Bactericidal protein as well as coding gene and application thereof

Abstract

The invention discloses a bactericidal protein as well as a coding gene and application thereof, and belongs to the technical field of gene engineering. The amino acid sequence of the bactericidal protein is a sequence as shown in any one of SEQ ID NO.1-12, or a sequence with the same function after any one of SEQ ID NO.1-12 is substituted and / or deleted and / or added with 1-30 amino acids, or a sequence with the same function after any one of SEQ ID NO.1-12 is added with 1-20 amino acids at the C tail end and / or N tail end. The bactericidal protein produced by the invention can hydrolyze cell walls of bacteria to cause dissolution of micrococcus luteus. Through verification, the bactericidal protein can significantly inhibit or kill Gram-positive and Gram-negative bacteria and saccharomycetes, and a novel bactericidal protein preparation and a preparation method thereof are provided for daily chemical, food, feed and other industries.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a bactericidal protein and its encoding gene and application. Background Art

[0002] The cell wall of microorganisms is composed of polysaccharides, mainly peptidoglycan, glucan, mannan, chitin and cellulose. The fungal genome contains a wealth of unknown proteins with glycoside hydrolase activity. If the above polysaccharides can be hydrolyzed, the cell wall of microorganisms can be hydrolyzed to achieve a bactericidal effect. No related research on fungal bactericidal proteins has been reported in the known academic research field.

[0003] Trichoderma reesei is an important industrial production strain that meets the GRAS (Generally Regarded as Safe) standard, and the enzyme preparations produced by its fermentation have been widely used in food, feed and other industries. It is known that there is no report in the academic research field that Trichoderma reesei strains can ferment and produce fungal bactericidal proteins.

[0004] The present invention intends to utilize Trichoderma reesei to secrete and express fungicidal proteins, thereby developing new technical ideas and providing new technical solutions in the field of pathogen killing. Summary of the invention

[0005] The purpose of the present invention is to provide a bactericidal protein and its encoding gene and application to solve the problems existing in the above-mentioned prior art. The bactericidal protein produced by the present invention can hydrolyze the cell wall of bacteria and cause the dissolution of Micrococcus luteus. It has been verified that the bactericidal protein can significantly inhibit or kill Gram-positive, Gram-negative bacteria and yeast, and provides a new bactericidal protein preparation and its preparation method for daily chemical, food, feed and other industries.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a bactericidal protein, the amino acid sequence of the bactericidal protein is a sequence shown in any one of SEQ ID NOs. 1 to 12, or a functionally identical sequence after 1 to 30 amino acids are substituted and / or deleted and / or added to any one of SEQ ID NOs. 1 to 12, or a functionally identical sequence after 1 to 20 amino acids are added to the C-terminus and / or N-terminus of any one of SEQ ID NOs. 1 to 12.

[0008] In the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed; adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. Therefore, in the present invention, the amino acid sequence of the bactericidal protein also includes variant forms of any of the sequences of SEQ ID NO.1 to 12 that have the same function as the bactericidal protein. These variant forms include: substitution and / or deletion and / or addition of several (1 to 30, preferably 1 to 20, more preferably 1 to 10, and more preferably 1 to 5) amino acids, and adding one or several (1 to 20, preferably 1 to 10, and more preferably 1 to 5) amino acids at the C-terminus and / or N-terminus of any of the sequences of SEQ ID NO.1 to 12.

[0009] The present invention also provides a gene encoding the above-mentioned bactericidal protein, wherein the nucleotide sequence of the gene is a sequence shown in any one of SEQ ID NOs. 13 to 24, or a sequence having more than 85% homology with any one of SEQ ID NOs. 13 to 24.

[0010] Due to the existence of codon degeneracy (degeneracy refers to a sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid), in the present invention, the nucleotide sequence of the gene encoding the bactericidal protein also includes a sequence having more than 85% homology with any one of SEQ ID NOs. 13 to 24.

[0011] The present invention also provides a recombinant expression vector, which contains a nucleotide sequence encoding the bactericidal protein or contains the gene.

[0012] The present invention also provides a recombinant microorganism, wherein the recombinant microorganism comprises the above-mentioned recombinant expression vector.

[0013] Preferably, the chassis microorganism of the recombinant microorganism is Trichoderma.

[0014] Preferably, the Trichoderma is Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

[0015] The present invention also provides a use of the above gene, the above recombinant expression vector or the above recombinant microorganism in producing bactericidal protein.

[0016] The present invention also provides a method for producing the bactericidal protein, which comprises fermenting the above-mentioned recombinant microorganism and collecting the fermentation liquid to obtain the bactericidal protein.

[0017] The present invention also provides an application of the bactericidal protein prepared by the production method in the preparation of a bactericide.

