coded, the DNA, cDNA or staining The genetic DNA can be inserted between the promoter and terminator. target gene is the plasmid pAMH110 between the Cbh1 promoter and terminator. can be inserted into the cbh1 expression cassette in.

If it is desired not to use one component of cbh, transcriptional regulatory elements of other genes can be used. can be used. For example, 3-phosphoglycerate kinase gene (pgk) transcriptional regulation Vector constructs containing node regions are key enzymes involved in ATP production by glycolysis. Can be used as 3-phosphoglycerate kinase, when glucose is present and the cell It is expressed under conditions in which the synthesis of the enzyme is suppressed.

Although the inventors do not intend to be tied to any particular theory, the effectiveness of the expression of the target gene is , the number of copies of the target gene integrated into the Trichoderma genome and the genome It seems that the difference depends on both the location of the gene and the location of the gene integration.

The DNA constructs produced by this invention can be used to develop any Trichoderma strain. Can be transformed. These strains include, for example, Trichoderma reesei strain Q M9414 (ATCC26921), RUT-C-30 (ATCC56765) , and advanced production such as VTT-D-79125, a descendant of QM9414. There are sexual mutant strains (Nevalainen 1985, Technical Research Center ・In Finland Publications 3ns 26 (1985), Nispo, Finland. land). Transformation of Trichoderma can be performed using techniques known in the art and in particular 198 Technology disclosed in United States Application No. 071044077 dated April 29, 2007 This can be done by

Trichoderma host cells are cultured, and the enzyme of interest is grown under conditions that allow expression of the enzyme of interest. can be produced by culturing a host strain with the desired properties. For example, Trichoderma host strains with desirable properties are, for example, 6% Sorca flots cellulo. 3% distilled liquor lees, 0.5% KH2PO4, 0.5% (NH4) ,S.

4. Can be cultured in a liquid culture medium that can contain 0.1% Struktol. objective group The recombinant gene is placed under the control of the cbh1 promoter in Trichoderma if it is sensitive to glucose repression and inducers such as cellulose, latin Cutose or sophorose is required (Allen et al., “Biotechnological and Bioengineering”, 33:650-656 (1989)). to The pH in Lycoderma culture can be adjusted to approximately p by adding phosphoric acid or ammonia. H5 and the temperature can be kept at 30°C during the culture. however, Temperature and pH should be adjusted according to the strain and protein produced ( Meliboori et al., Biotechnology Letters, 12:117-120 (1 990)).

The vector system can be used in the method for producing recombinant proteins and especially immunoglobulins of the present invention. can be used. The vector system includes (a) in the first vector, ).Lycoderma at least one gene of interest, such as an immunoglobulin, integrated into the genome of A gene encoding a chain or an immunologically active fragment thereof; and (b) in the second vector, at least Both encode one gene of interest and another gene, e.g. another immunoglobulin chain or or a gene encoding an immunoactive fragment thereof, and (CXa) and/or or (b) to provide a selectable marker that provides selectivity for Trichoderma containing It can be constructed as follows. Alternatively, the selectable marker may be provided on a separate vector. It can be done.

In another example, two genes encode two different genes of interest. The sets are integrated at different sites on the same vector.

The cloned DNA used in the host of the present invention also includes cases where it contains natural introns. It is possible that there is none. Furthermore, the genomic DNA is a natural 5 of the DNA gene sequence. “The promoter region and/or said sequence may function in Trichoderma In case 3, it is obtained with an accompanying transcription termination region. Furthermore, the genomic DNA is Gene sequence encoding the 5' untranslated region of RNA and/or 3' untranslated region It is obtained in a form accompanied by a gene sequence encoding the region. Trichoderma host cells are transcriptional and/or translational regulatory signals associated with mRNA and protein expression. to an appreciable extent, the 5' and/or 3' non-transcribed regions of the native gene, and /or 5° and/or 3° untranslated regions of mRNA are involved in transcriptional and translational regulation. can be kept and used for Genomic DNA is obtained using methods well known in the art. (e.g., “Guided Tow Molecular Cloning Techniques”) Nix, S., L., Berger Isoline, Academic Press (1987)) . Alternatively, mRNA can be obtained from any cell that produces or expresses the protein of interest. can be isolated and used for the production of cDNA by techniques well known in the art (e.g. For example, "Guide to Molecular Cloning Techniques", S, L , Berger Isoline, Academic Press (1987)). Preferably, use lllRNA products are isolated from cells that produce large quantities of proteins. Techniques commonly used for enrichment of mRNA products naturally or for specific sequences, e.g. Target proteins can be isolated in vitro by sucrose gradient centrifugation, or both. It is enriched for the encoding a+RNA.

For cloning into a vector, the appropriate NA product (genomic DNA or cDNA) is optionally sheared or enzymatically cleaved, and an appropriate vector ) - to create a recombinant gene (genome or cDNA) library. Lee was formed.

The DNA sequence encoding the protein of interest has either a blunt end or a star for ligation. Tugard termination, restriction enzyme digestion to provide suitable ends, as appropriate Cohesive end filling, alkaline phospha to avoid undesired binding by conventional techniques including tase treatment and ligation with appropriate gauze. Therefore, it can be inserted into a DNA vector. Techniques related to the operation are described by Maniatis et al. "Molecular Cloning (A Laboratory Manual)" (Cold ・Spring Harbor Laboratory-No. 11982), and is disclosed in the industry. It is well known.

Coding the protein of interest, e.g. immunoglobulin chains and especially the chains of monoclonal antibodies. Libraries containing clones to be coded can be screened to A clone containing, for example, a) a suitable nucleic acid containing a sequence specific to the DNA of this protein; b) hybridization with the probe(s) in question; The natural mRNA that hybridizes to the clone is translated in vitro, and the translation product is Hybridization selection translation analysis for further characterization, or C) clones. If the cloned gene sequences are themselves capable of expressing mRNA, The DNA of the protein is determined by immunoprecipitation of the translated protein product produced by can be identified by means of specific selection.

Target protein-specific oligos that can be used to identify clones for the target protein Nucleotide probes are designed from information about the amino acid sequence of a protein and are It can be purchased if desired.

Because the genetic code is synonymous, multiple codons can be used to code for specific amino acids. acids can be encoded (Watson, J.D., Molecular Biology In 3rd edition of ``The Genes'', by W. A. Benjamin, Incorporated, Me. Nguchi Park, California (1977) pp. 356-357). peptide fura analysis of the oligonucleotides with the lowest degree of synonymy. The encoded amino acid sequence is identified. This is preferably only a single codon This is done by identifying sequences that contain the amino acids encoded by.

Amino acid sequences may be encoded by only a single oligonucleotide sequence. However, in many cases the amino acid sequence is determined by one of a set of similar oligonucleotides. can be coded by Importantly, the components of this set all contain the same peptide. contains an oligonucleotide sequence capable of encoding a fragment, i.e. potentially Contains the same oligonucleotide sequence as the gene encoding the peptide fragment, but with a set Only one component of the gene has a nucleotide sequence identical to the encoding sequence of the gene. include. This component is present in the set and can interact with DNA even in the presence of other components of the set. A single oligonucleotide can be used to encode a peptide because it can be hybridized. Generate a non-fragmented set of oligonucleotides in the same manner as when cloning a gene. It is possible to use

Genetic Code (Watson, J.D., "Molecular Biology in the ・Gene” 3rd edition, W, A, Benjamin, Incoholated, Menguchi・ Park, CA (1977)). The different oligonucleotides above can be identified from the amino acid sequences, and they each May encode proteins. In fact, specific oligonucleotides make up the actual protein sequence. The probability of this occurring is due to abnormal base pairing relationships in eukaryotic cells and specific codons. By considering the frequency with which it is actually used (to code for a particular amino acid) It can be estimated. Such "codon usage rules" are described in Rays, R1, etc., "Journal. Disclosed in In Molecular Biology J, 183:1-12 (1985). It is. By using Ray's "codon usage rules," protein sequences can be codified. A single oligonucleotide containing a theoretical “most likely” nucleotide sequence that could be coded A set of nucleotide or oligonucleotide sequences is identified.

A suitable oligonucleotide or oligonucleotide capable of encoding a gene fragment of a protein. a set of leotides (or a set of said oligonucleotides or oligonucleotides) complementary to the set) are synthesized by means well known in the art (e.g. , “Synthesis and Application in DNA and RNAJ (S , A.

Edited by Narang, 1987, Academic Press, San Diego, California. ), identify and isolate the cloned gene of interest using techniques known in the art. It can be used as a probe for Nucleic acid hybridization and cloning Maniatis, T3, etc. (``Molecular cloning, A. Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, New York (1982) and Bath - female, B, D, etc. ・Practical Approach”, IRL Press, Washington, DC (198 5)). Then, the hybridization is possible. By analyzing the components of the above gene library, it was found that The extent and nature of the genome-encoding sequences of interest they contain are determined.

The above DNA probe can be detected to facilitate the detection of the target DNA coding sequence. Labeled with a group. The detectable group has a detectable physical or chemical characteristic. Any substance that has a property is sufficient. The substance has already been used in the field of nucleic acid hybridization. In general, most labels useful in the above methods are suitable for the present invention. can be used. Particularly useful are radioactive labels, eg! ! p, 3H, 140, 35 3% 125I, etc. those that provide adequate signal and have sufficient half-life Any radioactive label can be used. Oligonucleotides are analogous to Barigby, P., J., W., et al., Journal in Molecular Biology. 113:237 (1977), for example, by known means. 'Analytical Biochemistry Translation' and 'Analytical Biochemistry The T4 DNA polymer described in "Mistry", 135:456 (1983) can be radiolabeled with a substituted conjugate.

Alternatively, the polynucleotide can also be used with non-radioactive markers such as biotin, enzymes, etc. Nucleic acid hybridization probes, when labeled with a natural or fluorescent group. It is useful as For example, Leary, J. J., et al., Proceedings The National Academy in Sciences in the Unite 80:4045 (1983), Lentz, M. 9 etc., “Nucleitsc Acids Research J, 12:3435 (1984), and Lenz, M9, EMBO Journal, 6:817 (1983).

That is, in summary, the actual identification of a protein sequence (or a partial sequence of a protein) A theoretical [most likely JDNA] that could encode such a peptide A sequence or a set of said sequences may be identified. Oligos complementary to this theoretical sequence By constructing nucleotides (or "most likely" oligonucleotides) by constructing a set of oligonucleotides complementary to a set of octides) , probe(s) to identify and isolate clones containing the protein gene. A DNA molecule (or set of DNA molecules) that can function as a Ru.

An alternative method of gene cloning uses expression vectors to express the protein of interest. into an expression vector. A library is produced by cloning. Then, e.g. antibodies that recognize the target immunoglobulin or fragment thereof, particularly in this case By screening the library with Screen the library.

Therefore, by the methods discussed above, the target immunoglobulin or this protein can be Gene sequences capable of encoding fragments can be identified. Furthermore, characteristic analysis of the gene sequence This sequence is used to identify and produce recombinant immunoglobulins. It is desirable to express the protein coded for. As a result of the above expression, the target protein and Specifically, clones expressing proteins with the properties of the target immunoglobulin are identified. . The properties include the ability to specifically bind to antibodies directed against the protein, and the ability to bind to the protein. ability to induce the production of compatible antibodies, and among others, specific functions for proteins. This may include the ability to provide

A cloned protein encoding sequence, preferably in double-stranded form, obtained through the above method. The sequence is operably linked to sequences that control transcriptional expression in the expression vector. The recombinant protein or its functional derivative is introduced into the Trichoderma host cell. Can be manufactured.

A nucleic acid molecule, e.g. ``operably linked'' sequence to a nucleotide sequence encoding a polypeptide. A polypeptide is said to be "capable of being expressed" if it is "expressible."

Operable linkage is such that expression of the sequence is under the influence or control of the regulatory sequence. A bond by which a sequence is joined to regulatory sequence(s). two dna sequences (e.g., protein coding sequences and promoters attached to the 5' end of the coding sequence). As a result of induction of promoter function, the protein coding sequence m When RNA is transcribed, and the nature of the bond between the two DNA sequences, (1) (2) introducing a frame shift mutation; (2) inhibiting the expression of RNA or protein; (3) interfere with the ability of the expression control sequences to direct the expression of the template; – to be able to function if the region sequence does not interfere with the possibility of being transcribed. are said to be connected. That is, the promoter directs the transcription of that DNA sequence. Where practicable, the promoter region is operably linked to the DNA sequence. ing.

The exact nature of regulatory sequences required for gene expression varies between species or cell types However, generally the 5° non-inverters involved in the initiation of transcription and translation, respectively, are Contains transcripts and 5° untranslated (non-coding) sequences.

Expression of proteins in a Trichoderma host relies on the use of regulatory regions that function in said host. Requires. Trichoderma generally recognizes transcriptional regulation, e.g. those of other filamentous fungi. A variety of transcriptional and translational control sequences can be used to identify the target. In eukaryotes, translocation If the transcript is not linked to translation, the control region is methionine (AUG) codon. It may or may not provide a single codon. Generally, said region is a host cell. It contains a promoter region sufficient to direct the initiation of RNA synthesis in cells.

As is widely known, eukaryotic mRNA transcription encodes the primary methionine. It starts with a codon that For this reason, eukaryotic promoters and protein A bond between DNA sequences encoding methionine or a functional derivative thereof It is preferred to ensure that there are no intervening codons that can be used. Said code The presence of the fusion protein (AUG codon in the same sequence as the protein-encoding DNA sequence) (if the AUG codon is in the coding frame) or a frameshift mutation (if the AUG codon is in the protein dark (if it is not in the same reading frame as the coding sequence) is formed.

The secretion of the expression product of the gene encoding the desired product is preferably carried out through the secretory pathway of the cell. This is achieved by a front region that ensures efficient targeting of the expression product of the molecule. Preferred specific example In this case, since the homologous Trichoderma secretion signal sequence exists, the target protein is isolated from the surrounding area. Secreted into the medium.

The anterior region may provide the signal sequence of the immunoglobulin molecule or may contain a different can provide a signal sequence for a protein. There are many heterologous signal sequences that can be used. It can exist. The front region contains a natural signal or leader peptide or part thereof or Synthetic signal/leader sequences, especially synthetic sequences containing preferred codons in the desired host. It can be a column. The anterior region is generally cleaved from the desired product during secretion, where the mature protein is can be separated from the culture medium. The signal/leader sequence encodes the protein of interest. may be derived from the signal/leader sequence of the Trichoderma or Trichoderma gene. It may be derived from genes encoding other secreted proteins from other biological sources.

