CN114015676A - Construction method of cellulase suitable for traditional Chinese medicine feed additive - Google Patents
Construction method of cellulase suitable for traditional Chinese medicine feed additive Download PDFInfo
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Abstract
The invention discloses a construction method of cellulase suitable for a traditional Chinese medicine feed additive, and relates to the technical field of genetic engineering. The cellulase nucleotide sequence is shown as SEQ ID NO. 3; the amino acid sequence is shown as SEQ ID NO. 4. The monomer cellulase with higher enzyme catalytic activity at the gastrointestinal environment temperature is obtained. The constructed enzyme is a novel cellulase which can efficiently promote the dissolution of the traditional Chinese medicine cellulose at the gastrointestinal environment temperature, and further promote the quick release of the effective components.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for constructing cellulase suitable for a traditional Chinese medicine feed additive.
Background
The traditional Chinese medicine fermented feed has great advantages in China. The traditional Chinese medicine is safe, non-toxic and multiple in target spot, and has wide prospect as a feed additive.
The traditional Chinese medicine fermentation can improve the dissolution efficiency of the effective components of the traditional Chinese medicine, improve the drug effect, improve the taste, realize the synergistic effect of the bacteria and the enzyme, and better improve the immunity of animals and the absorption efficiency of nutrition.
Cellulase is often required for fermentation of Chinese herbs. The optimum enzyme activity of the existing cellulase is about 50 ℃. When traditional Chinese medicine is fermented, the temperature is about 37 ℃, and the existing cellulose exonuclease is not suitable for traditional Chinese medicine fermentation.
In the gastrointestinal tract of animals, the temperature of the digestive environment is typically around 37 ℃. The cellulase degrades fiber components in plants in vivo, and requires higher enzyme activity at about 37 ℃.
Therefore, how to provide a cellulase suitable for a traditional Chinese medicine feed additive is a problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a construction method of cellulase suitable for a traditional Chinese medicine feed additive.
In order to achieve the purpose, the invention adopts the following technical scheme:
a cellulase suitable for Chinese medicinal feed additive has nucleotide sequence shown in SEQ ID NO. 3.
Preferably: the amino acid sequence is shown as SEQ ID NO. 4.
The invention also provides a construction method of the cellulase suitable for the traditional Chinese medicine feed additive, which comprises the following steps:
(1) carrying out error-prone PCR amplification by using a synthetic sequence with a nucleotide sequence shown as SEQ ID NO.1 as a template;
(2) carrying out double enzyme digestion on the amplified product by using restriction enzymes BamHI and EcoRI, and recovering a gene fragment;
(3) carrying out double digestion on the expression vector pRSFduet-1 by using the same restriction enzyme as the restriction enzyme in the step (2);
(4) connecting the gene fragment recovered in the step (2) with the vector subjected to double enzyme digestion in the step (3) to obtain a recombinant expression vector pRSFduet-1-SQ, then transforming the recombinant expression vector pRSFduet-1-SQ into an Escherichia coli E.coli DH5 alpha competent cell, extracting an expression vector plasmid, identifying and sequentially numbering the transformed strains;
(5) and the transformed strain is subjected to protein expression, purification and enzyme activity analysis to screen target cellulase.
Preferably: the error-prone PCR reaction system in the step (1): error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, 5mM MnCl23.0 μ L, 10 μ g/μ L DNA template 1 μ L, 10 μ M PCR primer 2 μ L, Taq DNA polymerase 1 μ L, sterilized double distilled water 17 μ L; the procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1 min; annealing at 45 deg.C for 1 min; extending at 72 ℃ for 4 min; after 32 cycles, extension was carried out for 10min at 72 ℃.
Preferably: enzyme digestion system in steps (2) and (3): 11 μ L of ultrapure water, 1 μ L of BamHI, 1 μ L of EcoRI, 3 μ L of Buffer, and 14 μ L of target gene.
The invention also provides application of the cellulase suitable for the traditional Chinese medicine feed additive in fermentation.
According to the technical scheme, compared with the prior art, the invention discloses the construction method of the cellulase suitable for the traditional Chinese medicine feed additive, and the obtained technical effect is that the monomer cellulase with higher enzyme catalytic activity at the gastrointestinal environment temperature is obtained. The constructed enzyme is a novel cellulase which can efficiently promote the dissolution of the traditional Chinese medicine cellulose at the gastrointestinal environment temperature, and further promote the quick release of the effective components.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a construction method of cellulase suitable for a traditional Chinese medicine feed additive.