[0018] The present invention has the following beneficial effects:

[0019] The present invention discloses an amino acid sequence of a bactericidal protein and a gene sequence encoding the bactericidal protein. The gene encoding the bactericidal protein is transformed into the Trichoderma genome using the Trichoderma expression vector CBH1T1.0, and a recombinant Trichoderma strain that secretes and expresses the bactericidal protein is successfully constructed. The recombinant Trichoderma strain can be used for the production of bactericidal proteins. Experimental verification shows that the bactericidal protein produced by the present invention can significantly kill Gram-positive, Gram-negative bacteria and yeasts, and its effect is better than the common bactericidal proteins in the prior art. The present invention provides a new bactericidal protein preparation and a preparation method thereof for daily chemical, food, feed and other industries, and its application in killing pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a schematic diagram of the structure of the recombinant expression vector of Example 1;

[0022] Figure 2 This is a schematic diagram of the construction process of the recombinant Trichoderma strain in Example 2;

[0023] Figure 3 The results of the inhibition zone test of the bactericidal protein F5 and commercial lysozyme in Example 4 are shown. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] In an embodiment of the method of the present invention, the formula of the culture medium and reagents is as follows:

[0030] (1) Luria Bertani (LB) medium formula: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, dilute to 1 L with tap water, natural pH.

[0031] (2) PDA medium formula: 200 g potato, 20 g glucose (or xylose), 20 g agar, dilute to 1 L with tap water, natural pH.

[0032] (3) Formula of Trichoderma fermentation medium (1 L): glucose 30 g, lactose 10 g, cellulose powder 10 g, bran 5 g, corn steep liquor 5 g, peptone 3 g, yeast powder 2 g, KH2PO4 2.0 g, CaCl2 0.34 g, MgSO4·7H2O 0.3 g, Mandels trace element solution 1 mL, Tween 80 1 mL.

[0033] (4) Formula of Mandels trace element solution (1000×): FeSO4·7H2O 5 g, CoCl·6H2O 2 g, ZnSO4·7H2O 1.4 g, MnSO4·H2O 1.6 g, and pure water to make up to 1 L.

[0034] (5) Agrobacterium conjugation to transfer Trichoderma: The expression vector was electroporated into Agrobacterium tumefaciens, and then the successfully electroporated Agrobacterium was inoculated with Trichoderma strains QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT-C30 (ATCC56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), and PC-3-7 (ATCC 66589) on IM plates (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3): 259-264) were co-cultivated for Agrobacterium tumefaciens-mediated conjugative transfer. After two days of co-cultivation, the cells were added to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening until mycelium and spores grew. PCR verification was then performed to confirm that the grown cells were correct transformants.

[0035] The plasmid extraction kit was purchased from AXYGEN, the gel recovery kit was purchased from MAGEN, the seamless cloning kit was purchased from Quanshijin, and the DNA restriction endonuclease and ligase were purchased from NEB. Similar products from other companies can also be used instead.

[0036] The experimental methods in the following examples where specific conditions are not specified were generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0037] The amino acid sequences of the bactericidal proteins in the following examples are shown in SEQ ID NOs. 1-12.

[0038] SEQ ID NO.1:

[0039] MKTTFAALIVSLTSIVSAYPITGDVVNCRTGPGTSYAIKTSYKKSHDISISCQTTGTSVNGNNIWDKTADGCYVADYYVKTGSSGFVTKKCSASGGGGSSSGSYCKTINGAGVDLIAKWEGFVASPKPDPIGLPTVGYGHLCQQKNCAEVKYKFPLTKTTAKELLLDDLPKYTKCLADVLNSKPKLNANQWAALSSWVFNVGCGNAKTSTLVKRLNNGEAPNTVAAQELPKWRMAGGKVMPGLEARRKDEVKLFKTASSKEAYPKCQA。

[0040] SEQ ID NO.2:

[0041] MSSSLKFLGVLLPALAVACTGPPVNQNGLNLIKSFESFQPSVYDDGFGNPTIGYGHLCGDATCSEVTYPKPLSEADASRLLADDLVSYQDALTNALADPVTLNDNQYAALVSWTFNIGNGNMQKSDLVARMNKGENVATVAHDELPQWNKANGQVVNGLTRRRKAELDLFDAPAIYGALPVPC。

[0042] SEQ ID NO.3:

[0043] MPSNTIKFILAALPALAAAACSGPDVNEATIKLMKGYESWEADVYDDGYGNPTVGYGHLCDDWSCSDVSYDIPLSESDGEKLFAEDIVAYQNGVVAALSDDVTLNDNQYGALVSWCFNVGTGAVAESTLAKRLNNGEDPDTVAEEELPKWVYANGAPSEGLKNRRAAELKLFTTSSDTEALPVDC。

[0044] SEQ ID NO.4:

[0045] MQRTVIAALAALASVASAQCNIPDVNTATINLITEFEGFRANIYTDPTGNPTVGIGHLCRDSRCSDVSYPIPLSQANGKKLLADDLGTYEECLTMMTNSRVVLNLNEFGALVSWAFNMGCGAAKGSTLIKRLNAGEKPQTVLPQELPKWVNGNGQVLPGLVRRRNAEVELARTATSSPALPACS。

[0046] SEQ ID NO.5:

[0047] MKFLTPLAVQLAALASMASATVPGFDISHYQATVDFAKAYADGARFVIIKATEGTTYTDPSFSDHYTKATNAGFIRGGYHFAQPASSSGAAQANYFLKHGGGWSADGITLPGMLDLEYAPSGDSCYGLSASAMVSWINDFVNTYHAATTQYPLIYTSTSWWQLCTGNNGSFGSKSPLVIARYASSVGALPNGWSVYTIWQNSDASPWGGDNDIFNGNLAQLQKIARGS。

[0048] SEQ ID NO.6:

[0049] MKFSIAAIALFTLSAVALPATVVRRGASAATVNLISEVEDFRADFYDMMGHKTIGYGHDCVAKKDCDSINAPISNAQGDEILHKDLVGFEQCVCALPNANALNANQYGALVSYAFNTGCGGLQQAWTAAMTSKNFDSICADFPHTNTLNGVLDNRRKKEAALCSTPTTQMSGC。

[0050] SEQ ID NO.7:

[0051] MKFSIAAIALFTLSAVALPATVVRRGASAATVSLIGEVEGFRADFYDMMGHKTIGYGHDCVAKQDCDSIKAPISNAQGDEILQKDLAGFEQCVCALPNAKALNANQYGALVSYAFNTGCGGLQQAWTAAMTSKNFDSICADLPHTNTLNGVLDNRRKKEAALCSTPTTQMCGC。

[0052] SEQ ID NO.8:

[0053] MKTTFAALIVSLTSIVSAYPITGDVVNCRTGPGTSYAIKTSYKKSHDISISCQTTGTSVNGNNIWDKTADGCYVADYYVKTGSSGFVTKKCSASGGGGGGSSSGSYCKTINGAGVDLIAKWEGFVASPKPDPIGLPTVGYGHLCQQKNCAEVKYKFPLTKTTAKELLLDDLPKYTKCLAEVLNSKPKLNANQWAALSSWVFNVGCGNAKTSTLVKRLNNGEAPNTVAAQELPKWRMAGGKVMPGLEARRKDEVKLFKTASSKEAYPKCQA。

[0054] SEQ ID NO.9:

[0055] MKTAFAALVFSLASIVSAYPITGDVVNCRTGPGTSYAIKKSYKKNQDISISCQTAGTSVNGNSIWDKTADGCYVADYYVKTGSSGYVTKKCSASSGGGSSSSSYCKTINTAGVDLIAKWEGFVASPKPDPIGLPTVGYGHLCQQKNCREVKYKFPLTKTTAKELLLDDLPKYTKCLADYLNDKPKLNANQWAALTSWVFNVGCGNAKTSTLVKRLNNGEAANTVAAEELPKWRMAGGKVLPGLEARRKDEVKLFKTASSKQAYPKCQ。

[0056] SEQ ID NO.10:

[0057] MKTAFAALVFSLASIVSAYPITGDVVNCRTGPGTSYAIKKSYKKNQDISISCQTAGTSVNGNSIWDKTADGCYVADYYVKTGSSGYVTKKCTASSGGGSSSSSYCKTINTAGVDLIAKWEGFVASPKPDPIGLPTVGYGHLCQQKNCREVKYKFPLTKTTAKELLLDDLPKYTKCLADYLNDKPKLNANQWAALTSWVFNVGCGNAKTSTLVKRLNNGEAANTVAAEELPKWRMAGGKVLPGLEARRKDEVKLFKTASSKQAYPKCQ。

[0058] SEQ ID NO.11:

[0059] MSSFKLLAALLPALAVACTGPPVNQNGLNLIKSFESFQPSVYDDGFGNPTIGYGHLCGDATCSEVTYPKPLSEADASALLAGDLVSYQDALTNALADPVTLNDNQYAALVSWTYNVGNGNMQKSDLVARMNKGEDVAAVAHDELPQWNKANGQVVNGLTRRRNAELGLFDAPAIYGALPVPC.

[0060] SEQ ID NO.12:

[0061] MKAISLLALPSLAYAAVQGFDISNWQPTVDYDGAYASGARFAMIKATEGTTYIDPLFNTHYPGATSAGLIRGGYHFAHPDSSSGSAQASYFLAHGGGWSADGLTLPGM LDLEAGCYGLSPSAMTSWIRDFGETYKDATGRYPMIYTTTSWWTECTANNADFGDYPLVLARWASSPGTLPASWDYYSFWQNSDQYAFGGDSQVWNGSEERLRVFASG.

[0062] The method for synthesizing the bactericidal protein encoding gene provided in the embodiments of the present invention is as follows:

[0063] Provide amino acid sequences (SEQ ID NO.1-12), and the corresponding nucleotide coding sequences (SEQ ID NO.13-24) are synthesized by a gene company. The synthesis rules are based on the codon preference of the host. Because of the difference in hosts, the codon preference is also different (SEQ ID NO.13-24 are bactericidal protein coding genes expressed by Trichoderma). Provide an empty expression plasmid without a gene connected to the gene company, and inform the gene company of the restriction site that needs to be connected to the expression gene, as well as the DNA sequence upstream and downstream of the restriction site to avoid frameshift mutations. The gene can be directly synthesized on the provided plasmid to construct an expression vector for expressing reducing sugar oxidase, eliminating the steps of PCR, restriction digestion, and connection.

[0064] It should be noted that the N-termini of the fungicidal protein amino acid sequences (SEQ ID NOs. 1 to 12) provided by the present invention all contain their own secretory peptide sequences, and can be secreted and expressed in Trichoderma.