Signals derived from genes encoding proteins secreted by Trichoderma/ Examples of leader sequences are the cbh1 signal sequence or the egll signal sequence or the like. is part of. From a gene encoding a secreted protein heterologous to Trichoderma The induced signal/leader sequence is Aspergillus amylase or glucoa. It can be derived from genes related to mylases. The purpose coding sequence of the protein is Derma is linked to a signal sequence, such as a yeast sequence, that allows the protein to be secreted from the host. (Wood, C.R., et al., Nature, 314:446-449 (198 5), Kaiser, C.A., et al., “Science”, 235+312-317 (19 87)).

repression or activation so that the expression of an operably linked gene can be modulated Transcription initiation regulatory signals can be selected that cause transcription to occur. Interestingly, temperature sensitivity Therefore, expression can be inhibited or initiated by changing the temperature, or expression can be inhibited or A regulatory signal that is subjected to biological regulation, such as substrate or metabolic regulation.

If desired, untranscribed and/or untranslated regions up to the protein coding sequence3 ° can be obtained by the cloning method described above. 3゛-The non-transcribed region is located at the end of its transcription. may be retained with respect to the control sequence elements. 3-Untranslated region is the end of its translation Regulatory sequence elements or elements that direct polyadenylation in eukaryotic cells can be held regarding the ment.

In a preferred embodiment, a genetically stable transformant of Trichoderma is constructed; At this time, the DNA of the monoclonal antibody of interest is integrated into the host chromosome. purpose thing Coding sequences for clonal antibodies can be derived from any source. said built-in The staining is performed de novo within the cell or, in highly preferred embodiments, in the host stain. Integration of a DNA sequence in a vector, e.g. a chromosome, that is functionally inserted into a chromoplast This can be aided by transformation by DNA elements that promote growth.

Cells in which the DNA introduced into the chromosome is stably integrated are the same (chromosome one or more markers that allow selection of host cells containing the expression vector; The choice is made by introducing the car. A selectable marker gene is expressed or co-transfected into a DNA gene sequence or co-transfected Better to introduce the same cells. Various selectable markers, such as argB (Asp. Lugilus nidulans or I. lycodylmer 1-sei) and Supergillus nidulans) may be used. In a preferred embodiment, the dominant choice is selection markers, such as aIlldS (Kelly, JMl et al., EMBO Journal, 4 :475-479 (1985), Taipan, J. et al., "Gene", 26:20 5-221 (1983)) or Phleoma inone resistance gene (Mattern, 1 ．． E. et al., “Abstracts on the Nineteenth Lecture.” Yards on Molecular Chemicals on Easts & filamentous fange and its impact on biotechno (1987)) is used. Sakae An auxotrophic selection marker, such as trpC or argB (Pentik, M. et al. Gene, 61:155-164 (1987)) is also available. the purpose Monoclonal antibodies are expressed in two different plasmids on two different plasmids. When expressed from a set (one cassette expresses the light chain and one cassette expresses the heavy chain) The use of two different selectable markers ensures that the host is In some cases, it may be desirable to be transformed by a plasmid.

Trichoderma reesei grows poorly on acetamide as the sole nitrogen source Instead, growth can be further inhibited by adding CsC1 to the medium. Therefore , using acetamide as the sole nitrogen source in the presence of CsC1 as a selective medium. By growing the A+dS transformants, A+dS transformants can be identified.

Important factors in selecting a particular plasmid include the recipient cell containing the vector. Ease of identification when vectors are recognized and sorted from vector-free recipient cells the desired copy number of the vector in the host and the ability to transfer the vector between heterologous host cells. This includes whether it is desirable to be able to "shuttle."

Once the vector or DNA sequence containing the construct(s) is manufactured for expression Once the DNA construct(s) have been transformed, the DNA construct(s) can be used in various It is introduced into a suitable host cell by any suitable means. After introduction of the vector, recipient cells The transformed cells are grown in selective media that select for growth. cloned gene The expression of the sequence(s) results in the production of the protein of interest or protein fragments are produced. This expression can be carried out in a continuous manner or in transformed cells. or in a controlled manner.

By using the methods described herein, antibodies of a given specificity can be isolated. ras and with the same specificity, but whether human or non-human, It is possible to switch (exchange) classes of antibodies belonging to different species. Bivalent , bispecific (i.e. with different variable regions), chimeric heavy chain and non-chimeric light chain. molecules with chains or variable binding domains attached to peptide moieties with desired functions. Various types of immunoglobulin molecules can be provided, including molecules with conjugates.

Chimeric heavy chains and non-chimeric (i.e. Antibodies with light chains (all human or all non-human) have the required polypeptide chain. can be produced by appropriate association of These chains are present in the two Trichoderma species of the present invention. Immunoglobulin-expressing clones (one expressing heavy chain and one expressing light chain) ) or by using one clone expressing both chains of interest. can be manufactured. The monoclonal antibodies, light and heavy, are used in Trichoderma host cells. a disulfide which can be subjected to association in cells or which characterizes the chain association of said antibody. can be combined in vitro by forming a bridge. light chain and heavy Methods for forming functional immunoglobulin molecules by reassembling molecules are known in the art. It is known (Boss, M.A., et al., Nuclear Acids Research J, 12 : 3791-3806 (1984), Cabin, Sl. et al., ``Proceedings on the national academy on the sciences on the u Knighted Stave on America”, 81:3273-3277 (198 4)).

Association, aggregation or other characteristics of antibodies obtained from cell fusion or hybridoma technology Class switching is useful when changing the gender is desired.

For production of intact and functional immunoglobulin molecules by Trichoderma host cells Balanced synthesis of both light and heavy end proteins within the host cell is preferred. stomach. One method is to place light and heavy genes in separate cassettes in separate vectors. In this method, both mirrors are expressed using the same promoter.

Alternatively, when using true promoters, they are one bell compared to the other because the promoter is stronger or weaker than the other. A balance should be maintained so that unbalanced synthesis of chains does not occur.

Efficient secretion of immunoglobulin chains functions as a secretion signal in Trichoderma. fusion proteins in which one or both immunoglobulin chains are preceded by a functional amino acid sequence. This can be achieved by building white matter. In a preferred embodiment, the amino acid sequence is derived from an effectively secreted Trichoderma protein or a portion of said protein. Ru. The effectively secreted cellulases of Trichoderma are well suited for fusion constructs. do. After secretion, the Trichoderma protein portion of the fusion is e.g. proteolytic cleavage sites are added or removed by other engineering treatments. can be done.

For those skilled in the art, the entire specifically mentioned DNA sequence is correction of a pair of bases that are non-essential to the produced product or encoded fusion substance or It is self-evident that it can be modified by deletion. For example, cbh 1 or eg A DNA sequence substantially similar to a l1 signal or promoter sequence is defined as may be used if they exhibit the intended function in Trichoderma.

In addition, genes related to the target protein may have a detrimental effect on the activity of the protein. It may be modified if it has no effect.

VI, Transformation of l-lycoderma hosts Any method for transforming Trichoderma with the constructs of the invention may be used.

In a preferred embodiment, the transformation technique comprises transformation of Aspergillus nidulans. Adapted from methods (Balance, Jl et al., ``Biochemical and Physical Communications”, 112+284-289 (1983), Tail I Kupart, J9 etc., ``Gene'', 26, 205-2201983)). child Venti, Sola, M. et al., Gene, 61:155-161 (1987). It is described in. Protoplasts are produced by growing mycelium on agar plates. By a known method by suspending the mycelium in a buffered solution of Bojim™ 234. Manufactured. Instead of the conventional sucrose solution of Novozym™ 234, advantageously Sorbitol solutions may be used. The transforming DNA is then transformed into the material described herein. Add to the protoplast solution described in more detail in the experimental part. This transformation is usually a selectable marker inserted into another plasmid (e.g. in p3SR2). using the a-layer dS or argB in psa143) to co-transformation in which Trichoderma reesei is simultaneously transformed into Trichoderma reesei at high frequency. It is done. When equimolar amounts of DNA are used for transformation, a large proportion of the trait The transformant contains a non-selective plasmid integrated into the genome. argB- ) a Lycoderma reesei strain containing equimolar amounts of plasmids p3SR2 and psa1 When used in transformation with 43, transformants are grown on dual selective media (minimal, Acetamide, obtained in CsC1). Alternatively, a single plasmid It can also be used in transformations where selectable markers are inserted.

The transformant is then cultured in an appropriate medium. The desired product is produced in a simple defined medium. The glucose-inducible gene (or its glucose-inducible promoter) ) may be used. Grow Trichoderma in medium containing 2% glucose In this case, secretion of all extracellular proteins, including proteases, is strongly suppressed. Target protein Genes that feed proteins, e.g. immunoglobulins, are strongly enhanced in glucose-containing media. When linked to a promoter to be expressed, the protein of interest is preferably added to the growth medium. secreted. Because other proteins are not produced under these conditions, the protein of interest is It is the main protein secreted into the culture medium.

When the fungal mycelium is removed from said culture, the protein of interest is in very pure form. present in the solution produced. If necessary, said protein can be added to almost the only protein present. Since it is a single protein, it can be further purified very easily.

Using solid media allows you to test the transformed cells for the presence or absence of specific proteins. Rapid screening of thousands of colonies arising from Trichoderma conidia was performed. quantitative evaluation of the amount of the protein produced can be performed. Detection of said protein Several types of solid media are known in the art for commercial use.

When grown on solid media, many fungi form large, diffuse colonies. Therefore, 1 To generate multiple colonies (less than 100) per plate, Addition of growth-limiting chemical agents may be desired. Drugs used for this purpose include , Rose Bengal, Bull's Bile and Phosphon D1 Triton X-100 and There are saponins. Some fungi have replica plates similar to those developed for bacteria. In some cases, by using techniques, bacterial colonies in different growth media can be Some properties can be tested.

Typically, after screening in plates, shake flasks are placed in liquid production medium. The selected colonies are cultured and protein production is measured. trichodel The plant is well suited for fermentor culture and allows the use of inexpensive raw materials for growth. . If it is desired to express multiple recombinant proteins in the host, shaking flasks can be used. The best isolates exhibiting high recombinant protein production at Scoscale were selected for the second round. can be used in the transformation of a host. Alternatively, the desired Trichoderma host can be simultaneously Can be co-transformed with multiple genes of interest.

VII, immunoglobulin products In order to obtain the proteins and especially the immunoglobulins of the invention, the above-mentioned proteins having the desired properties The recombinant host is cultured under appropriate conditions. Target protein is cultured from Trichoderma host The protein is secreted into the culture medium and recovered from the culture medium by methods known in the art. Ru.

Protein products can be produced by tricodelation, e.g. in liquid culture media, in fermenters with the desired properties. It can be produced by culturing the Luma strain, and the medium may be, for example, 6% Solca Flour. cellulose (BW40, James River Corporation, Harake Nsak, New Chassis), 3% distilled liquor residue (alcohol distillation Waste, Arko Limited, Koskenkorva, Finland), 0.5% KH2 PO4,0,5% (NH4)zSOa and 0.1% Stork as antifoaming agent Thor (Stroktor 5B2023, Sill and Seirachiel, Hambu rug, Germany). Expression of recombinant protein is caused by cbh1 promoter. When under instructions, Trichoderma strains are sensitive to glucose repression and quality expression requires an inducer (cellulose, lactose or sophorose) (Allen et al., “Biotechnology and Bioengineering”, 3 3:650-656 (1989)). pH is preferably phosphoric acid or ammonia The pH should be maintained at approximately 5 by adding be. However, temperature and pH should be adjusted according to the strain and protein being produced. (Merivuori et al., Biotechnology Letters, 12(2)+1 17-120 (1990)).

Recombinant protein products can be prepared from culture media using methods well known in the art. will be collected. The host of the present invention partially or completely lacks cellulase activity. or if the product is produced by a promoter that functions in the presence of glucose. If the protein is purified, the recombinant protein is sufficiently pure that it can be cultured without further purification. Can be utilized directly from the culture medium. If desired, the product is lyophilized or Immune activity may be concentrated and/or stabilized for storage in other ways. Protein is cultured. It is secreted into the culture medium and the target protein product can be obtained by collecting only the culture medium. The recombinant protein products of the present invention are very economically supplied and used. trichodel There is no need to extract the protein from the host.

If desired, the expressed protein may be subjected to conventional conditions such as extraction, precipitation, chromatography, etc. Further refinements can be made by chromatography, affinity chromatography, electrophoresis, etc. can be manufactured.

VIIl, pharmaceutical composition The protein of the present invention can be produced by chemically or genetically engineering techniques to target the desired action site. It may be conjugated to fragments of other agents that target the protein. With another method chemically or genetically engineer other compounds into recombinant proteins or their Additional properties of said protein by conjugation to active fragments, In particular, properties that enhance the function of a protein, or its undesirable or physiological conditions. The ability to treat or alleviate symptoms may be improved or imparted.

The amounts and methods of administration of proteins, and particularly the immunoglobulins of the invention, include: It can be easily determined by one of ordinary skill in the clinical or industrial arts. In general, protein The amount of white matter and especially immunoglobulins is a consideration, e.g. type of application, Type of monoclonal antibody used, age and health of the patient being treated the condition, the type of concurrent treatment, if any, the frequency of treatment and the nature of the desired effect; quality, counterindications (if any) and individual physician or scientist. Varies depending on other variables.

To obtain the desired results, the dose may be administered in one or more applications. obtain. The recombinant protein of the present invention can be administered with an agricultural or pharmaceutical carrier suitable for administration. can be given. Recombinant proteins can be used to prevent, palliate, prevent or cure a desired condition. Forms that act on the condition being treated or their intended function, such as immunoglobulin products It can be administered in a functionally preserved form.

Recombinant protein products of the invention for parenteral administration can be prepared in sterile aqueous or non-aqueous solvents, suspensions, etc. Includes suspensions and emulsions. An example of a non-aqueous solvent is propylene glycol. polyethylene glycol, vegetable oil, fish oil and injectable organic esters. Ru. Aqueous carriers include water, water-alcoholic solutions, emulsions or suspensions, such as Examples include saline and buffered pharmaceutical parenteral excipients, such as sodium chloride solution, phosphorus Contains gar dextrose solution, dextrose + sodium chloride solution, lactose Available in Ringer solution or fixed oils. Intravenous vehicles include fluid and nutrient replenishment electrolyte replenishers, such as those based on Ringer's dextrose. .

The recombinant proteins of the invention can also be used in pumps, spray means and in particular for certain applications. Alternatively, it may be administered in sustained release form if it is desired to prolong the effect of the dose. Also , the protein of the present invention can be targeted to specific organs by appropriately inserted catheters or by targeting specific organs. those molecules as part of a chimeric molecule (or complex) designed to can be delivered to specific organs in high concentrations.

Administration in sustained-release form is recommended for patients when repeated injections are indicated over a long period of time. is more convenient. For example, genetic or chronic diseases such as tumors or neoplasms Maximize patient comfort when the method of the invention is used to treat plastic conditions In order to It is desirable to do so.