The biological agents referred to in the examples are all commercially available, for example:
coli DH5 a, purchased commercially, deposited in the laboratory; restriction enzymes, Takara; plasmid extraction kit, Tiangen Biochemical technology (Beijing) Ltd; ready-to-use error-prone PCR kit, Beijing Tianenzze technologies, Inc.; BCA protein concentration determination kit, Fuzhou Ao research laboratory equipment, LLC; plasmid small quantity extraction kit, gel recovery kit, Tiangen Biotechnology (Beijing) Ltd; plasmid pRSFduet-1 was purchased from Korea Biotechnology Ltd; the sequence SQ2-1 gene is synthesized completely by Scophthal limited company;
Primer F:GGATCCATGAAACGTAACCTGTT；
PrimerR GAATTC TTAATGGTGGTGGTGGT, available from Ongko Biometrics Ltd;
media and buffers used during protein expression and purification:
LB culture medium: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast extract:
washing buffer solution: 10mM imidazole, 50mM K2HPO4-KH2PO4300mM NaCl, 10% glycerol, pH 8.0:
elution buffer: 200mM imidazole, 50mM K2HPO4-KH2PO4300mM NaCl, 10% glycerol, pH 8.0:
preservation buffer solution: 50mM K2HPO4-KH2PO4300mM NaCl, 10% glycerol, pH 8.0.
Example 1
Error prone PCR
Sequence synthesized with primers Primer F and Primer R as primers SQ1-1(GGATCCATGAAACGTAACCTGTTCCGCATCGTTTCTCGTGTTGTACTGATTGCTTTCATCGCATCCATCTCTCTGGTGGGTGCTATGAGCTATTTCCCGGTAGAAACCCAGGCGGCCCCGGATTGGTCTATTCCGTCCCTGTGCGAATCTTATAAAGATGACTTTATGATCGGTGTTGCGATCCCTGCCCGTTGTCTGAGCAACGATACCGACAAACGCATGGTTCTGAAACATTTCAACTCTATCACCGCCGAAAACGAAATGAAGCCAGAAAGCCTGCTGGCCGGTCAGACTAGCACCGGCCTGTCTTACCGCTTTTCCACTGCAGACGCTTTTGTGGATTTCGCGAGCACTAACAAAATTGGCATCCGCGGCCATACCCTGGTTTGGCACAACCAGACCCCGGATTGGTTCTTCAAAGATTCTAACGGTCAGCGTCTGTCCAAGGACGCACTGCTGGCTCGTCTGAAACAGTACATCTATGATGTTGTGGGCCGTTACAAGGGCAAAGTGTATGCGTGGGATGTAGTGAACGAGGCAATCGACGAAAACCAACCGGACAGCTACCGTCGCTCTACCTGGTACGAGATCTGTGGTCCGGAATACATTGAGAAAGCCTTCATCTGGGCCCACGAGGCTGACCCGAATGCGAAACTGTTTTACAACGATTATAATACCGAGATCAGCAAGAAACGTGACTTTATCTACAACATGGTTAAAAACCTGAAATCCAAGGGCATCCCTATTCACGGCATCGGTATGCAGTGTCATATTAATGTGAACTGGCCGTCCGTTAGCGAAATTGAAAACTCTATCAAACTGTTCTCCAGCATCCCGGGTATCGAAATTCATATCACTGAACTGGACATGTCTCTGTATAACTACGGCTCTAGCGAAAACTACTCCACCCCGCCGCAGGACCTGCTGCAGAAGCAAAGCCAGAAATACAAGGAAATTTTTACTATGCTGAAAAAATACAAAAACGTTGTTAAGAGCGTTACCTTCTGGGGTCTGAAAGACGACTACTCTTGGCTGCGCAGCTTCTACGGCAAAAACGACTGGCCGCTGCTGTTCTTCGAAGACTACTCTGCTAAACCGGCTTATTGGGCGGTCATCGAAGCTAGCGGCGTGACTACCTCCTCCCCGACCCCGACTCCGACCCCGACCGTAACCGTTACTCCGACCCCGACTCCGACTCCGACCCCAACTGTGACCGCGACTCCTACTCCGACCCCTACCCCGGTCTCCACTCCGGCAACTGGCGGCCAAATCAAAGTGCTGTACGCTAATAAGGAAACCAACTCCACCACGAATACCATCCGTCCGTGGCTGAAGGTTGTTAACAGCGGCTCCAGCAGCATCGACCTGTCTCGCGTTACTATCCGCTATTGGTATACCGTTGACGGTGAACGTGCCCAGTCCGCAGTCTCTGATTGGGCGCAGATCGGTGCTTCCAACGTCACTTTCAAATTTGTAAAACTGAGCTCTAGCGTGAGCGGTGCTGACTACTACCTGGAAATCGGTTTCAAGAGCGGTGCTGGTCAGCTGCAGCCGGGTAAAGACACTGGCGAGATCCAGATCCGTTTCAACAAATCTGACTGGTCTAACTATAACCAGGGCAACGACTGGTCTTGGCTGCAGTCCATGACCTCTTACGGTGAAAACGAGAAAGTTACTGCGTACATTGATGGTGTTCTGGTGTGGGGCCAGGAACCGTCTTGGGACATTAGCGAACTGTCTATTTCTGGTGAATACGTTCGCAGCCGTATCAAAGGTATCCCATACCAGCCGATCGAACGTACCCTGAAAATCTCCCAGGACCAGGTGGCATGTGCACCGATTGGCCAGCCGATCCTGCCGTCTGACTTCGAGGACGGTACCCGTCAGGGCTGGGATTGGGACGGCCCGTCCGGTGTTAAAGGTGCTCTGACCATTGAAGAAGCGAATGGCTCCAACGCTCTGAGCTGGGAAGTTGAGTACCCGGAAAAAAAACTGCAGGATGGCTGGGCATCTGCTCCTCGCCTGATCCTGCGTAATATTAACACCACGCGTGGTGATTGTAAATACCTGTGCTTCGACTTCTATCTGAAGCCGAAGCAGGCGACCAAAGGCGAACTGGCGATCTTCCTGGCGTTTGCTCCTCCGAGCCTGAACTACTGGGCTCAAGCTGAAGACTCCTTCAACATCGACCTGACTAATCTGTCTACTCTGAAGAAAACCCCGGATGATCTGTACTCTTTCAAAATTTCTTTCGATCTGGATAAGATCAAAGAAGGCAAAATCATCGGCCCTGACACCCACCTGCGTGACATCATCATCGTTGTAGCAGACGTGAACTCTGACTTCAAAGGTCGTATGTATCTGGACAATGTGCGTTTCACGAACATGCTGTTCGAAGATGTAACGCCGCAGACTACGGGTTACGAAGCGATCTCTAAACTGTACTCTAAGAAAATTGTTAACGGCATCAGCACCAACCTGTTCGGCCCGGAGAAAGCTGTTACTCGCGCAGAAGTGGCGGCTATGGCAGTCCGTCTGCTGGACCTGCAAGAAGAATCTTACAACGGTGAGTTTGTAGACGTATCTAAAAACTCCTGGTACGCGAACGAAGTATCTACTGCGTACAAAGCAGGTATCATTCTGGGCGACGGTAAGTACATCAAACCGGAGAAAGCCGTTACTCGCGAGGAAATGGCGGTCTTCGCGATGCGTATCTACCGTATCCTGACTGACGAAAAAGCAGAAGCGACCGAAGAAATTGCAATTTCTGACAAAAACAGCATTTCTTCCTGGGCGCGTCAAGATGTAAACGCTGCTATCTCTCTGGGCCTGATGGACGTCTTCACTGACGGTTCTTTCGGCCCGAAGGTGAAAGTGACTCGTGCCGAAGCGACTCAGATTATTTATAAGATCCTGGAACTGACTGGTAAAATGCACCACCACCACCACCATTAAGAATTCAs shown in SEQ ID NO.1) as a template (including a restriction enzyme cutting site and a terminator TAA), error-prone PCR is carried out, and the error-prone PCR reaction system is as follows:
error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, 5mM MnCl23.0. mu.L, 10. mu.g/. mu.L DNA template 1. mu.L, 10. mu.M PCR primer 2. mu.L, Taq DNA polymerase 1. mu.L, sterilized double distilled water 17. mu.L.
The error-prone PCR procedure was as follows:
pre-denaturation at 95 ℃ for 3 min;
denaturation at 94 deg.C for 1 min;
annealing at 45 deg.C for 1 min;
extending at 72 ℃ for 4 min;
after 32 cycles, extension was carried out for 10min at 72 ℃.
Enzyme digestion and transformation:
and respectively adding double distilled water, an endonuclease buffer solution, an enzyme digestion substrate and a restriction endonuclease into the PCR tubule to perform double enzyme digestion reaction, wherein the adding sequence is more or less. The target gene and pRSFduet-1 are placed at 37 ℃ for double enzyme digestion, and the reaction system is as follows:
synthesizing a SQ1-1 sequence double enzyme digestion system:
11 μ L of ultrapure water, 1 μ L of BamHI, 1 μ L of EcoRI, 3 μ L of Buffer, and 14 μ L of target gene.
pRSFduet-1 double enzyme digestion system:
pRSFduet-1 plasmid 43. mu.L, BamHI 1. mu.L, EcoRI 1. mu.L, Buffer 5. mu.L.
After carrying out double digestion reaction for 3h at 37 ℃ by using BamHI and EcoRI restriction enzymes, adding Loading Buffer to terminate the reaction, and purifying and recovering the double digestion product according to the instructions of a gel recovery kit.