[0065] The gene sequences encoding the bactericidal proteins in the following examples are shown in SEQ ID NOs. 13 to 24:

[0066] SEQ ID NO.13:

[0067] ATGAAGACGACGTTTGCGGCTCTTATTGTGTCATTGACCTCCATTGTCTCTGCATACCCCATCACCGGCGACGTCGTCAACTGCCGCACTGGCCCTGGCACCAGCTACGCCATCAAGACCAGCTACAAGAAGTCCCACGACATCAGCATCAGCTGCCAGACCACCGGCACCAGCGTCAACGGCAACAACATCTGGGACAAGACCGCCGACGGCTGCTACGTCGCCGACTACTACGTCAAGACCGGCAGCAGCGGCTTCGTCACCAAGAAGTGCAGCGCCTCTGGCGGCGGCGGCAGCTCCAGCGGCAGCTACTGCAAGACCATCAACGGCGCTGGCGTCGACCTGATCGCCAAGTGGGAGGGCTTCGTCGCTAGCCCCAAGCCTGATCCTATCGGCCTGCCTACCGTCGGCTACGGCCACCTCTGCCAGCAGAAGAACTGCGCCGAGGTCAAGTACAAGTTTCCCCTGACCAAGACGACCGCCAAGGAGCTGCTCCTCGACGACCTGCCTAAGTACACCAAGTGCCTCGCCGACGTCCTCAACAGCAAGCCCAAGCTCAACGCCAACCAGTGGGCCGCTCTCAGCAGCTGGGTCTTTAACGTCGGCTGCGGCAACGCCAAGACGAGCACCCTCGTCAAGCGCCTCAACAACGGCGAGGCCCCTAACACCGTCGCCGCTCAGGAGCTGCCCAAGTGGCGCATGGCTGGCGGCAAGGTCATGCCCGGCCTTGAGGCCCGACGCAAGGACGAGGTGAAGCTGTTCAAGACGGCCAGCTCCAAGGAGGCTTACCCCAAGTGCCAGGCC。

[0068] SEQ ID NO.14:

[0069] ATGTCATCCAGCCTCAAGTTCCTGGGGGTCCTGCTGCCGGCGCTCGCCGTCGCTTGCACCGGCCCTCCTGTCAACCAGAACGGCCTCAACCTCATCAAGTCCTTCGAGAGCTTCCAGCCCAGCGTCTACGACGACGGCTTCGGCAACCCCACCATCGGCTACGGCCACCTCTGCGGCGACGCTACTTGCAGCGAGGTCACCTACCCCAAGCCCCTGTCCGAGGCCGACGCTTCTCGCCTCCTGGCTGACGACCTCGTCTCCTACCAGGACGCCCTCACCAACGCCCTGGCCGATCCTGTCACCCTCAACGACAACCAGTACGCCGCCCTCGTCAGCTGGACCTTCAACATCGGCAACGGCAACATGCAGAAGAGCGACCTGGTCGCCCGCATGAACAAGGGCGAGAACGTCGCCACCGTCGCCCACGATGAGCTGCCTCAGTGGAACAAGGCCAACGGCCAGGTCGTCAACGGCCTGACCCGCCGACGAAAGGCCGAGCTCGACCTGTTCGACGCCCCCGCTATCTACGGCGCCCTGCCTGTCCCTTGC。

[0070] SEQ ID NO.15:

[0071] ATGCCTTCGAACACCATTAAGTTCATCCTCGCTGCGTTGCCCGCCCTGGCGGCAGCGGCTTGCAGCGGACCCGACGTCAACGAGGCCACCATCAAGCTCATGAAGGGTTATGAGAGCTGGGAGGCCGACGTTTACGACGACGGCTACGGGAACCCGACAGTGGGATATGGCCACCTCTGCGATGACTGGTCCTGTTCGGACGTATCTTACGACATTCCGCTCTCGGAGTCAGATGGCGAGAAGCTGTTTGCGGAAGATATCGTGGCATACCAAAACGGCGTCGTGGCTGCTCTAAGCGACGATGTGACGCTGAACGATAATCAGTACGGCGCCTTAGTCAGTTGGTGCTTCAACGTCGGCACGGGGGCCGTTGCGGAATCTACACTTGCCAAGAGGCTCAACAACGGCGAAGATCCGGACACCGTCGCCGAGGAGGAGCTCCCCAAGTGGGTCTACGCCAATGGTGCCCCATCCGAAGGCTTGAAGAATCGGCGCGCAGCAGAGCTGAAACTGTTTACTACTAGCTCCGACACGGAGGCCCTGCCTGTCGACTGC。

[0072] SEQ ID NO.16:

[0073] ATGCAGCGCACCGTCATTGCCGCCCTTGCTGCTCTGGCCTCCGTGGCGTCTGCTCAGTGCAACATCCCGGATGTGAACACTGCAACGATCAACCTGATCACCGAGTTTGAGGGCTTCCGTGCTAATATCTACACGGATCCGACGGGCAACCCGACAGTCGGCATTGGCCACCTCTGCCGCGACAGCCGGTGCTCTGACGTCTCGTATCCCATACCTCTCTCCCAAGCCAACGGCAAGAAGCTGCTTGCCGACGACCTGGGCACATACGAGGAATGCCTGACCATGATGACCAACAGCCGAGTTGTTCTGAATCTCAACGAGTTTGGGGCCCTCGTGAGCTGGGCGTTCAACATGGGCTGCGGTGCGGCCAAAGGCTCCACGCTCATCAAGCGGTTGAATGCAGGAGAGAAGCCCCAGACGGTGCTCCCTCAAGAACTACCCAAGTGGGTCAATGGGAACGGACAGGTCCTGCCAGGTCTCGTAAGAAGGCGCAACGCGGAAGTCGAGTTGGCCCGAACCGCGACTTCAAGTCCAGCACTGCCCGCCTGTTCG。