If the biological activity of the protein is not destroyed by the digestive process and the properties of the compound If the protein can be absorbed throughout the intestinal tissue, the recombinant protein of the present invention Doses such as tablets, capsules, powder packets or liquid solutions for oral administration It can be used in any form.

Pharmaceutical compositions providing the recombinant proteins of the present invention can be prepared by methods known per se, such as conventional methods. manufactured by suitable mixing, granulating, dragee-making, dissolving, freeze-drying means or similar processes. be done. The compositions of the present invention can be used by themselves and alone, among many other uses. diagnosis and control of physiological and agricultural conditions, industrial treatment of waste, sewage, and and in the industrial production of biologics.

In intravenous dosage form, the composition of the invention has a sufficiently rapid onset of action that it provides immediate improvement. Useful for acute treatment of physiological conditions requiring

Additionally, lower potency versions are useful in treating mild or chronic physiological diseases.

Said composition is conveniently provided in kit form, said kit being suitable for a given application. Contains buffers and mixed solutions suitable for use with recombinant proteins. For example, the present invention The composition may contain a diagnostic kit or an antigen recognized by the recombinant protein of the present invention. The reagents needed for the test assay can be provided.

1X, i! ! Advantages of the present invention Trichoderma can secrete large amounts of protein, e.g. at concentrations as high as 40 g/L (culture solution). Trichoderma has been shown to be useful for recombinant protein synthesis and especially 3, a particularly useful and practical host for the synthesis of robins, Derma cbb 1-promoter is a protein secreted from the total Trichoderma host. Because it is a strong single copy promoter that directs synthesis of less than 60% of the quality, It provides a very convenient promoter for the expression of target genes. Transformation system is highly flexible and can be adapted to any gene of interest. Toriko The Derma host provides an "animal cell type" high mannose glycosylation pattern. Ma In addition, the cultivation of Trichoderma is based on sufficient previous experience in fermentation technology on an industrial scale. It is supported by

Proteins and especially immunoglobulins produced by the method of the present invention and their inducers The conductor has applications in the agricultural, food and pharmaceutical industries, for example. immunoglobe Applications of phosphorus include as an immunotoxin and as an immunocontrol medium for tumor localization. as milk processing, large-scale purification of enzymes, stereospecific drug substances and other molecules. It in purification, sewage purification and development of sensitive and specific biosensors This includes uses such as

The invention is described in more detail in the Examples below. These examples have a slight It merely shows a specific example of application of the invention. For those skilled in the industry, It is self-evident that several similar applications will be devised. Therefore, narrowing the scope of the invention The examples should be construed only as clarifications of the uses of the invention, rather than as It is possible.

Example raw materials plasmid 1, p285 is ATCC number 20681.

2, pcAMG91 Boer, Eo et al., rEMBo Journal, 3:15 81-1585 (1984).

3, pic19R Marsh, J.L., et al., Gene, 32:481-48 5 (1984).

4, PSA 143, John, M. A. et al., “Enzyme and Micro "Vial Technology", 6: 386-389 (1984), base, B1 etc. , Scene, 25:109-117 (1983).

5, p3sR2, Tilburn, J8, etc., "Gene", 26+205-221 (1983), Kelly, J. M. et al., rEMBO Journal, 4, 475-47. 9 (1985).

6, pDJB2 Balance, D. J., et al., Gene, 36:321-331 (1985).

7, pMC187] in Casadaban, M. J., et al. Imology, 100B: 293-308 (1983).

s, p”r”rol and pTTll are described in patent application WO35104672 has been done.

9, pUC9 and pUc13, Vieira, Jl et al., Gene, 19:25 9-268 (1982).

lQ, pUc18 and pUc19 have been described by Janisuberon, C9 et al. ”, 33:103-119 (1985).

11, pTZ18R and I) TZ19R are available from Pharmacia .

12, pAN5-41B, van Garkom, R.F., M. et al., “Gene” , 48:211-217 (1986).

13, pAN8-1 was produced by Mattern, 1. E., Punto, P. J., Rankless , S.L., Bowels, P., H., and van den Hondel, C.A., M. , J, J,, “Transformation on Aspergillus oryza ``Abstracts on the Nineteenth Runteren Lecture'' on Molecular Genetics on East and Fi Lamentus Fungi and Its Impact on Biotechnology -, Runteren, Netherlands (1987), 34.

14, pAMH102, Harutski, A1, Ushitaro, Jo, / (Illy, M o, Bentik, M. and Knolls, J., C., r. Nobel Fan. GULL EXPRESSION SYSTEM: SECTION ON ACTIVE CAR Fu Chymosin from the Filamentus Fungus Trichoderma Lee Sei, Bio/Technology, 7 (1989), 596-603. It is.

15, pAMHll, Harutski, A3, Manteira, A2, Venti Tsuku , Ml, Mutstilainen, So, Pula-1R1, Suominen, Pl, Nord. Su, J.

and Nevalainen, H., rGenetics Engineering on Tri. Coderma to Prodeuse Strains with Nobel Cellulase ・Promoter”, “Enzyme and Micronomical Technology” ” (1990, currently being submitted).

16, p3SR2, Kelly, J. M., & Nokan-Ins, M. J., “Trans. Sformation on Aspergillus niger pi aIld S.G. 'On Aspergillus niger', 'rEMBo Journal', 4 (1985) ): 475-479, Tilburn, J9, Shatzocchio, C5, Tyro, G., T., Zabitskiyzisman, J. H., Robkinton, R. A., and D. Bis, R, W,, rTransformation Pi Integration I "Aspergillus nidulans", "Gene", 26 (1983), 205 -221.

17, pUAOl from FIG. 26 is also depicted in FIG. 8 and Example 9. listed in IV It is.

18, pAN924-21 in FIG. , Bowels, P., H., Goin, T1, Bitsser, J2, van den. ・Bloek, HlW, J., Hammer, J.E., Timberlake, W.E., O. and van den Hondel, C.A., M., J.J., rExpression. on an Escherichia coli β-galactosidase fusion g. 'In Aspergillus nidulans', 'Gene', 40:99-106 (1985)! : Described.

19, pML5, Takkinen, Ko, Lautkanen, M-L, Sisman, B. 6. Alftan, K1, Imohon, T1, Vanne, Lo, Karl Teikhon, M9 , Knolls, J., C., and Thiry, T.T., “An active system. Single Chain Antibody Containing a Cellulase Linn 'Car Domain Is Secret Pie Escherichia Colli', 'Professional Tin Engineering” (submitted).

host strain Escherichia coli strains JM101, JM103, JM109 and DHI (Year Nishupon, cl et al., “Scene”, 33:103-119 (1980)) and DH5α (BRL) are host and DH5α (BRL) in Escherichia coli cloning. used. Trichoderma reesei strain QM9414 (ATCC26921 ) and Trichoderma reesei strain RUT-C-30 (ATCC56765X model Ntencourt, B.S., and Eberleif, D.E., r Selective Sk. Leaning Methods for Isolation on High Yildane cellulase mutants on Trichoderma reesei”, “Atopan Seeds in Chemistry Series J (1979), 289-301) Used in fungal transformation and expression studies.

Trichoderma minimal medium Gelcose 20g/1 (NHa)zs04 5g/I KHzPCL 15g/l Mg504 0.6g/l CaC11o, 6g/l Fe50*X7H205mg/l Mn5CLXHzO1,56mg/l Zn5O,x7H! 0 1.4+g/l CoCl2 2mg/l The purpose of the study was to investigate the immunoglobulin content of the form occurring in Trichoderma reesei. The purpose was to produce children. For this specific study, IgG antioxacilone (anti-O X) cDNA for kappa light chain IgG3 and IgG1 anti-Ox heavyweight genes, Obtained from mice immunized against 2-phenyloxacillone (Cartiphon , M2, Griffiths, G. M., , Malcolm, A. F., and Milstein, C1, rmRNA sequences define an abnormality. Restrictive TgG Response Tow 2-phenyloxacylone And It's Early Diversification J, "Nature", 3 04 (1983), 320-324).

Example 1 Induction, enrichment and isolation of auxotrophic Trichoderma reesei mutant strains Using the filtration concentration method described above by Hon. We isolated auxotrophic mutant strains that require luginine (Nebaraihon, Hl, Technical Research Center in Finland, Publication 26 (1985) ). The mutagen used was UV light. Nutrition that requires arginine The auxotrophic strain produces a series of minimal pre-replenishers supplemented with different intermediates in arginine biosynthesis. By using this method, a mutant strain lacking the argB gene was identified. (Nevaraihon, Ho, Technical Research Center in Finland , Publication 26 (1985)). Mutant strains that require citrulline for growth are , was considered as a possible argB-mutant strain. Isolated mutant strain a The rgB-characteristic is a plus containing the Aspergillus nidulans argB gene. By transforming them into prototrophs using mido psa143 It was confirmed (Example 4).

Tryptophan auxotrophic mutants contain anthranilic acid, indole or tryptophan. Characterized in a series of minimal medium plates supplemented with Tophane (Sanchez F7 et al., Gene, 51+97-102 (1987)). to grow considered indole-requiring colonies to be putative trpC mutants, e.g. Transformation of Supergillus nidulans with pHY101 plasmid confirmed their trpC-characteristics (van Gorkom, R.F., M. et al., “G. 40:99-106 (1985)).

Example 2 Production of protoplasts Sepharose in Votate-dextrose agar plates (Difco) Mycelium was grown on the disks. The mycelium obtained from the 5-Sepharose culture was 1 or 2 moles of sorbitol buffered at pH 5,6 with 0.1 moles of potassium phosphate The suspension was suspended in 15 μl of a 5+eg/ml solution of Novozym® 234 in a 500ml solution. mixture The samples were incubated at 30°C for 1°5-2 hours. 4000g for 5 minutes Protoplasts are extracted from mycelium by filtration through rounded sintered glass (porosity 1). Separated, 1.2 mol sorbitol - 10 mmol Tris, MCI (pH 7, 5) and washed twice.

More advanced purification of protoplasts can be achieved by the method described by Tilburn et al. Irburn, Jl et al., Gene, 26:205-221 (1983)). This can be achieved by in 1.2M Mg5Oa buffered at pH 5.8 A 5 mg/al solution of Novozym™ 234 was used for protoplast production. Ta. After incubation and filtration, the protoplast suspension was diluted with an equal volume of 0. 6 moles of sorbitol - 100 mmol of Tris, HCI (pH 7.0) Centrifuged with Burley at 4000g for 15 minutes. Protoplasts are tubes A clear band was formed in the middle. Protoplasts were added to 1 volume of 1.2 mol Thor - suspended in 10 mmol Tris, HCI (pH 7,5) and spun down. and 1.2 mol sorbitol - 10 mmol Tris, HCI (pH 7.5) Washed twice inside. Protoplasts, 5X10'-5X10' protoplasts /■ 1.2 mol sorbitol-10 mmol CaCl2.10 mmol at a concentration of 1 Limol Tris, HCI (pH 7,5) 11: suspended.

Protoplast viability was determined by treatment with 1 molar sorbitol as an osmotic stabilizer. It was evaluated in Lycoderma minimal medium. Overlay protoplasts with 3% agar I placed it on. The regeneration frequency is 40-8 for strain QM9414 (ATCC26921) It was 0%.

Example 3 Using Aspergillus nidulans acetamidase (amdS) gene Transformation of Trichoderma reesei Plasmid p3SR2 was used in the transformation. It is the entire acetamidase structure Aspergillus nidyu with gene aid S and its regulatory region amd I (Hines, M. J., et al., “Molecular & Cellular Biology, 3:1430-1439 (1983), Tilver. J9 et al., Gene, 26:205-221 (1983)).

20 μm (4-10 μg) of transforming DNA was added to 200 μl of protoplasts. added to the solution. 50μl 25% PEG6000 - 50mmol CaClz Add 10 mmol Tris, MCI (pH 7,5) and keep the mixture on ice for 20 min. Incubated. 2+111 PEG solution was added and the mixture was further heated to room temperature. and incubated for 5 minutes. 4ml of 1.2M sorbitol-10M Add 1 g of Limol CaC, 10 mmol of Tris, MCI (pH 7.5), and Rotoplasts were placed on a 3% agar overlay. The selective medium was (NHn)zsO Contains 10 mmol of acetamide as the sole nitrogen source in place of n 1 molar sorbitol as a fixing agent and to suppress background growth. Trichoderma minimal medium supplemented with 12.5 mmol CsC1.

In the case of 0M9414 strain (ATCC26921), the transformation frequency per 1 μg of DNA was Transformants with a frequency of 40-600 were obtained. DNA was subjected to two cycles of CsC1/bromination. Purification by ethidium centrifugation gave the highest frequency.

Sporulation was rarely observed on selective media, but on potato dextrose. - When transferred to agar, transformants sporulated as usual for 21i. to acetamide Their ability to grow has changed. The diameter of colonies on selective medium is lll It was in the range of 11 to 20 degrees.

The mitotic stability of the transformants was investigated. 10 large shapes with various sizes The transformants were subcultured on acetamide-CsC1 plates and Sporulation was performed on xistrose (PD) and recultured on PD. hetero power riosi To eliminate heterogeneity induced by spores, individual colonies arising from spores are Tested for A■dS+ phenotype. Unicornential clones from each prototransformant were Subjected to continuous culture (5 growth cycles) on non-selective medium and after each generation on selective medium, the table The current model was tested. Of the 10 large transformants tested, 1 on non-selective medium After cycling, 6 out of 10 transformants gave 100% AmdS+ conidia and 3 47-87%, with one transformant giving no Aa+dS+ conidia.

This result remains the same through five non-selective growth cycles, with initially unstable showed that the A+dS+ phenotype could be stabilized later.

From 10 small original colonies, 3 had the AmdS+ phenotype ( 94%, 44% and 5% of the spores. The other seven colonies are Only spores could not be grown on cetamide. Interestingly, the total A ndS+ spores are grown on acetamide plates, characteristic of large colony transformants. It showed vigorous growth on the top.

The presence of plasmid DNA in the transformants was determined by separating them from the transformants (Rae et al. Dell, C9 et al., Letters in Applied Microbiology, 1: 17-20 (1985)), cut with XhoI or 5ail and EcoRI. The whole DNA was analyzed by Southern plot. Vector pUc1 8 and Sal I-Eco containing the and S gene of plasmid p3sR2 The RI fragment was used as a probe. Transformation is performed using Trichoderma geno It was shown that recombination occurred at slightly different sites in the mu DNA. (1 to several copies per genome).

Example 4 Aspergillus nidulans arg B and arg by trpC genes B- and trpC-transformation of Trichoderma reesei in birds with heterologous DNA. Complementation of auxotrophic mutants in Coderma reesei was demonstrated. Asper Plasmid psa143 containing the argB gene from Gillus nidulans was used to transform Trichoderma reesei arg B-mutant strain. As Plasmid pHY101 containing the pC gene from Pergillus nidulans ( Van Gorkom, R.F., M. et al., Gene, 40:99-106 (198 5)) was used to transform the Trichoderma reesei trpC-mutant strain. .