[0074] SEQ ID NO.17:

[0075] ATGAAGTTCTTGACTCCGCTTGCTGTGCAGCTCGCCGCCCTGGCGTCCATGGCCTCGGCGACAGTTCCCGGCTTCGACATTAGCCATTACCAGGCAACCGTCGACTTCGCCAAGGCGTACGCCGACGGCGCCCGCTTCGTCATCATCAAGGCCACAGAAGGCACCACCTACACCGACCCCAGCTTTAGTGACCACTATACGAAAGCTACCAACGCCGGTTTCATCCGAGGAGGCTACCACTTTGCCCAGCCCGCATCTTCTAGCGGGGCTGCACAGGCCAACTACTTCCTAAAGCACGGCGGTGGCTGGTCCGCCGATGGCATAACCCTGCCGGGCATGCTCGACCTGGAGTATGCACCTTCTGGAGATTCATGCTACGGCCTCAGTGCATCAGCCATGGTCTCCTGGATCAACGACTTTGTCAACACGTACCATGCCGCGACGACGCAGTACCCTCTCATCTACACATCGACGAGCTGGTGGCAATTGTGCACTGGCAACAACGGGTCCTTTGGCAGCAAGAGCCCACTTGTTATTGCCAGGTATGCTTCGAGCGTGGGAGCTCTGCCCAATGGATGGTCGGTATATACCATCTGGCAGAACTCGGATGCGTCCCCGTGGGGCGGCGACAACGATATCTTCAATGGGAATCTCGCTCAACTGCAGAAGATTGCGCGGGGTTCA。

[0076] SEQ ID NO.18:

[0077] ATGAAGTTCTCGATAGCTGCCATTGCCTTGTTCACCCTGAGCGCCGTGGCGCTGCCGGCAACTGTTGTTCGGAGGGGCGCCTCTGCCGCCACCGTCAACCTCATCAGCGAGGTCGAGGACTTCCGCGCCGACTTCTACGACATGATGGGCCACAAGACCATCGGCTACGGCCACGACTGCGTCGCCAAGAAGGACTGCGACAGCATCAACGCCCCTATCAGCAACGCCCAGGGCGACGAGATCCTCCACAAGGACCTCGTCGGCTTCGAGCAGTGCGTCTGCGCCCTGCCTAACGCCAACGCTCTCAACGCCAACCAGTACGGCGCCCTCGTCAGCTACGCCTTCAACACCGGCTGCGGCGGCCTCCAGCAGGCCTGGACCGCCGCCATGACCAGCAAGAACTTCGACTCCATCTGCGCCGATTTTCCCCACACCAACACGCTCAACGGCGTCCTCGACAACCGCCGCAAGAAGGAGGCCGCTCTCTGCAGCACGCCCACCACGCAGATGAGCGGCTGC。

[0078] SEQ ID NO.19:

[0079] ATGAAGTTCAGCATAGCTGCCATCGCCCTCTTCACCCTCTCAGCCGTCGCCTTGCCTGCCACCGTGGTTCGACGGGGCGCCTCTGCCGCCACCGTCAGCCTCATCGGCGAGGTCGAGGGCTTCCGCGCCGACTTCTACGACATGATGGGCCACAAGACCATCGGCTACGGCCACGACTGCGTCGCCAAGCAGGACTGCGACAGCATCAAGGCCCCTATCAGCAACGCCCAGGGCGACGAGATCCTCCAGAAGGACCTGGCCGGCTTCGAGCAGTGCGTCTGCGCCCTGCCTAACGCCAAGGCTCTCAACGCCAACCAGTACGGCGCCCTCGTCAGCTACGCCTTCAACACCGGCTGCGGCGGCCTCCAGCAGGCCTGGACCGCCGCCATGACCAGCAAGAACTTCGACTCCATCTGCGCCGACCTGCCTCACACCAACACGCTCAACGGCGTCCTCGACAACCGCCGCAAGAAGGAGGCCGCTCTCTGCAGCACGCCCACCACGCAGATGTGCGGCTGC。

[0080] SEQ ID NO.20:

[0081] ATGAAGACTACCTTTGCCGCTCTTATTGTTTCGTTGACGTCCATCGTGTCAGCATATCCCATCACGGGAGATGTCGTCAACTGCCGCACGGGACCCGGCACATCGTACGCCATTAAGACCAGCTACAAGAAGAGCCACGACATCAGCATATCCTGCCAAACCACGGGGACCAGCGTCAATGGCAACAACATCTGGGACAAAACTGCTGATGGCTGCTACGTGGCCGACTACTACGTCAAGACGGGGTCCTCGGGCTTCGTCACCAAGAAGTGCTCGGCGTCTGGCGGCGGGGGTGGTGGATCCTCTTCCGGGAGCTACTGCAAGACAATCAACGGCGCCGGAGTGGATCTCATCGCGAAGTGGGAGGGCTTTGTTGCCTCACCTAAGCCCGACCCGATTGGCTTGCCGACAGTTGGCTACGGCCATCTATGCCAGCAGAAGAACTGTGCTGAGGTAAAGTACAAGTTCCCTCTGACCAAAACGACCGCCAAGGAACTGCTGCTCGACGACCTGCCGAAGTATACGAAATGCCTGGCCGAGGTGTTGAATAGCAAACCAAAGCTGAACGCAAACCAGTGGGCTGCCCTGTCGAGCTGGGTCTTCAACGTGGGATGCGGCAATGCGAAGACCTCTACACTTGTCAAGCGACTCAACAATGGTGAGGCTCCCAACACCGTGGCAGCGCAGGAGCTCCCAAAATGGCGCATGGCCGGCGGCAAGGTCATGCCCGGCCTCGAGGCGAGGCGGAAGGACGAAGTCAAGCTCTTCAAGACGGCCAGTTCAAAGGAAGCATATCCGAAATGTCAAGCC。

[0082] SEQ ID NO.21:

[0083] ATGAAGACGGCCTTTGCTGCTCTTGTTTTTAGCCTGGCAAGCATTGTCTCCGCCTACCCCATCACAGGCGATGTCGTGAATTGTCGAACTGGTCCGGGAACCTCCTACGCTATCAAGAAAAGCTACAAGAAGAACCAAGATATTAGCATCTCGTGCCAGACCGCAGGGACGTCCGTCAACGGCAACTCCATCTGGGACAAGACTGCGGACGGCTGCTACGTGGCCGACTACTACGTCAAGACCGGTAGTTCTGGATATGTCACCAAGAAGTGCAGCGCCTCGTCCGGAGGTGGCTCATCTAGTTCATCGTACTGCAAGACCATCAACACTGCTGGAGTCGACTTGATAGCCAAATGGGAGGGCTTCGTAGCCAGCCCCAAGCCCGATCCTATTGGGCTGCCAACAGTGGGCTATGGGCACTTGTGCCAGCAGAAGAACTGCAGAGAAGTGAAATACAAGTTCCCGCTGACTAAAACGACGGCGAAGGAGCTGCTGCTCGACGACCTTCCAAAGTACACGAAGTGCCTCGCCGATTATCTAAACGACAAGCCGAAGCTGAATGCCAACCAGTGGGCGGCGCTCACATCCTGGGTGTTCAACGTCGGCTGTGGCAATGCCAAAACCTCTACCCTCGTCAAGCGGCTCAACAACGGCGAGGCGGCAAACACGGTTGCCGCCGAGGAGCTCCCCAAGTGGCGCATGGCCGGCGGCAAGGTCCTGCCTGGCCTGGAAGCGAGGCGCAAGGACGAGGTTAAGCTCTTCAAGACGGCATCGTCGAAGCAGGCTTATCCCAAGTGCCAA。

[0084] SEQ ID NO.22:

[0085] ATGAAGACGGCCTTTGCCGCGCTGGTGTTTTCCCTTGCCTCGATTGTCTCGGCGTATCCCATCACCGGCGACGTCGTCAACTGCAGGACGGGGCCGGGGACAAGCTATGCCATCAAGAAATCGTACAAGAAGAACCAAGACATATCCATCAGCTGCCAGACGGCTGGCACGTCTGTCAATGGCAACAGCATCTGGGACAAGACTGCGGATGGCTGCTACGTCGCTGACTACTATGTGAAAACTGGTAGTAGTGGATACGTGACGAAGAAGTGCACCGCATCTTCAGGTGGAGGAAGCTCGTCATCTTCCTACTGCAAGACCATCAACACGGCGGGAGTCGACCTCATTGCCAAGTGGGAGGGCTTCGTGGCCAGCCCGAAGCCCGATCCTATTGGGCTCCCCACGGTTGGCTACGGCCACCTCTGCCAGCAGAAGAATTGCCGCGAAGTTAAGTACAAGTTCCCTCTGACCAAGACCACAGCAAAGGAGCTCCTCCTGGACGATTTGCCGAAATACACCAAATGCTTGGCCGATTATCTCAACGACAAGCCAAAGCTGAACGCCAACCAGTGGGCTGCTCTTACTAGCTGGGTCTTCAACGTTGGCTGTGGCAATGCCAAGACGTCGACCCTCGTCAAGCGACTTAACAACGGCGAGGCAGCAAACACCGTGGCGGCCGAGGAGCTGCCAAAATGGCGCATGGCCGGCGGCAAGGTCCTGCCCGGTCTGGAAGCCAGACGGAAGGACGAGGTAAAGCTCTTCAAGACAGCTTCCTCCAAGCAGGCGTACCCTAAGTGTCAA。

[0086] SEQ ID NO.23:

[0087] ATGAGCTCATTCAAGCTGCTTGCTGCATTGCTCCCAGCACTGGCTGTGGCCTGCACTGGACCTCCCGTCAACCAAAACGGTCTGAACCTCATCAAGTCGTTTGAGTCCTTCCAGCCGTCTGTATACGACGACGGCTTCGGCAATCCCACCATTGGCTATGGCCATCTTTGCGGCGACGCCACCTGCTCCGAAGTGACGTATCCCAAGCCCTTGTCGGAAGCGGATGCATCCGCACTACTGGCCGGGGATCTGGTCAGCTACCAGGATGCTCTCACCAACGCGCTCGCCGACCCCGTCACGCTGAACGACAACCAGTACGCCGCGCTTGTGAGTTGGACGTACAACGTGGGTAACGGAAATATGCAGAAGAGCGACCTCGTCGCGCGCATGAACAAGGGCGAGGACGTCGCCGCCGTTGCGCACGACGAGCTCCCTCAATGGAACAAAGCTAACGGCCAGGTCGTCAATGGGCTCACAAGGCGACGGAATGCCGAGCTGGGACTGTTTGATGCTCCAGCCATCTACGGCGCCTTGCCGGTTCCGTGT。

[0088] SEQ ID NO.24:

[0089] .