Select transformants on minimal medium without arginine or tryptophan. I chose. Depending on the plasmid, the transformation frequency can be as low as per microgram of DNA. It varied from 10 to 300 transformants.

Isolate chromosomal DNA from transformants and perform Southern Nosobridization experiments Plasmids into the chromosomal DNA of Trichoderma reesei by using Proper incorporation of the DNA was demonstrated. Multiple tandem copies of psal143 are present in the genome. incorporated into the system. Southern and dot plot 1 hybridization In the analyzed transformants, the copy number of argB increased from about 2 to more than 100. showed that it changes. Argf transformants express through at least three generations It was shown to be 100% type stable. This is a conidia from S transformant. Tested by continuous culture of offspring and then testing the Arg phenotype on minimal medium (50-80 colonies/transformant).

Example 5 Co-transformation of Trichoderma reesei with adS and argB plasmids The possibility of co-transformation of Trichoderma with non-selective plasmids was first proposed by Al. demonstrated by the ginine auxotrophic mutant strain.

arg B-Trichoderma reesei strain, equimolar amount of Aspergillus nidyu Lance plasmids psa143 (argB) and p3SR2 (amds) transformants were selected for the Arg-phenotype, and the acetamide - Regarding acquisition of amd S by streaking with CsC1 plate Tested. Among the ArgB transformants, 86% were identical (AmdS+; Southern analysis of transformants revealed that both plasmids were located at several different locations in the genome. It was shown that it is incorporated as a variable amount of copies.

Double selection in minimal acetamide-CsC1 medium resulted in amdS transformation. ~100 Amd”Arg+ transformants and and some small colonies were formed.

When testing the stability of the ArgB+ phenotype of argBamds cotransformants, 3 out of 5 were found to be 100% stable, the rest were analyzed progeny In 7% or 80% of cases, ArgB+ characteristics were lost. the same individual colony - had also lost the AmdS+ phenotype. If this instability occurs on the same chromosome, e.g. It is unknown whether this was induced by the simultaneous incorporation of andS and argB into the position. Ru.

In addition, Aspergillus nidulans glycerol phosphate dehydrogenase gene (gpd) (van Gorkom, R, F, M, et al., “Gene”, 48:211 -217 (1988)) promoter and N-terminal protein coding region Plasmid pAN5- containing the Escherichia coli 1acZ genes linked in phase. 41B was used for co-transformation. In addition, this plasmid can be used in Aspergillus - Contains the P. nidulans argB gene and pBR322 sequences. Prototrophic strain Toriko Delma reesei (0M9414) was plasmidized with 3SR2 (amds) and Co-transformed with pAN5-4IB and select transformants for Ad+ phenotype. and β-gal expression on acetamide-CsC1-plates containing X-gal. and screened. Glucose is present in the medium and p If H is neutral, endogenous Trichoderma reesei β-galactosidase activity could not be detected at all.

After 1-4 days of growth, blue color was recognizable. 1.010.7 molar ratio of plasmid (p 3SR2/pAN5-4IB) was used for transformation, the Amd clone Of these, 13% of large clones and 6% of small clones were 1.0/2.6 models. 39% of X gal clones and 7 %was gotten. The presence of both plasmids in And+ transformants showing blue color indicates that Proven by Southern hybridization.

Example 6 cbh 1-) Construction of Lycoderma reesei strains Different cells by the described method Activation of the cellulase gene triggers a series of processes that produce a specific combination of cellulases. A Trichoderma reesei strain can be constructed. In addition, cbh 1- strain is cbh 1 is important for efficient production of heterologous proteins when using a promoter. Ru. Trichoderma reesei strain QM9414 (ATCC26921) has cbh Cbh by Southern /X hybridization using 1-specific probe Since it has been shown to involve only one chromosomal copy of one gene - the recombination event It is thought to inactivate the child. The inactivated gene was constructed as follows. Vector pU Plasmid p'r' containing a full-length cDNA clone of the cbh1 gene in C8 r.

1 (W085104672) was cut with restriction enzymes Bgll and BglII. . A 0.8 kb fragment from the 5′ terminal region of the cbh1 gene was extracted using conventional techniques. It was separated from the agarose gel by surgery. Fragment using S1 nuclease blunt-ended and ligated into EcoRI cut blunt-ended pUc18 vector. and transformed into Escherichia coIJJM109. cbh 1 gene hula The DNA from the clone containing the component was isolated, and the BglI-Bgl of cbh1 was isolated. It was digested with the restriction enzyme EcoRI, which cuts in the middle of the II fragment. Eco The RI-generated ends were filled in and ligated with truncated cbh 1 residue. Plasmid pMS4 containing a frameshift mutation in the middle of the gene fragment It was regenerated (Figure 2).

Trichoderma was incubated with plasmid pM along with a 3-4 fold molar excess of plasmid pMS4. S4 and Aspergillus nidulans amds-containing plasmid p3SR2 and co-transformed.

Transformants were selected based on the amd S+ phenotype described above (Example 3), and a single carbon Purification was performed on a selective medium containing acetamide as a source. Purified transformant as carbon source and 1% solka flotu cellulose as nitrogen source. microorganisms in 200 μm Trichoderma minimal medium containing 0.2% proteose peptone. Grown on black titer plates. reported (Nunmi, Ml et al., “Bio Chemical Journal, 215+677-683 (1983)) CBHI specific Ophthalmogenic immunity by using undiluted growth medium against antiserum (sheep) Cellulase phenotype was tested by diffusion method. Produces normal amounts of CBHII Some transformants were identified that did not produce detectable CBHI.

Example 7 ■, Production of plasmid 285'proC containing the prochymosin gene (Figure 3) Prochymosin cDNA is an example of a heterologous protein in Coderma reesei. Those that were expressed were selected (Figure 3). The preprochymosin gene was isolated from calf stomach cDNA. It was isolated from the library and inserted into the Pst1 site of pBR322 by G-C tailing. (Solazzo, M9 et al., Gene, 37:199-206 (1985 ), Kunkel, T.A., r Proceedings in the National Academy. Demy in Sciences in the United Stave in a Merica, 8.2:48g-492 (1985)), pR26 was obtained. pU C9 was cut with 5alI, completed with Flenow polymerase, and added with T4 ligator. Ligation was carried out using zeolase. The obtained plasmid was digested with BamHI-EcoRI. Cut the 2.7 kb large fragment into a 0.47 kb BamHI- When lined with the EcoRI fragment 'f'/a, the H of the prochymosin gene pUC9' containing an indITI site at the N-terminus was generated. pUc13 to Ba Cut with 1HI-Nar I and Nar I-Xma I, and The small fragment was labeled with the 0.64 kb XmaI-BclI fragment of pR26. When igated, a platy containing the Xba1 site of the prochymosin gene at the C-terminus. Sumid pUc13' was obtained. 0.65kbXmal-Xba of pUc13' The l fragment was inserted into the O146kb HindIII-XmaI fragment of pUC9'. and the 1lkb Xba l-H1ndIII fragment of p285. Upon ligation, the plasmid p containing the prochymosin gene shown in FIG. 285'proC was generated.

II. Construction of plasmid pAMG/Ten1 pcAMG91 was converted into 5ail and Digested with PstI restriction endonuclease.

A 698 bp fragment was separated from the digest on an agarose gel.

This SalI-PstI fragment is the 14-molecule of glucoamylase mRNA. 0bp 3' untranslated portion encodes 540bp 3° up to the poly(A) addition site Contains areas. When this 3゛ fragment is treated with T4-DNA polymerase, Addition of an XbaI linker and the XbaI restriction enzyme ensure that the restriction site is It was made into a "slip end". This 3' end of the glucoamylase gene was When ligated to linearized pUc13, the glucoamylase gene polypeptide (A) Plasmid pAMG/Tenn containing additional regions was generated.

IIl, Plasmid I) Construction of MT837 promoter and glucoamylase pcAMG91 containing sequences encoding the (AMC) signal and leader peptide The 0.5 kb S+5aII-BssHII fragment of Digested pUc13 and synthetic ss encoding the first 6 amino acids of prochymosin HII-BamHI adapter was ligated. Generated plasmid pM A 0.8 kb Nde1-BamHI fragment was isolated from T626 and pro 0,5k from p285'proC containing sequences for the N-terminal half of C bBamHI-Ec.

pMT6 containing the R1 fragment and the C-terminal part of the sequence for proC Twenty-two 3.0 kb EcoRI-Ndel fragments were ligated. ( pMT622 is simply EcoRI-Xba of p285' in pUC13. I subcloning). The generated plasmid pM7648 (see Figure 4) was The entire prochymosin gene sequence is preceded by the MC promoter and signal/lead coding sequence. Contains genes. p to include the arg B gene of Aspergillus nidulans MT648 was further modified (John, M.A., et al., Enzyme & Microvial Technology, 6:386-389 (1984)). pM 1,8 kb Nar I-filled XbaI7 fragment of 7648, psa1 When the 6.8 kb C1aI-filled EcoRI fragment of 43 was ligated , pMT651 was obtained. Sequences further upstream of the AMC promoter (upstream activity The conjugated sequence, UAS) was further inserted. This is the first clone pcAMG91 The 3.7 kb C1a I-Bs5HII fragment from a 1.1 kbBssHII-filled XbaI fragment of pMT 648 containing and pMT813 (pMT813 is an EeoRV-BglII cut piC19R He cloned 3, 1 kb BaiHI-Nru I fragment and The 3.1 kb C1al-filled argB gene was cloned as ArgB gene heridation as a loaded EeoRI fragment more accomplished. The generated plasmid pMT830 was a UAS, pMT830 from AMC. Contains all genes for promoter, signal and leader sequences and proC had acquired an expression unit. Finally, add the AMC terminator sequence to the plasmid p Taken as a 0.6kb Xbal-XbaI fragment from AMG/Ten*. , cut with XbaI and inserted into dephosphorylated pMT830, resulting in pMT837. It will be done. The construction of pMT837 is shown in Figure 5.

Example 8 Production of chymosin using pMT837 Trichoderma reesei strains QM9414 and RUT-C-30 were transformed into plasmid Co-transformed with pMT837 and p3SR2. Plasmid pMT837 is , the prochymosin gene preceded by the AMG promoter and signal/leader sequence. including. The construction of plasmid pMT837 was described in Example 7 (Figure 3 (See also 5). Co-transformation and selection were performed as in Example 5.

To produce chymosin, 1% Solca chlorocellulose and 0.2% Pro Transformants were cultured in minimal medium containing theosepebutone.

Collect the mycelia, concentrate the supernatant by TCA precipitation if necessary, and dilute with 2 molar NaOH1 Diluted with 0 mmol Tris (pH 8.5). The sample was subjected to SDS-page ( 7,5%-15% polyacrylamide gradient) and nitrocellulose filter. The electrical plot was plotted against the target. Incubate the filter with rabbit prochymosin antiserum. α-Usagi-1g G-peroxidase, purchased from Sigma. The conjugate was used to stain with 4-chloro-1-naphthol.

Chymosin was shown to be secreted into the medium at a level of approximately 1 μg/l. AMC The promoter apparently functions inefficiently in Trichoderma reesei. for the homologous Trichoderma reesei promoter-terminator vector. The construction improved the production level of chymosin.

Example 9 Calf prochymo in Trichoderma reesei using cbh I promoter Construction of heterologous expression vector for syn production I, zygotic calf prochymosin of prochymosin gene to cbhl terminator region The gene was obtained from plasmid pR27 (Figure 6). Contains the entire habo gene The Pst I-Pst I fragment (~1110 bp) was extracted by conventional techniques. It was separated from agarose gel and ligated to the PstI site of pUc19.

This plasmid was partially digested with Pstl, and the ends were flattened with 81 nuclease. The Ava II terminator fragment of the cbh I gene (~75 0 bpX Klenow fragment) was ligated into this plasmid. . End 1. of cbhl cDNA subcloned in pBR322. gk The terminator region of the cbh I gene was obtained from the bBamHI fragment. (Teiri, T. et al., “Io/Technology”, 1:696-699 ( 1983)).

pro linked to the terminator region of the cbh I gene in pUc19. This plasmid containing the C' fragment was named pAMHloo (Figure 7) .

cbh I promoter using Il, Bgl I-5acII adapter Fusion of regions and prochymosin-encrypted regions 5acII-PstI fragment encoding the N-terminal region of prochymosin (80 bp) was isolated from plasmid pR27 (see Figure 6). First, cb h　I promoter region was converted into a 2.8 kb long EcoRI-EcoRI fragment. pUc18 (plasmid pUAO1, Figure 8) from genomic clone λ44A as and then subcloned the ~2°2 kb long EcoRI-BglI fragment into was isolated from this subclone. The BglI site is a signal of the cbhI gene. located in the middle of the file array. cbh I gene promoter and signal peptide ~2.2 kb long EcoRI-BglI fragment containing approximately half of the coded region and the ~80 bp long 5acII-PstI prochymosin fragment. Secure binding is mediated by the BglI-5acII adapter (NOR202 +N0R203, Figure 10). These fragments were combined with adapters into Ec pUC19 digested with oRI-PstI was ligated. This plasmid is the complete signal sequence of CBHI fused to the first amino acid of prochymosin; encodes some of its N-terminal sequences. Finally, from this construct, Eco RI-pcbh], 5s-proc-Pstl fragment was separated, and part 3 It was ligated to pAMH100 which had been digested with PstI from the ' end. Approximately 6.7 The kb fragment was separated from the ligation mixture by gel electrophoresis and the 5′ end of the fragment (EcoRI end) and the 3′ end of the plasmid pAMH100. ′- end with S! processed and ligated.

That is, the promoter of the cbh I gene and pro C' fragment , proC in front, followed by the cbh I terminator region. this The construct was named pAMH102 (Figure 11).

IIl, Addition of a selectable marker to the chymosin expression plasmid Selectable markers include Aspergillus nidulans aldS Genes (Hines, M. J., et al., “Molecular and Cellular Biology Zee, 3:1430-1439 (1983)) or Aspergillus nidyu. The argB gene of Lance can be used (Zijin, M.A., et al. and Microvial Technology”, 6:386-389 (1984) ).

and S gene was isolated from pL3SR2 as a PvuI-SalI fragment. and ligation of the 6k14PvuI-8alI fragment of pAMH102. I did. This selectable vector was named pAMH104 (Figure 11).