[0090] Bactericidal protein, like lysozyme extracted from commercial egg white, will hydrolyze the cell wall of indicator bacteria, causing the indicator bacteria to rupture and die. The process is manifested as the absorbance of the bacterial suspension of the indicator bacteria gradually decreases, and the bacterial suspension slowly becomes clear. Therefore, in the following embodiments, the national standard GB1886.257-2016 determination method is used to determine the bactericidal activity of bactericidal protein. The bactericidal activity is simply defined as follows: One bactericidal activity unit is defined as the volume or mass of the bactericidal protein solution required to cause a decrease in absorbance of 0.001 per minute at 450nm using a suspension of Micrococcus luteus at 25°C and pH 6.2. The level of bactericidal activity can also directly reflect the level of expression of the bactericidal protein.

[0091] Example 1 Construction of bactericidal protein expression vector

[0092] The present invention utilizes the previously constructed Trichoderma reesei expression plasmid CBH1T1.0 (recorded in the patent document "202211596367.5 Construction and application of a xylanase and its secretory expression strain") to construct a recombinant Trichoderma strain, so that the bactericidal protein is secreted and expressed in the recombinant Trichoderma strain.

[0093] The present invention commissioned Shanghai Jierui Gene Synthesis Company to directly synthesize 12 bactericidal protein coding gene sequences (named F1 (SEQ ID NO.13), F2 (SEQ ID NO.14), F3 (SEQ ID NO.15), F4 (SEQ ID NO.16), F5 (SEQ ID NO.17), F6 (SEQ ID NO.18), F7 (SEQ ID NO.19), F8 (SEQ ID NO.20), F9 (SEQ ID NO.21), F10 (SEQ ID NO.22), F11 (SEQ ID NO.23), F12 (SEQ ID NO.24)) at the XbaI restriction site (TCTAGA) of the CBH1T1.0 plasmid, that is, to construct 12 corresponding recombinant expression vectors, named CBH1T1.0-F1~12 in sequence (the schematic diagram of the recombinant expression vector structure is shown in the figure). Figure 1 ).

[0094] The upstream and downstream sequences near the XbaI restriction site of the CBH1T1.0 plasmid (the insertion position of the target gene) are shown in SEQ ID NO. 25, the gene insertion site TCTAGA is underlined, and the insertion position is indicated by " / ". The ATG at the front end of the bactericidal protein encoding gene sequence is the start codon of the encoding gene, and the TAG in the second half of the XbaI restriction site of the CBH1T1.0 plasmid is the stop codon.

[0095] SEQ ID NO.25:

[0096] TTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGCGCA TC / TAGATAACTTCGTATAATGTATGCTATACGAAGTTATACTAGAGAAGTTCCTATACTTTTTAGAGAATAGGAACTTCGGAATAGGAACTTCACTAGAGATATCAAGCAACT ACGTAAAACTCCATGAGATTGCAGATGCGGCCCACTGGAATACAACATCCTCCGCAAGT CCGA.

[0097] Example 2 Construction of a recombinant Trichoderma strain expressing bactericidal protein

[0098] The 12 recombinant expression vectors constructed in Example 1 were transformed into Trichoderma strains QM6a (ATCC 13631), QM9414 (ATCC26921), RUT-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), and PC-3-7 (ATCC66589) using the Agrobacterium tumefaciens-mediated transformation method (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264) to obtain recombinant Trichoderma strains.

[0099] This example uses Trichoderma RUT-C30 (ATCC 56765) as a host strain to illustrate the detailed process of constructing a recombinant Trichoderma strain, RUT-C30-CBH1T1.0-F1-12; when other Trichoderma strains are used as host strains, the construction process is exactly the same.

[0100] The process is as follows:

[0101] The recombinant expression vector CBH1T1.0-F1~12 was transformed into Trichoderma strain RUT-C30 by Agrobacterium tumefaciens-mediated method, and the endogenous cellulase gene cbh1 was replaced by homologous recombination. Twelve recombinant Trichoderma strains expressing bactericidal proteins were successfully constructed (the schematic diagram of the construction process is shown in the figure). Figure 2); the recombinant Trichoderma strain eliminated the hygromycin resistance marker according to the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761).

[0102] Example 3 Production of bactericidal protein

[0103] The recombinant Trichoderma strain was fermented and cultured in Trichoderma fermentation medium to induce the secretion and expression of the bactericidal protein. The fermentation tank culture method refers to the literature "Chen et al. Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3. Biotechnol Biofuels. 2020, 13: 62". After 8 days of fermentation, the fermentation broth was taken, centrifuged at 12500rpm for 10min, and the supernatant was taken, which was the crude extract of the bactericidal protein. The crude extract was spray-dried, and then a universal protective agent (starch or cyclodextrin) was added at a weight ratio of 1:1 to prepare a solid fungicidal protein powder. The activity of the crude extract and the fungicidal protein powder was determined respectively by the national standard GB1886.257-2016. As shown in Table 1, the activity of the fungicidal protein powder reached 100-400U / mg.