IV, cbh I promoter and preprochymo using oligonucleotides Accurate fusion of thin cryptographic sequences The amino-terminal Pstl fragment (~150 bp) of preprochymosin was Starting 12 bp upstream from G, it was inserted into the PstI site in pBR322. isolated from pH26 (Figure 3) containing the complete preprochymosin cDNA clone. Ta. This fragment contains the same <cbh1 signal sequence encoding region. cbhlEcoRI-5acI (~2.6kb) promoter fragment ( Figure 8) was subcloned into the polylinker of pTZ19R. cbh　l The EcoRI-Sac I fragment was derived from the original λ44A clone (Thieli , T6 et al., Bio/Technology, 1:696-699 (1983)) p. 2,8 kb EcoRI-EcoRI sub in UC18 (pUAol, Figure 8) Obtained from clones. pTZ19R (Pharmacia) contains the F1 origin of replication and T7 that can be subjected to oligonucleotide mutagenesis using the and SS-templates (single stranded) A pUc19-based plasmid containing a promoter. Generated plasmid The construction method of (pAMH105) is shown in FIG. From this plasmid, cbh l The sequence between the promoter region and proCATG is a specific oligonucleotide. , was deleted by loop mutagenesis using OAMH3 (Figure 10). oligo The process of performing nucleotide-directed mutagenesis is shown in FIG. origin of the problem Phosphorylate the oligonucleotide and sspAMH105 DNA (Marsh, J.L., et al. , Gene, 32:481-485 (1984)) hair kneeling, 5s- DNA from JM103/pAMH105 (as reported by Pharmacia) They were separated at a molar ratio of 20:1.

Eftidarzab, M., et al. [Nukreitsk Acids Research J, 14 :5115 (1986), prepropolymerase and Oligonucleotide primers were extended using T4 ligase. Escheri H. coli l1ut I mispairing repair deletion strain, BMH71,18 (Vieira, J. Gene, 19:259-268 (1982)). 5eal (present in the polylinker of pTZ19R, see Figure 9) It was digested by The pool of transformants was grown in 5111 liquid culture (Miller, J, H ,, in ``Experiments in Molecular Genetics'' (cold ・Described in Spring Harbor Laboratory, New York (1972)) The cells were grown overnight in Luria broth (containing 100 μg/ml ampicillin). . Plasmid DNA was isolated from the transformant pool and re-quenched by S. al. and retransformed into Escherichia coli JM109 strain. Possible deletion Clones were screened by digestion with various restriction enzymes and The specificity of the deletion was confirmed by sequencing. The generated plasmid is pAMHl It was named lol (Figure 12). This plasmid was further injected with EcoRI and Pst The pcbh1-preproC fragment produced was separated by digestion with Figure 14), the Pstl end of pAM was digested with Pstl and 5alI. H100 plasmid was ligated. Generated ligation fragment The ends of the two were made blunt with prepro and ligated. Generated plasmid The code was named pAMHlol. It is fused to the signal sequence of prochymosin. cbh1 promoter region and terminator region of cbh1 gene contains the entire prochymosin coding region fused to (Figure 14).

■, by signal sequence fusion performed by using oligonucleotides Precise fusion plasmid of cbh1 promoter and prepro C gene Construction of pAMH1103 (Figure 12) was performed using pAMH1103 (Figure 12) as described in detail in Section IV above. It was done in essentially the same way as llol, but was used for this construction as an exception. The specific oligonucleotide was OAMHl (Figures 10 and 9). generated Plasmid pAMH1103 contains the amino terminal part of the coding region of the proC gene. amino acids from the prepro C signal sequence preceding the minute (~140 bp) Amino acid 1- from the cbh l signal sequence fused to (aa)-12-16 (Figure 12). Plasmid pAMH1103? et al., pcbh 1-5so-proC' (.

= fusion) fragment is isolated from pAMHlo in essentially the same manner as described in Section IV above. (See Figure 12). Generated plasmid pAMH103 is the promoter region of cbhl, the signal of cbhl and preproC Sequence fusion, then proC coding region fused to cbh1 terminator region Including area. .

VI, cbh1 coding to proC coding region by use of oligonucleotides Accurate fusion of areas The construction of plasmid pAMH1106 (Figure 13) was described in detail in section IV above. was carried out in essentially the same manner as pAMHllol, with the exception of this construction number. the specific oligonucleotide used was OAMH2 (Figures 10 and 9 ). Promoter region of cbhl, signal sequence of cbh1 and proc The raw material containing the first 1-208a encryption region of the mature CBHI fused to the encryption region From the generated plasmid pAMH1106, pcbh l-mature o-proC' A fragment containing pAMHloo (Fig. 12).

The generated plasmid pAMH106 contains the promoter region of cbh1, cbh The signal sequence of 1, the aal of mature CBHI fused to the coding region of proC. - Contains 20 encrypted areas.

Example 10 Production of chymosin using pAMH102 and pAMH104 Trichoderma li -Sei strains QM9414 and RUT-C-30 were transformed into plasmids pAMH102 and and p3SR2 at a molar ratio of 5:1, respectively. Plasmid pAMH The construction of 102 is described in Example QII section. Co-transformation and selection , was carried out in the same manner as in Example 5. Transformants were selected as described in Example 3. Purified on selective acetamide.

To produce chymosin, transformants were treated with 1% Solca chlorocellulose and Culture in minimal medium (10-50+l1l) containing 0.2% proteose bebutone. did.

The mycelium was collected, the supernatant was concentrated by TCA precipitation, and the 2 molar NaOH/10 molar IJ mole was added. diluted in Tris (pH 8.5). The mycelium is destroyed in the presence of liquid nitrogen, The disrupted cells were pelleted and the supernatant was treated as for the culture medium. Sample S Fractionated on DS-page (7.5%-15% polyacrylamide gradient) and nitro Electrical plots were applied to cellulose filters. filter, rabbit prochymosin Antiserum and α-rabbit-IgG-AP (alkaline phosphatase) conjugate Gate and incubate with nitro blue tetrazolium and 5-butyl Lomo-4-chloro-3-indolylphosphate (purchased from Promega Biotique) stained with). The amount of chymosin inside and outside the mycelium was compared (Figure 17) . More copies of plasmid pAMH102 integrated into bacterial chromosomal DNA The clones containing Southern hybrids were also able to produce more chymosin. Indicated by dization. When one copy of one transformant is used, secreted The amount of chymosin was estimated from Western gel to be 100 μg/l (cultured Medium) and Ayada (Figure 17). Secreted prochymosin was analyzed by Western gel and and milk coagulation activity (chymosin coagulates milk by cleavage of β-casein determinants) ) was shown to be processed into active chymosin by (Bailey, M. , J. et al., Biotechnology Letters, 10:161-166 (198 8)). Various forms of chymosin found inside the mycelium (decomposed products inside the cell) To measure the amount of preproC, proC, C and chymosin) induced from The amount of chymosin secreted is 0.04% of the total mycelium protein (Figure 17). was 60% of the total chymosin produced. This is Trichoderma reesei This shows that even foreign gene products outside the mycelium can be secreted effectively.

Plasmid pAMH104 (Fig. 1) is capable of secreting large amounts of prochymosin. Transformants containing several copies of 1) are measured for milk clotting activity in the growth medium. Screening was carried out by The best of the 40 transformants tested 500 μg/l as determined by Western gel and milk coagulation activity. (culture medium).

Cultures grown in 5-liter laboratory fermenters and small-scale cultures (10-5 01111), the amount of active chymosin secreted was 200 times greater. Due to the increase, the amount of chymosin produced by this type of strain is 100 mg/1 Before and after.

Example 11 Universal expression vector for homologous and heterologous protein production in Trichoderma reesei construction of Construction of universal vectors for production of various proteins in Trichoderma lee sei The promoter and terminator regions of the ebh1 gene were A universal expression vector was constructed containing: ebh i terminator, 3 transcriptions into the Pst1 site of pUc19 together with an adapter containing the stop codon TAA in the reading frame. Upon subcloning, plasmid pAMH110' was generated (Figure 15). P The stI terminator fragment was isolated from pAMH110' and cbh 1 promoter region, EcoRI-Pst containing the amino terminus of the Pr0C gene. I fragments from pAMH102 and these fragments were The 1l-NdeN site has already been blunted by Frenow before this subcloning step. It was subcloned into EcoRI-PstI digested pUc19★. Generate The resulting plasmid pAMH110 contains the promoter and terminus of the ebhl gene. Noter and between these sequences were removed by 5acII and NdeI digestion. Contains the resulting stuffer fragment. After the ends are blunted, the cD of the gene The NA or chromosomal copy can be inserted between the promoter and terminator. (Figure 15).

Example 12 Trichoderma reesei endoglucanar under the cbh l promoter expression of ze To increase the amount of endoglucanase produced, the egll gene was Bound to the strong cbh l promoter. Trichoderma reesei endog The cDNA of lucanase ■ is universally available as an ECORI-BamHI fragment. Subcloned into the expression vector pAMH110 (described in Example 11). this The vector was first digested with 5acII-Ndel to remove the stuffer. - The fragment was deleted (Fig. 16). cbh l gene promoter and The generated plasmid pAMH11 containing the egl l gene between the 1 and Trichoderma reesei QM941 together with p3SR2 in a 5:1 molar ratio, respectively. 4 (ATCC26921). Transformants have AmdS+ phenotype. and further purified on selective media. 50 ml of each of the six transformants Cellulase induction medium in liquid culture medium (Solca Flotu, 1% as carbon source) It was grown for 4 days. Then, hydroxyethylcellulose (0.1%, Ref. By measuring the release of reducing sugars from the culture supernatant (12), the culture supernatant was Tested for lucanase activity. The EG I activity of the transformant was controlled (QM94 14), the best transformants had 4 lower endoglucanase activities than the control strain. It has been shown to secrete twice as much. This example shows that by changing each promoter The amount of various cellulolytic enzymes in Trichoderma reesei can be modified. to show that

Example 13 Construction of expression vector for kappa light chain The construction is a 4 step process: 9-pEN301, pEN302, pEN30. 3, pEN304 and pEN305 were generated (Figure 22).

I7 Plasmid pE ligation of bacterial expression cassette to Escherichia coli vector To assemble N301, the chymosin expression cassette (~420°bp) was Excised from plasmid pAMH102 by cI and partial 5phI digestion. . Plasmid pTZ19R (2860 bp, Pharmacia) was digested with PstI. It became linear. The ends of both fragments were flattened by treatment with Namium nuclease. It was smooth. The blunt-ended 5acI-8phl fragment was converted into a blunt-ended Ps The tI open chain vector was ligated. The generated plasmid was called pEN301. I named it.

Il, replacement of chymosin cDNA by light chain cDNA plasmid pEN301 Cutting with stI and Be11 yielded a 5.9 kb hector. light chain The cDNA insert was digested with Xhol-8acI (896 bp) into pLTI (Fig. 18). To blunt the fragment ends , upon ligation, formed plasmid pEN302 (6,9 kb) .

IIl, Precise bond formation: cbhl signal sequence-light chain cDNA polymerase linkage cbh by creating a new Apa1 site using chain reaction (PCR) method. 198 bp between the 1 signal sequence and the light chain cDNA was deleted. used The primers are depicted on plasmid pEN303 in Figure 22 and their nucleotides are Identified by size in squares. Can hybridize to the promoter region 1 oligo (19 nt) with Ksp1 site and signal sequence and light chain cDNA Using another oligo (28 nt) with an Apa1 site at the junction between A An 83 bp fragment was amplified. ApaI site is cbh1 signal created by changing the sequence CGTGCT at the end of the file sequence to CGGGCC. The codons for Arg and Ala were retained. Other fragments (88 bp) is one oligo (29 nt) from the junction region with Apa1 site and Ps By using another oligo (18 nt) with a t1 site (in the light chain cDNA). Created by The 5abp fragment was digested with Ksp■ and Apal and 88 The bp fragment was digested with Apal and Pstl.

Ligation of the fragments at their Apa1 sites yields a 132 bp fragment. The KspI-PstI open-chain vector pEN 302 was ligated. New junctions were checked by sequencing. P The lasmid was named pEN303.

rv, a bacterial marker, and a phleomycin resistance cassette added to the light chain expression vector. Lasmid pAN8-H Mattern, 1. E,,Punto,P,J,,Lankres, S9, Pauwels, P., H., and Van Tan Hondel, C.A.M. J, J,, “Transformation on Aspergillus oryzae ”, “Abstracts on the Nineteenth Runteren Lecture on molecular genetics on yeast and filament ``Ntas Fafzi and Iic Impact on Biotechnology'' In, Runteren, Netherlands (1987), 34) to EcoRI-NdeI. The 3.5 kb phleomycin resistance cassette was released from the hectare by further cutting. Ta. Plasmid pEN303 was cut with EcoRI and the 5Jkb fragment was separated. I let go. Fragments were first ligated at their EcoR1 site. 1 The ends of the 0.3kb product were filled in with Flenow, and the fragment was created by ligation. was made into a ring. The resulting plasmid pEN304 was oriented in the same direction as the light chain expression cassette. It had a phleomycin cassette. Plasmid pEN305 is had the expression cassette in the opposite orientation.

Example 14 Construction of an expression vector for cbh l-heavy chain fusion protein This construction was performed in nine steps. , plasmids pEN201 to pEN209 were generated (Fig. 23, Fig. 24).

■, Binding of heavy chain cDNA to vector The gene encoding the heavy hitter (1gG3) was transfected with HindIII and EcoRI. When cut from plasmid pLT2 (Figure 49), an 870 bp insert was generated. , this was transformed into H1ndIII-EcoRI open chain plasmid pTZ18R (2,8kb , Pharmacia). The generated 3.7kb plasmid was transferred to pE It was named N201.

Il, addition of the chromosomal cbh 1 gene to the 5' site of the heavy chain cDNA AvaI (2 , 1kb) 'QENIOI (Figure 21) by cutting the chromosome cbh l gene. Plasmid pEN202 (5.8 kb) was assembled by. (Plasmid pEN101 is a subclone, and the chromosome cbh1 gene is cloned into the λ44A clone. (Thiri, T9, Salvuori, ■, and Knowles, Jl, “The Molecule Larger Cloning in the Major Cellulase Gene from Tri Coderma Reese J1 “Bio/Technology J1: 696-699 (198 3)) into plasmid pT218R. Cut lambda DNA with ApaI , PTZ18R was cleaved with BglI, and the fragment was dyed before ligation. (treated with enary nuclease).

Plasmid pEN201 was cleaved with HindIH (3,7 kb). Reige Both fragments were made blunt-ended using Namium nuclease prior to lysis.

The goal of precise junction formation between IIl, cbhl and heavy chains is to efficiently The majority of the secreted CBHI protein (corresponding to amino acids 1-484) is fused to by facilitating the secretion of heavy-duty drugs from Trichoderma host cells. Ta. All fusion protein constructs in which the signal sequence is joined to the immunoglobulin sequence. construct fusions in a manner that does not alter the translational decoding phase of immunoglobulin RNA sequences. It is necessary to Furthermore, this CBHI fragment is a secretory "carrier", i.e. In other words, it is intended to function only as a secretory signal; Since the functional function is not required in the final product, a factor Xa recognition site was added between the two regions. As a result, the CBHI portion can be easily converted into proteins after secretion by methods known in the art. It became possible to remove it hydrolytically. Correct joining deletes 265 bp It was done by One tJ of pEN202 [DNA product is phage: M Manufactured with the help of 13KO7. 36 nt long oligo containing precise junctions The cleotide was hybridized to the single end strand DNA. Oligonu as a primer By using cleotide, the plasmid is dNTP.

It was made into double strands by Klenow polymerase and ligase. double stranded DNA mixture The compound was digested with 5phl (site in the loop, single site in the plasmid). The deletion plasmids were concentrated using Transform the digested DNA mixture into JM109 The whole pool of transformants was grown in liquid medium. DNAI! pool items The correct plasmid was reconcentrated by digesting the mixture with 5 phl.

The digested mixture was transformed into JM109. Corrected by sequencing the junction region A new clone was identified. The generated plasmid was called pEN203 (5.5 kb). Name it.

Iv, transfer of the junction region to a non-contaminated background. Errors can easily occur in the long term. To avoid errors in construction, sequenced The exact junction region (190 bp) was extracted into pE by BstEII-Narl digestion. Cut from N203, clean pack ground: BstEII-Narl Transferred to open plasmid pEN202. The generated plasmid is pEN204 It was named.

■ Addition of bacterial terminator to the construct 476 bp long cbh l terminator was excised from plasmid pAMH111 by BglI digestion. plasmid pEN204 (5.5 kb) was cleaved with KpnI.

Fragment ends were flattened by treatment with Salmonella nuclease prior to ligation. I made it a smooth end. The plasmid is named pEN205.

Construct for forming a precise junction between V10 heavy chain and cbh1 terminator Until now, the heavy chain cDNA in the v, cl, hinge and C2 regions has been It consists of When producing Fab molecules, add a translation stop signal after the 01 region. must be placed in Violet hinge and C2 region deletion (235 bp) and Fusion of the cbh1 transcription terminator with a translation stop signal to the C1 terminus This was achieved by using the PCR method. The primers used are shown in Figure 23, plus depicted in midd pEN206 and identified by their nucleotide size. Ru.

30 amounts that hybridize to the cbh l gene and have a BstEII site there. and another 3 that hybridizes to the heavy C1 terminus and has a BclI site therein. By using the 0-mer, an 890 bp long sequence was amplified by PCR. c. 4 that hybridizes to the bhl terminator region and has a BdI site there. Hybridizes further down in the heptamer and terminator, where N By using a mono-30mer with a si1 site, another ~240 bp fragment created an item. Cut the first amplified fragment with BstEII and BdI. The other end was cut with BclI and N5il. fragments of them ligated at the Be11 site, and the deleted sequence was located between these sites), and was formed. The ~1100 bp fragment was added to the BstEII-NsH open chain plasmid pEN20. 5 cells were ligated. Sequencing of the PCR region in the generated plasmid The plasmid was named pEN206.

VTl, IgG 3 heavy chain cDNA to IgG1 heavy chain cDNA exchange IgG3 To exchange heavy chain cDNA to IgG1 heavy chain cDNA, a 640 bp long Ig G1 heavy chain cDNA was digested with NarI-NdeI to create plasmid pTI8 ( Figure 20). Plasmid pEN206 into NarI and BclI (5.2 kb). Ligate the fragments at their Nar1 sites. It was done. To blunt the ends of the fragment, Upon reaction, a 5.8 kb plasmid pEN207 was formed.

nIl, addition of cbhl promoter to the construct plasmid pEN207, It was degraded by Ksp1 (=SacII) and N5iI digestion. cbh The l promoter was transformed into plasmid pUJ10 (Fig. 26). The 2.5 kb fragment from pEN207 was transformed into pUJl Ligation of the 5.0 kb fragment from o resulted in a 7.5 kb plasmid. pEN208 was formed.

Xl, fungal marker, and addition of S aid S expression cassette to the construct, 5 Plasmid p3SR2 (Cabin, S9, etc., EP125023, Kaiser, C.A., et al., Science, 235:312-317 (1987) ), a 2.7 kb insert was generated, which was inserted into the 5spl open-chain plasmid. pEN208 (7.5kb) was ligated. Generated 10.2kb plastic The sumid was named pEN209.

Example 15 Construction of expression vector for IgG1 heavyweight I9 fusion vector 1) Deletion of cbh1 gene from EN207 using PCR By this, cbh1 gene (1533bp) was extracted from plasmid pEN207. Deleted it. The primers are depicted in Figure 25, plasmid pAJ201, and their They were identified by their nucleotide size.

An oligo containing a Ksp1 site that hybridizes to the cbh l promoter region ( 19nt) and an Apa1 site at the junction between the signal sequence and key genes. Amplify the 83 bp fragment by using another oligo (28 nt) with did. cbh 1 sig so that the codons for Arg and Ala are retained By changing the sequence CGTGCT at the end of the null sequence to CGGGCC, A A paI site was created. One oligo (29 nt ) and another oligo with Nsi 1 site (within cbh 1 terminator) ( Another fragment (943 bp) was created by using 19 nt).

The 83bp fragment was digested with Kspl and ApaI and the other (943bp The fragment of p) was digested with ApaI and NsiI. fragment ligation at their Apa1 site yields an approximately 1ooobp fragment was created, and then converted into the KspI-NsiI open-chain vector pUJ10 ( Figure 26) Heridation was performed. The new junction was checked by sequencing. . The plasmid is named pAJ201 (6,0 kb).

Il, addition of bacterial marker, aa + d S to the construct plasmid pA J 201 (6,0 kb) was cleaved by SalI-XhoI digestion. p3SR2 The amdS expression cassette from kb).

The fragments were ligated at their 5alI sites. 9.8kb product After filling the ends with Flenow and circularizing the plasmid by ligation, the plasmid A lasmid pAJ202 (9.8 kb) was generated.

Example 16 Light chain expression Trichoderma reesei strain R1JT-C-30 (ATCC56765) was expression vectors, pEN304 and pEN305, both containing phleomycin ( trademark) marker). Similarly, Katanuki conversion was also carried out at the same time. , plasmid pEN303 (no marker 2) and plasmid pAN8-1 ( (containing the phleomynon resistance gene) were simultaneously transformed into 62 RUT-C~30. I changed it. Transformation in Trichoderma is carried out using the selection markers amdS and ar Trichoderma reesei transformation method based on the use of gB (Pentilla, M. 1 Nevalainen, H1 Rat-1M, % Salmiphon, E., and Knowles, J. l “A Versatile Transforming System for the Self” Llorithic filamentus fungus trichoderma reesei”, “ Gene, 61, 155-164 (1987)). Same si By using the stem, mutant strains with t, rpC markers can be selected. Ru. In this experiment, transformants were selected by phleomycin resistance.

Two different types of plate/'overlay media were used. On the other hand, K) (2 P04 was replaced by 2HPO4 and 0.44 mol of sucrose was used as an osmotic stabilizer. It was based on Trichoderma reesei minimal medium supplemented with the other dark The reduced medium (MnR) consisted of 0.3 g monophthalate, 2.5 g yeast extract, 2. It consisted of 5g glucose and 150g sucrose/liter. both In the culture medium, the pH was adjusted to 7 to avoid destruction of phleomycin. coagulation 1.5% agar for the basal medium and 0.6% agarose for the overlay. I used

After transformation, protoplasts were cultured and incubated for 2-3 hours at room temperature. After that, apply an overlay containing 5 ml of phleomycin (Caila, France). Added to list. Two phleomycin concentrates, 35 μg/ml or 50 μg/m 1 (total plate medium) was used. Fresh stock of Phleomycin (30mg /ml) in sterile water.

Transformation plates were incubated at +28°C for 5-7 days before transformation. The moults were harvested and cultured on slants in MnR + 100 μg/ml phleomycin for 3 cycles. 1 week each (pEN304 and pEN305) or 1 cycle/week (pEN303-pAN81) Select stable transformants by growing I chose.

Before screening, transformants were cultured on PD slants (potato seedlings) to promote sporulation. Transferred to Kistolose Aka-). 5 spores per slant with 9% NaC 1 and suspended in 50n1 of liquid medium (2% Solca Flotu Cellulose and 1% The cells were transferred to Trichoderma reesei minimal medium (pH 5.0) supplemented with malt residue. shape The transformant was incubated at +28°C for 4 days while shaking at 160 rpm + n in a shaking flask. Made it grow. The best producers were placed in a 500 ml medium (inoculum from two slants). The cells were cultured at 250 rpm for 5 days. Remove the culture by filtration or centrifugation (3000g). The nutrient medium was separated from the mycelium. Mycelium was collected from the filtrate for Zazan analysis.

Light chain production was screened from the culture medium by using the ELI SA test. I clicked. Place the microtiter plate with anti-Ox IgG3 Fab antiserum (deposit). Part on Biotechnology and Immunology, Univerge The membrane was coated with purified 1gG fraction from T. on Helsinki). 100μl anti-Ox - IFab antibody, 7.5 μg/ml in 0.1 molar bicarbonate buffer, pH 9.6, Pipette into wells and incubate at +4°C. Wells between each stage 100 μl of 50 mmol phosphate buffer, pH 7.4-0.9% NaCl (PB Washed with S). by using 0.5% BSA in PBS for 1 hour at room temperature. The uncoated areas were blocked.

Culture supernatant samples both undiluted and diluted 1=20 in 0.5% BSA in PBS (100 μm) were added to the microtiter wells. Purification from mouse ascites The Fab molecule, 11-1On, was used as a standard in RUT-C-30 culture medium. Diluted in 5% BSA or PBS medium. Incubate the sample for 2 hours at room temperature. I turned on.

Secondary antibody: goat anti-mouse kappa alpha (alkaline phosphatase/saza Biotechnology Associates, Inc.) 1:5 00 dilution to the wells and incubated for 1 hour at room temperature. The substrate is p-nitrophe per ml of ethanolamine-MgC1□ buffer (Orion) Di-Na-Salt of Nilphosphate (Orion Company, Helsinki) 2 mg Met. The wavelength used for absorbance measurements was 405nl11.

As estimated from the ELISA test, the secreted light chain (pEN304/RUT-C- The amount of 30) was 200 gg per liter of culture medium (500i 1 culture) . The light chain producing Trichoderma reesei strain was purchased from 3740AG Baarn, The Netherlands. , central view of Oostilstrad 1, B-O Box 273 1 as accession number CB525290 to Low Fall Schimmelkurzless Deposited on June 25, 990.

Perform a preliminary incorporation test by blotting the culture supernatant onto a nitrocellulose filter. went. Filter with TBS (20 mmol Tris, pH 7,5-500 mmol) Blocked with 0.5% milk in molar NaC1), monoclonal anti-CBHI anti (1:50 in TBS) (Vienna, Austria). Institut für biohemosche technologie rant microbi CH5) provided by Professor C, P, Kubitschek of Ology. use The secondary antibody was anti-mouse polyvalent immunoglobulin alkaline phosphatase. Conjugate (Sigma). Protoplot/Immunoblotting/ Filters were stained using the system (Promega Biotic). Control strain (RU The fact that a positive signal was found only for T-C-30) indicates that two positive light chains Both productive transformants (out of 130 transformants screened) were c. It is shown to have a light chain expression cassette integrated at the bh1 locus.

Example 17 Expression of cbh 1-heavy chain fusion The heavy chain construct pEN209 (aids marker) was transferred to Trichoderma reesei strain R. Transform into UT-C-30. Plasmid pEN208 (no marker) and and p3SR2 are co-transformed into RUT-C-30. As a single nitrogen source Transformants are selected by their ability to use cetamide.

The plates and overlay medium used in the transformation are Trichoderma reesei A minimal medium in which 0.5% ammonium sulfate is mixed with 12 mmol acetamide. has been replaced. Basal growth was inhibited by adding 15 mmol of CsC1. Stop. The osmotic stabilizer used is 1 molar sorbitol.

After 8-10 days of culture, transfer the transformants from the transformation plate to PD slants. , forming spores.

Transformants were grown in shaking culture and subjected to ELISA tests and tests as described in Example 4. and Western analysis.

Example 18 Emergence of heavyweights The heavy chain construct pAJ202 was transformed into RUT-C-30 strain and the AmdS+ phenotype was obtained. Select about.

The method used is as described in Examples 16 and 17.

Example 19 Expression of different forms of antibodies ■, Bacterial transformation Transformation of heavy chain constructs pEN209 and pAJ202 into light chain expressing parent strain did.

The ability to utilize acetamide as a sole nitrogen source in the AmdS+ phenotype and screened for transformants. Trichoderma reesei transformant p EN304/pEN209 and pEN304/pAJ202 were obtained from the Netherlands, 3740AG Varun, Austilstrad 1, P.O. Box 273 Each of the Central Bureau Fall and Schimmel Kurzless has a number on it. CB5287.91 (dated June 3, 1991) and CB5288.91 (19 (dated June 3, 1991).

Co-transformation was also performed, pEN208 (no fungal marker)/pEN304 (marker: phleomycin resistance) and pAJ201 (no bacterial marker)/p Transforming EN304 and screening them for phleomycin resistance did.

Transformations were performed as described in Examples 16 and 17 above.

Il, Screening for antibody-producing transformants Transfer the transformants to 50 Otori liquid medium (Trichoderma reesei minimal medium supplemented with 3% whey and 1.5% malt grains) , pH 5,0). Transformants were incubated in shake flasks at 28°C, 20Or pm for 4 days.

Antibody production was cultured by using an ELISA test as described in Example 16. Screening was performed from the supernatant.

Measure the immunoreactivity of the Fab produced by using the same <EIjSA. Ta. Microtiter plate conjugated to hapten, namely BSA phenyloxacylone (Markeller-100, Carltihon, Ml, Perco Hong, J.L., and Karjaraihon, K., rInheritance on An. TiBody Specificity Buoy Aunty 2-Phenyloxacillo 'In the Mouth', 'Journal on Experimental Medicine' 148:1644-1660 (1978)). 0x-BSA , used at a concentration of 50 μg/ml in 0.1 molar bicarbonate buffer (pH 9,6). other Meanwhile, an ELISA test was conducted as described in Example 16.

A rough estimate of immunoreactivity from the ELISA test shows that the fraction of best transformants The yields of secreted Fab and CBHI-Fab were approximately 0.8 mg/l and CBHI-Fab, respectively. It was about 45 g/l.

IIl, pre-fermentation Two transformants producing Fab and CBHI-Fab were fermented. The medium is , 60% whey, 30% malt residue, (NH4)! SO4 6, KH!　P　O It contained 2 to 3 parts (g/l). The initial pH without adjustment was 4.8.