[0104] Table 1 Bactericidal activity of bactericidal proteins expressed by recombinant Trichoderma strains; Staphylococcus aureus a It is the abbreviation of Staphylococcus aureus.

[0105]

[0106]

[0107] Table 1 shows that when Trichoderma RUT-C30 (ATCC 56765) is used as the host strain, the activity of the produced bactericidal protein is higher.

[0108] Example 4 Application of bactericidal protein

[0109] The bactericidal protein powder prepared in Example 3 was used for the inhibition zone experiment, and commercial egg white extracted lysozyme powder (from Ningxia Xiasheng Industrial Group Co., Ltd., with an activity of 3000 U / mg) was used as a control. The inhibition zone experiment method was used to evaluate the inhibitory effect of the bactericidal protein on common pathogens.

[0110] The experimental method is as follows:

[0111] Taking the bactericidal protein powder F5 produced by RUT-C30-CBH1T1.0-F5 as an example, dissolve an appropriate amount of F5 and commercial lysozyme powder in ultrapure water to prepare a test liquid and a control liquid with a concentration of 40U / μL, and filter and sterilize. Select three indicator bacteria: Candida albicans, Staphylococcus aureus, and Escherichia coli. Streak the indicator bacteria on the LB plate and culture at 37℃ for about 12h. Pick a single colony from the plate and culture it in an LB test tube at 37℃ for about 12h to form the indicator bacteria seed liquid, which is placed in the refrigerator for use.

[0112] After the LB medium is sterilized, the medium is cooled to slightly above body temperature. 100 μL of each of the indicator bacteria seed solutions are pipetted into the unsolidified LB medium and shaken well. The medium is quickly poured into a plate, with 15 mL of medium per plate, and waited for solidification.

[0113] Cut filter paper pieces with a diameter of 6 mm, sterilize at high temperature, and dry aseptically. Allow each filter paper piece to absorb 10 μL of bactericidal protein test liquid or commercial lysozyme control liquid. Use sterilized tweezers to pick up the filter paper piece and stick it to the surface of the plate. Turn the plate over, mark it, and place it in a 37°C incubator for incubation. The inhibition zone can be observed after about 12 to 24 hours.

[0114] The results of the inhibition zone test are as follows Figure 3 As shown, it can be seen that bactericidal protein can significantly kill or inhibit the growth of Gram-positive (Staphylococcus aureus tolerant to egg white lysozyme) and yeast (Candida albicans), producing a significant inhibition zone, while egg white lysozyme cannot inhibit these two strains of bacteria; bactericidal protein can significantly kill or inhibit Gram-negative bacteria (Escherichia coli), and its effect exceeds that of egg white lysozyme, and can produce a larger inhibition zone. The size of the inhibition zone produced by other bactericidal protein powders is shown in Table 1.

[0115] In summary, the present invention successfully constructed a recombinant Trichoderma strain that secretes and expresses bactericidal proteins after genetic recombination modification of Trichoderma. The bactericidal proteins produced by the recombinant Trichoderma strain can significantly kill or inhibit Gram-positive, Gram-negative bacteria and yeasts, providing a new bactericidal protein preparation and its preparation method for daily chemical, food, feed and other industries.

[0116] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bactericidal protein, It is characterized in that The amino acid sequence of the bactericidal protein is the sequence shown in any one of SEQ ID NOs. 1 to 12, or a functionally identical sequence after 1 to 30 amino acids are substituted and / or deleted and / or added to any one of SEQ ID NOs. 1 to 12, or a functionally identical sequence after 1 to 20 amino acids are added to the C-terminus and / or N-terminus of any one of SEQ ID NOs. 1 to 12.

2. A gene encoding the bactericidal protein according to claim 1, It is characterized in that The nucleotide sequence of the gene is a sequence shown in any one of SEQ ID NOs. 13 to 24, or a sequence having more than 85% homology with any one of SEQ ID NOs. 13 to 24.

3. A recombinant expression vector, It is characterized in that The recombinant expression vector contains a nucleotide sequence encoding the bactericidal protein according to claim 1, or contains the gene according to claim 2.

4. A recombinant microorganism, It is characterized in that The recombinant microorganism comprises the recombinant expression vector according to claim 3.

5. The recombinant microorganism according to claim 4, It is characterized in that The chassis microorganism of the recombinant microorganism is Trichoderma.

6. The recombinant microorganism according to claim 5, It is characterized in that The Trichoderma is Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

7. Use of the gene according to claim 2, the recombinant expression vector according to claim 3 or the recombinant microorganism according to any one of claims 4 to 6 in producing bactericidal proteins.

8. A method for producing bactericidal protein, It is characterized in that The bactericidal protein is obtained by fermenting and culturing the recombinant microorganism according to any one of claims 4 to 6 and collecting the fermentation broth.

9. Use of the bactericidal protein produced by the production method of claim 8 in the preparation of a bactericide.

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