The inoculum was grown in two steps: 1 x 200 ml for 2 days and 5 x 2 QQ ml for 1 day. Grow in high strength medium (buffered with 15g/IKHz PO4). fermentation used The tank had a working volume of 15 liters + 1 liter inoculum and a temperature (33°C for 48 hours, followed by pH (4.0 to 5.5, 4.0 to 5.5 with NH, OH and HsPOa) ．． 0 to 5.5) and I) Chemap C with automatic control means for Ox. It was an F2000 motorcycle actor.

Samples were collected daily, and the growth medium and mycelia were filtered through GF/B microfiber filters. It was separated by filtration through a filter. The mycelium was washed with 0.9% NaCl.

Samples were stored at -20°C.

Fermentation of Fab and CBHI-Fab producing strains was continued for 4 and 5 days, respectively.

The growth medium and mycelium were separated by centrifugation, 50 g/l silica was added and mixed. growth by filtering with a GF/B microfiber filter (Whatman). After cleaning the medium, it was stored at -20°C.

Screen the superior groove for antibody production by ELISA and Western analysis. I clicked. According to ELISA, the produced immunoreactive Fab and CBHI-F The amount of ab was 0.5 mg/l (4th day) and 150 mg/l (5th day, review), respectively. Bell improved slightly on the fourth day). For Fab/RUT-C-30 fermentation The amount of total protein (lowry) in the CBHI-Fab/RU was 9.3 g/l and In TC-30, it was 6.5 g/l.

IV, CBHI-Fab fusion protein has a high yield of purified antibodies, making it difficult to grow the fusion protein. Protein purification was performed only from the medium. Purification was carried out in three major steps. Anion exchange Separation was used as a crude purification step. Affiliates using Habten as a ligand Nity chromatography resulted in a pure antibody product. gel filtration By using it as a final step, the original fusion antibody can be autoproteolytically removed from the original fusion antibody. The cleaved Fa14-like anti-decommissioning product was isolated.

Fermentation growth medium containing CBHI-Fab protein was mixed with Sephadex G-25 (coarse) Transfer to starting buffer, 10 mmol sodium phosphate buffer (pH 7.2) through Then, it was subjected to ion exchange chromatography. For rough purification, DEA Anion exchange chromatography was performed using E-Sepharose FF. Na Increasing the Cl molar concentration from 100 mmol NaCl to 1 mol NaCl was eluted. CBHI-Fab is suitable for anion exchange chromatography. The fusion antibody reacts similarly to wild-type CBHI, i.e., the fusion antibody is soluble with high salt concentration. I let go.

The pooled and concentrated samples were treated with 10 mmol Tris-MCI, pH 7.5-0, Dialyzed against 9% NaCl, starting buffer for affinity chromatography. did.

Ox-BSA was added to CNBr-activated Cef7 at a ratio of 5 mg protein/ml (gel). 0-su4B (Pharmacia). affinity chromatography was carried out as described in the literature (Barlow, E. and Lane, Dl. ``Intibody's, A Laboratory Manual'', Cold Spring Harbour. Barr Laboratory, Cold Spring Harbor, New York (1988) ). The bound antibodies were purified with 100 mmol glycerol-HCI (pH 2,5). Elute and immediately neutralize the fraction with 0.25M Tris (pH 8.0) did.

CBHI-Fab products are self-proteolytically cleaved Fab-like antibodies. It was more contaminated. Two antiseptics were obtained by gel filtration using Sephacryl S-200. The body forms were separated and the buffer used was 0.1 molar Tris-HCl, pH 7.2. -0°2 mol NaCl. Tested against storage buffer, PBS (pH 7,4). The sample was dialyzed.

■ Characterization of purified fusion protein Calculated at 2800 ■ based on Trp and Tyr content of each protein sequence. Molar extinction coefficient (167,760 M-’C for fused and cleaved antibodies, respectively) ! +-' and 86670 cm- The quality concentration was determined from the absorbance at 280 nl (Uetraufer, D.E., " Atopansis in Protein Chemistry”, 17:303-390 ( 1962)).

Verification of antibody chain size by 5DS-PAGE electrophoresis and Western analysis did. Protein samples were treated with anti-1gG1 (Southern Biotechnology Associa- tion). Sheets, Inc.) and anti-CBHI core (CR2, C, P, (provided by Bicek). The size of the fusion heavyweight is approximately 86kD. , its cleavage counterpart is approximately 24 kD, the same size as the unfused Fab heavy chain. Ru. The size of the light chain is 23kD.

Immunoreactivity of fused and cleaved antibodies was tested by ELISA. autoprotein hydrolysis cleaved antibodies and authentic proteolytically digested Fabs derived from ascites. The activity against silon binding was 27 times higher than the original fusion antibody.

The N-terminal amino acid sequences of purified fusion and cleaved antibodies were prepared using Applied Biosystems. Measured with Muzu 477A Online 120A Modified Pulse Liquid Phase/Gas Phase Sequencer (Bauman, M., Comparative Gas Phase and Pulse ・Liquid Phase Sequencing on a Modified Apply De Biosystem Da 4 Fufu A Sequencer-11 “Analytical Biochemistry 190:198-208 (1990)). The N-terminus of the chain is It had been processed. Cleavage of the fusion removes two amino acids in front of the true N-terminus of the heavyweight. a noic acid residue was generated. Cleavage after tyrosine residues occurs at low levels in Trichoderma growth medium. Indicates the presence of chymotrypsin-like protease activity. Ion exchange fluxin According to ELISA results from only cleaved and the cleaved antibody was eluted in the first fraction.

Example 20 Construction and generation of single chain antibodies Both light and heavy variable regions are connected to each other by a flexible hinge region of CBHI. A single chain antibody (SCA) was used. Construction of functional genes was performed by Thierry, T6 , Takkihon, K6, Laukkahon, M, -L, , Alftan, K1, Sizman ,D.

and Knowles, J., C., Recombinant Secretary Fusillo. Protease” (U.S. Utility Patent Application No. 071552751, 1) (dated July 16, 990). The structure of a functional gene is shown in Figure 2. 9. Regulatory regions were reconstructed for Trichoderma expression.

15-mer oligo and V that bind to the heavy chain vH-region on either side of the NarI site.

24-mer with end of region, stop codon and Bc11 site created at end The SCA encoding gene was isolated from plasmid pML5 (Figure 29) by PCR using It was amplified. PCR amplified 730bp fragment was digested with Narl and BclI. I digested it.

Plasmid pAJ201 was cut by Narl-BclI digestion. pAJ20 The 5° 4kb fragment of 2 was ligated with the 730bp PCR fragment. By doing so, plasmid pEN401 (6.2 kb) was created. S The CA encoding gene is the cbh1 promoter in plasmid pEN401. and the terminator region.

Alternatively, the SCA-encoding gene can be expressed using a glucose-expressed promoter, i.e. Cloned under the Aspergillus gpd promoter. gpd promoter ( 1.2 kb) was amplified from plasmid pAN8-1. The 39-mer oligo is the final Published gpd protein with EcoRI and Xba1 sites created at the 5° end. lomotor (Punto, J.P., Dingemanse, M.A.1, Quivenhopen , A8, Soede, R.D., M., Pauwels, P.H.9 and Hwang-Jin-Ho. Ndell, C, A, M, J, J,, rFunctional Elements in. The Promoter Region in the Aspergillus nidulans g pdA encoding glyceraldehyde-3 to phosphate dehydrogen used from the 5” end of Naze J1 Gene, 93:101-109 (1990)). was used. Next, the 27jl: body oligo is ccccccc+151+. +156 ) to CCGCGG, and binds to the 3° end of the gpcl promoter modified from Ks A p1 site was created. Amplified XbaI-KspI digested gpd promoter is ligated with Xba I-Ksp I digested plasmid pEN401. by replacing the cbh1 promoter and plasmid pEN402(5, 9kb) was created.

Plasmids pEN401 and pEN402 were used as markers (pAN8-1). Transformed into RUT-C-30 strain by co-transformation with phleomycin resistance. I changed it.

Transformants were grown in shake flasks supplemented with 3% whey and 15% malt grains. The cells were grown in Lycoderma reesei minimal medium (pH 5,0). Also, pE N402/RUT-C-30 transformants were incubated with 2% glucose and 0.5% peptide. Cultured in 50 ml of Trichoderma reesei minimal medium (pH 5,0) supplemented with did.

Ox-BSA coated microtiter wells were used as described in Example 19. Expression of immunoreactive SCA was tested from culture supernatants by ELISA. rabbit anti Purified 1gG fracin3 of Ox IgG3 Fab antiserum was mixed with the first detection antibody (1: 300). The second detection antibody layer is a goat anti-rabbit IgG (H+L) antibody. with Lucalic Phosphatase Conjugate (Piorad Laboratories) there were.

Example 21 ■, Fab fragment of anti-2-phenyloxacylone IgG3 subclass antibody Cloning of light and heavy chain CDNA fragments encoding light and heavy chain O xIgG3 subclass antibody mRNA was transferred to hybridoma cell clone DHK- 2,6 (Finland, 00290 Helsinki 29, University Inn. Department of Bacteriology and Immunology, Helsinki , obtained from M. Karl Tikhon).

The first strand of cDNA was synthesized using specific primers. The light chain primer is C Light chain t containing Xbar restriction site used in cloning DNA fragments It was complementary to the 3' non-dark region of mRNA. Heavy duty primer has Xbal limit It was complementary to a certain two-domain region containing the site. First strand cDNA synthesis or Maniatis et al., “Journal in Molecular Cloning Near Laboratories” Cold Spring Harbor Laboratory, New York - (1982)). After alkaline hydrolysis of mRNA, Second strand synthesis for both light and heavy was performed using specific primers. light and The heavy chain second strand primer is identical in sequence to the 5' non-coding region of the mRNA; They both contained a 5phI restriction site. After second strand synthesis, Maniatis et al. (1982), the 3° ends of the cDNA fragments were injected with T4 DNAp. Trimmed with oll. First, the light and heavy chain blunt-ended CDNA fragments were 5phI and XbaI restriction enzymes, and then psP73 vector (P73 vector) Romega). The light chain cDNA clone was named pLTl, and the heavy chain c The DNA clone was named pLT2.

Il, variable and first variable of anti-2-phenyloxacillone IgG1 subclass antibody Cloning of heavy chain CDNA fragments encoding variable domains IgG1 sub Class antibody mRNA was transferred to hybridoma cell clone NQ-17°4.1 (class antibody). Separated from Rutihon et al., Nature, 304:320 (1983)) . Using oligo(dT) primers (Maniatis et al., 1982), heavy cDNA The first strand of A was synthesized. Direct PCR amplification of cDNA:mRNA hybrids Attached.

The 5° end PCR primer is the 5° end of the variable domain containing the EcoRI restriction site. The sequence was the same as that at the end. The 3° end PCR primer has a HindlII restriction region. It was complementary to the heavy chain invariant 2 domain coding region containing the position. Amplified Ig01 ps the key region cDNA as an EcoRl-H1ndIII restriction fragment. It was cloned into P73 vector (Promega). Escherichia coli expression vector The heavy chain cDNA was modified by PCR before cloning into vectors. heavyweights The final 3° end of the coding region of the variable 1 domain is divided into three molecules involved in disulfide bonds. by replacing the cysteine residue with other key players in the intact IgG1 molecule. (Figure 20). In addition, liposome-binding molecular sequences for light chain translation and Ndel restriction site used in frame cloning with light chain coding region , was introduced in the modified 3' sequence of the heavy chain (Figure 20). Ox IgG3 subclass Nar I-N of key coding regions in Fab-fragment expression vectors The deI restriction fragment was that of a modified 1gG1 subclass cDNA clone. The modified heavy chain cDNA of the OxIgG1 subclass was The vector was cloned into an E. coli expression vector. Ox1gG1 subclass Fab - The Escherichia coli expression vector of the fragment was named pTI8.

Only the heavy chains differ from each other in different IgG antibody subclasses. hybrid The tumor cell clones DHK-2,6 and NQ-17,4,1 have the same light chain Manufacture mRNA sequence for light chain from NQ-17,4,1 clone has already been published by Karl Tikhon et al. (“Nature J, 304:28 ( 1983)).

Ultimately, only IgGIFab was used in the Trichoderma expression test, but IgG triplex Strand CDNA is present at an early stage in the construction of heavyweight expression vectors (Example 1 (see 5).

All references cited herein are incorporated by reference in detail. About the present invention Although the invention has been described in detail in without conflicting with God or scope (within a broadly uniform range of conditions, parameters, etc.) It is to be understood that the scope of the claims may be practiced as follows.

EcoRI Cla/m5p pUC19EcoRT-PsLr FJG, 7 A = pψ flat region (~2.4 kb) OAIJH10AMH20AMH3 - Digestion with SLlar (Sa+al in polylinker) - Kataga conversion ■ Digestion by 18 mal A transformation ■ Screening of single light (chain) clones NOR202 5' 3' TG’rJCC’ACA　’GCT　’CGT　’GCNOR203 5' 3' ACG' A (iC' TGT' GGC' CAA' GA mature protein The promoter of oligo cbh1 was joined to chymosin SS to create a fusion between Oligo FIG, 10 −ligation■ FIG. 13 FIG. 14 View FfG, 16 Mockery ■---　・　・---− FIG. 17 FIG. 25 pAN92←21 day d engineering pLIc 18 sequence FIG. 28 Stuffing abstract A method for producing recombinant immunoglobulin in a Trichoderma host is described.

1°-+1. ae@+1m-w+-Per/Fl 91100218 International Research Report notice

Claims (1)

[Claims] (1) a. a gene encoding an expressible immunoglobulin gene, and b. to Trichoderma transformation containing selectable markers that can provide selectivity in Lycoderma replacement vector. (2) The gene encoding an expressible immunoglobulin gene is further promoted terminators, enhancers, signal/leader sequences, terminators, and their combinations. one or more DNA elements selected from the group consisting of combinations, said DNA sequences being operably linked to said gene to control the expression and/or secretion of the desired protein product. , the vector according to claim 1. (3) The vector according to claim 2, wherein the DNA element is heterologous to the Trichoderma genome sequence. (4) The vector according to claim 2, wherein the DNA element is homologous to the Trichoderma genome sequence. (5) The DNA element contains a gene expressed in glucose and a cellulase gene. 5. The vector according to claim 2, wherein the vector is selected from promoter and terminator regions of genes selected from the group consisting of genes. (6) The vector according to claim 5, wherein the cellulase gene is selected from the group consisting of cbh1 gene, cbh2 gene, eg11 gene, and eg12 gene. (7) The DNA regulatory element is the promoter and terminus of the cbh1 gene. 7. The vector according to claim 6, which is a vector region. (8) The vector of claim 1, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC and argB. (9) The vector according to claim 8, wherein the selection marker is amdS. (10) The vector according to claim 8, wherein the selection marker is phleomycin resistance. (11) The vector according to claim 8, wherein the selection marker is trpC. (12) The vector according to claim 8, wherein the selection marker is argB. (13) A vector system for Trichoderma transformation, comprising a. Expressable immunoglobulins a gene encoding the bulin gene, and b. Providing selectivity in Trichoderma the gene in part (a) above and the marker in part (b) above; A vector system in which the car is provided to Trichoderma in two separate DNA molecules. (14) The gene encoding the expressible immunoglucose gene is further promoted promoters, enhancers, signal/leader sequences, terminators and combinations thereof one or more DNA regulatory elements selected from a combination of By operably linking the DNA sequence to the gene, the target protein is produced. 14. The vector system according to claim 13, wherein the expression and/or secretion of the quality product is controlled. (15) DNA regulatory elements may be heterologous to the Trichoderma genome sequence. 15. The vector system of claim 14. (16) DNA regulatory elements are homologous to the Trichoderma genome sequence. 15. The vector system of claim 14. (17) The DNA regulatory elements are linked to glucose-expressed genes and cellular promoter and terminator of a gene selected from the group consisting of - region. (18) Cellulase gene is cbh1 gene, cbh2 gene, eg11 gene 20. The vector system of claim 17, wherein the vector system is selected from the group consisting of the eg12 gene and the eg12 gene. (19) DNA regulatory elements are located at the promoter and terminus of the cbh1 gene. 18. The vector system of claim 17, which is a Noether region. (20) The vector system of claim 14, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC, and argB. (21) The vector system according to claim 20, wherein the selection marker is amdS. (22) The vector system according to claim 20, wherein the selection marker is phleomycin resistance. (23) The vector system according to claim 20, wherein the selection marker is trpC. (24) The vector system according to claim 20, wherein the selection marker is argB. (25) A method for transforming Trichoderma, which comprises transforming a suitable Trichoderma strain with the vector according to claim 1. (26) The gene encoding the expressible immunoglucose gene is further promoted promoters, enhancers, signal/leader sequences, terminators and combinations thereof one or more DNA regulatory elements selected from a combination of By operably linking the DNA sequence to the gene, the target protein is produced. 26. The method of claim 25, wherein expression and/or secretion of the quality product is controlled. (27) The method of claim 26, wherein the DNA regulatory element is heterologous to the Trichoderma genome sequence. (28) The method of claim 26, wherein the DNA regulatory element is homologous to the Trichoderma genome sequence. (29) The DNA regulatory elements are linked to glucose-expressed genes and cellular 29. The method according to claim 26, wherein the method is selected from the promoter and terminator regions of the enzyme gene. (30) Cellulase gene is cbh1 gene, cbh2 gene, eg11 gene 30. The method of claim 29, wherein the eg12 gene is selected from the group consisting of the eg12 gene and the eg12 gene. (31) DNA regulatory elements are located at the promoter and terminus of the cbh1 gene. 31. The method of claim 30, wherein the method is a Noether region. (32) The method of claim 25, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC, and argB. (33) The method according to claim 32, wherein the selection marker is amdS. (34) The method according to claim 32, wherein the selection marker is phleomycin resistance. (35) The method according to claim 32, wherein the selection marker is trpC. (36) The method according to claim 32, wherein the selection marker is argB. (37) Transforming a suitable Trichoderma strain with the vector system according to claim 13. A method for transforming Trichoderma. (38) The gene encoding the expressible immunoglucose gene is further promoted promoters, enhancers, signal/leader sequences, terminators and combinations thereof one or more DNA regulatory elements selected from a combination of By operably linking the DNA sequence to the gene, the target protein is produced. 38. The method of claim 37, wherein expression and/or secretion of the quality product is controlled. (39) The method of claim 38, wherein the DNA regulatory element is heterologous to the Trichoderma genome sequence. (40) The method of claim 38, wherein the DNA regulatory element is homologous to the Trichoderma genome sequence. (41) The DNA regulatory elements are linked to glucose-expressed genes and cellular 41. The method according to any one of claims 8-40, wherein the method is selected from the promoter and terminator regions of the enzyme gene. (42) Cellulase gene is cbh1 gene, cbh2 gene, eg11 gene 42. The method of claim 41, wherein the eg12 gene is selected from the group consisting of the eg12 gene and the eg12 gene. (43) DNA regulatory elements are located at the promoter and terminus of the cbh1 gene. 43. The method of claim 42, wherein the method is a Noether region. (44) The method of claim 37, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC, and argB. (45) The method according to claim 44, wherein the selection marker is amdS. (46) The method according to claim 44, wherein the selection marker is phleomycin resistance. (47) The method according to claim 44, wherein the selection marker is trpC. (48) The method according to claim 44, wherein the selection marker is argB. (49) A method for producing immunoglobulin, comprising: a. Immunoglobulin heavy chain or transforming Trichoderma host cells with a recombinant construct encoding an immunoactive fragment of b. Coating an immunoglobulin light chain or its immunoactive fragment transforming a host cell with the recombinant construct containing the host cell; c. culturing the host cells under conditions that express the heavy and light chains; and d. c club inn A method comprising the step of recovering immunoglobulin produced by principal cells. (50) A method for producing immunoglobulin, comprising: a. Immunoglobulin heavy chain or transforming a first Trichoderma host cell with a first recombinant construct encoding an immunoactive fragment of b. Immunoglobulin light chain or its immunoactive fragment c. transforming a second host cell with a second recombinant construct encoding a. Heavy chain o culturing the first host cell and the second host cell under conditions that express the light chain and the light chain; and d. Immunoglobulin heavy chain or its immunoactive fraction produced by the first host cell immunoglobulin light chain or its activity produced by the immunoglobulin protein and the second host cell. collecting the sexual fragments, and e. Incubating the protein under conditions that promote reassociation of heavy and light chains into an immunoreactive conformation. (51) Recombinant encoding an immunoglobulin light chain or immunoactive fragment thereof constructs encoding immunoglobulin heavy chains or immunoactive fragments thereof. The recombinant constructs are each functional to a nucleotide sequence encoding a secretion signal. 51. The method of claim 49 or 50, wherein the method is (52) A method for producing an immunoglobulin light chain or an immunoactive fragment thereof, comprising: a. Recombinant encoding an immunoglobulin light chain or immunoactive fragment thereof transforming a Trichoderma host cell with the construct; b. culturing the host cell under conditions that express the light chain; and c. immunoglobulin light chain or The method includes the step of recovering the immunoactive fragment. (53) Recombinant encoding an immunoglobulin light chain or immunoactive fragment thereof The construct is operably linked to a nucleotide sequence encoding a secretion signal. 53. The method of claim 52, wherein: (54) A method for producing an immunoglobulin heavy chain or an immunoactive fragment thereof, comprising: a. Recombinant encoding an immunoglobulin heavy chain or immunoactive fragment thereof transforming a Trichoderma host cell with the construct; b. culturing the host cell under conditions that express the heavy chain; and c. immunoglobulin heavy chain or The method includes the step of collecting the immunoactive fragments. (55) Recombinant encoding an immunoglobulin light chain or immunoactive fragment thereof The construct is operably linked to a nucleotide sequence encoding a secretion signal. 55. The method of claim 54, wherein: (56) Transforming Trichoderma host cells with the vector of claim 1. A method for producing an immunoglobulin light chain or an immunoactive fragment thereof, comprising: (57 The gene encoding the expressible immunoglucose gene is further promoted terminators, enhancers, signal/leader sequences, terminators, and their combinations. one or more DNA regulatory elements selected from combinations thereof, wherein said DNA sequence is operably linked to said gene, thereby controlling the expression and/or secretion of the protein product of interest. The method according to item 56. (58) The method of claim 57, wherein the DNA regulatory element is heterologous to the Trichoderma genome sequence. (59) The method of claim 57, wherein the DNA regulatory element is homologous to the Trichoderma genome sequence. (60) The DNA regulatory elements are linked to glucose-expressed genes and cellular 60. The method according to any one of claims 57-59, wherein the method is selected from the promoter and terminator regions of the enzyme gene. (61) Cellulase gene is cbh1 gene, cbh2 gene, eg11 gene 61. The method of claim 60, wherein the eg12 gene is selected from the group consisting of eg12 and eg12 genes. (62) DNA regulatory elements are located at the promoter and terminus of the cbh1 gene. 62. The method of claim 61, wherein the method is a Noether region. (63) The method of claim 56, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC, and argB. (64) The method according to claim 63, wherein the selection marker is amdS. (65) The method of claim 63, wherein the selection marker is phleomycin resistance. (66) The method of claim 63, wherein the selection marker is trpC. (67) The method of claim 63, wherein the selection marker is argB. (68) A method for producing an immunoglobulin or an immunoactive fragment thereof, comprising transforming a Trichoderma host cell with the vector system according to claim 13. (69) A method for producing an immunoglobulin or an immunologically active fragment thereof, comprising transforming a Trichoderma host cell with the vector system according to claim 13. (69) A gene encoding an expressible immunoglucose gene is , more promos promoters, enhancers, signal/leader sequences, terminators and combinations thereof one or more DNA regulatory elements selected from a combination of By operably linking the DNA sequence to the gene, the target protein is produced. 69. The method of claim 68, wherein expression and/or secretion of the quality product is controlled. (70) The method of claim 69, wherein the DNA regulatory element is heterologous to the Trichoderma genome sequence. (71) The method of claim 69, wherein the DNA regulatory element is homologous to the Trichoderma genome sequence. (72) The DNA regulatory elements are linked to glucose-expressed genes and cellular 72. The method according to any one of claims 69-71, wherein the method is selected from the promoter and terminator regions of the enzyme gene. (73) Cellulase gene is cbh1 gene, cbh2 gene, eg11 gene 73. The method of claim 72, wherein the eg12 gene is selected from the group consisting of eg12 and eg12 genes. (74) DNA regulatory elements are located at the promoter and terminus of the cbh1 gene. 74. The method of claim 73, wherein the method is a Noether region. (75) The method of claim 68, wherein the selectable marker is selected from the group consisting of amdS, phleomycin resistance, trpC, and argB. (76) The method according to claim 75, wherein the selection marker is amdS. (77) The method according to claim 75, wherein the selection marker is phleomycin resistance. (78) The method according to claim 75, wherein the selection marker is trpC. (79) The method according to claim 75, wherein the selection marker is argB. (80) The method according to claim 56 or 68, wherein the immunoglobulin or immunoactive fragment thereof comprises a Fab region. (81) The immunoglobulin or immunoactive fragment thereof contains an Fc region, 69. The method according to claim 56 or 68. (82) The immunoglobulin or immunoactive fragment thereof is a light chain or an immunologically active fragment thereof. 69. The method of claim 56 or 68, comprising an infectiously active fragment. (83) The method according to claim 82, wherein the light chain is a λ chain. (84) The method according to claim 82, wherein the light chain is a κ chain. (85) The immunoglobulin or immunoactive fragment thereof is a heavy chain or an immunologically active fragment thereof. 69. The method of claim 56 or 68, comprising an infectiously active fragment. (86) The method according to claim 85, wherein the heavy chain is a μ chain or an immunoactive fragment thereof. (87) The method according to claim 85, wherein the heavy chain is a γ chain or an immunoactive fragment thereof. (88) The method according to claim 85, wherein the heavy chain is an α chain or an immunoactive fragment thereof. (89) The method according to claim 85, wherein the heavy chain is a δ chain or an immunoactive fragment thereof. (90) The method according to claim 85, wherein the heavy chain is an ε chain or an immunoactive fragment thereof. (91) A fusion protein containing an immunoglobulin or an immunologically active fragment thereof. Thus, the immunoglobulin or immunologically active fragment thereof has a beneficial function per se or is beneficial for immunoglobulin production or use and is a toxin. linked to an amino acid sequence capable of directing secretion in Lycoderma; A fusion protein in which the amino acid sequence can optionally be cleaved from an immunoglobulin or immunocleavable fragment thereof. (92) A fusion protein whose amino acid sequence is homologous to Trichoderma. (93) A fusion protein whose amino acid sequence is heterologous to Trichoderma. (94) The fusion protein according to any one of claims 91 to 93, wherein the amino acid sequence is a sequence of a cellulase or a fragment of a cellulase. (95) The fusion protein according to claim 94, wherein the cellulase is cellobiohydrolase I or a fragment thereof. (96) The fragment contains amino acids 1-484 of cellobiohydrolase I. 96. The fusion protein of claim 95. (97) The fusion protein according to claim 94, wherein the cellulase is cellobiohydrolase II or a fragment thereof. (98) The cellulase is endoglucanase I or a fragment thereof. 95. The fusion protein according to claim 94. (99) The fusion protein according to claim 94, wherein the cellulase is endoglucanase II or a fragment thereof. (100) Transformed with the fusion protein according to any one of claims 91 to 99. Trichoderma host cells. (101) Plasmid pEN304. (102) Trichoderma host transformed with the plasmid according to claim 101 Principal cell. (103) The host cell is 103. The Trichoderma host cell of claim 102 having accession number 252.90. (104) An immunoglobulin or an immunoactive fragment thereof, produced by the method according to claim 56 or the method according to claim 68. (105) The immunoglobulin according to claim 104, wherein the immunoglobulin is a chimera. (106) A composition comprising an immunoglobulin produced by the method according to claim 56 or the method according to claim 68. (107) A fusion protein comprising immunoactive fragments of light and heavy chains joined together by a linker selected from the group consisting of a synthetic linker, a Trichoderma-derived linker, and a heterologous linker. (108) The fusion protein according to claim 107, comprising a light chain or heavy chain variable domain. (109) Has a beneficial function in itself or is used for immunoglobulin production or use. The amino acid sequence is linked to an amino acid sequence that is beneficial for use and capable of directing secretion in Trichoderma, and that said amino acid sequence can be cleaved from the fusion protein if desired. 108. The fusion protein of claim 107. (110) selected from the group consisting of pEN303, pEN305, pEN208, pEN209, pEN401, pEN402, pAJ201 and pAJ202. plasmid. (111) Trichoderma host transformed with the plasmid according to claim 110 Principal cell. (112) Trichoderma reesei (pEN304/pEN209/RUT-C-30) with CBS accession number 287.91. (113) Trichoderma reesei (pEN304/pEN202/RUT-C-30) with CBS accession number 288.91. (114) Plasma in Escherichia coli with accession number DSM6584 Sumid pAJ202. (115) Plasma in Escherichia coli with accession number DSM6585 Sumid pAJ209. (116) Plasma in Escherichia coli with accession number DSM6586 Sumid pEN304. (117) Plasma in Escherichia coli with accession number DSM6587 Sumid pEN401. (118) Plasma in Escherichia coli with accession number DsM6588 Sumid pEN402.