After the double-enzyme digestion products are cut by the same restriction enzyme, the double-enzyme digestion products have the same cohesive end and can be connected into a complete plasmid through DNA ligase. Placing a synthetic SQ1-1 sequence containing the same cohesive end and a pRSFduet-1 double enzyme digestion product into the same PCR small tube, adopting a 10 mu L system for ligation reaction, and carrying out the following steps of cutting a target gene and a plasmid enzyme digestion product according to the ratio of 3: add 1 part, mix with 1. mu. L T4 DNA ligase and ligate overnight at 16 ℃. The ligated plasmid was designated pRSFduet-1-SQ.
A conversion step:
the ligation products were transformed into E.coli DH 5. alpha. competent cells by the following transformation procedure:
1) the temperature of the thermostatic water bath was first adjusted to 42 ℃.
2) A tube (100. mu.l) of the competent bacteria was taken out from the ultra-low temperature freezer at-80 ℃ and immediately warmed and melted with a finger, and then inserted into ice and subjected to ice-bath for 10 min.
3) Mu.l of the ligation product pRSFduet-1-SQ was added, gently shaken and then placed on ice for 20 min.
4) Shaking gently, inserting into 42 deg.C water bath, performing heat shock for 90s, rapidly placing back into ice, and standing for 5 min.
5) 900 μ L of LB medium without antibiotics was added to each tube and mixed gently, then fixed on the spring holder of the shaker, and shaken at 37 deg.C for 50 min.
6) To each of the solid LB plates containing the appropriate antibiotics, 300. mu.l of the above-mentioned conversion mixture taken out from the clean bench was dropped, and the plates were uniformly coated using a glass coating rod which was burned by an alcohol lamp and cooled.
7) The marked culture dish is placed in a constant temperature incubator at 37 ℃ for 30-60 min, and after all the liquid on the surface permeates into the culture medium, the culture dish is placed in the constant temperature incubator at 37 ℃ in a reversed mode for overnight.
And (3) carrying out recombinant plasmid identification on the grown single colony:
single colonies growing on the LB solid medium were picked, inoculated into 5mL of LB medium containing kanamycin, and incubated at 37 ℃ and 200 r.min-1Culturing overnight under the condition, collecting partial bacterial liquid, preserving strain with glycerol tube, and numbering as transformed strain.
Inoculating the transformed strain into 6mLLB medium (containing 50mg/L kanamycin sulfate), and culturing at 37 ℃ and 220rpm overnight for 14 h;
according to the following steps: 100 transfer seeds to 500mL LB medium, 37 degrees C150 rpm culture to OD6000.5; inducing expression of the gene with 0.7mM IPTG; culturing overnight at 18 ℃ and 150rpm for 24h after induction;
centrifuging at 4000g and 4 ℃ for 5min to collect thalli, re-suspending the thalli by using 30mL of preservation buffer solution, and then crushing the thalli by using an ultrasonic crusher (working for 3s, interval of 7s, time of 10min and ultrasonic power of about 200W);
the crushed cell suspension is centrifuged for 30min at 12,000rpm and 4 ℃, and the obtained supernatant is the crude enzyme solution of the protein.
Purification of
The protein was purified using Ni-NTA purification media from shanghai bio-works (south kyo jinsley biotechnology limited):
in step 1, 6mL of Ni-NTA purification medium was added to a 50mL empty chromatography column, the medium was allowed to settle freely, and the stock was drained.
Step 2, the chromatography medium is equilibrated by addition of 4 column volumes of wash buffer.
And 3, loading the crude protein enzyme solution into the column, and controlling the flow rate to be 0.5 mL/min.
In step 4, the column is washed with a washing buffer at a flow rate of 1mL/min, and the flow rate is 10 column volumes to remove the contaminating proteins.
And 5, eluting with 5 times of column volume elution buffer solution at the flow rate of 0.5-1 mL/min, and collecting the eluent.
At step 6, the target protein eluate is collected, the purified protein is desalted and concentrated using a Millipore ultrafiltration tube (15mL of 100kDa), and the imidazole-containing elution buffer is replaced with a storage buffer.
Enzyme Activity assay
Except for the determination temperature of 37 ℃, the enzyme activity of the cellulase is determined by other documents (von yue, Jianxin, Zhuli Wei, Dianhei Lei, research on the synergistic effect of the cellulase activity and the mixed cellulase, university of Beijing, 2009, volume 31, supplement 1).
Purified protein concentration the BCA protein concentration assay kit was used.
Sequence screening
Determining the specific enzyme activity of 0.051U/mg protein of initial cellulase with the amino acid sequence SQ1(MKRNLFRIVSRVVLIAFIASISLVGAMSYFPVETQAAPDWSIPSLCESYKDDFMIGVAIPARCLSNDTDKRMVLKHFNSITAENEMKPESLLAGQTSTGLSYRFSTADAFVDFASTNKIGIRGHTLVWHNQTPDWFFKDSNGQRLSKDALLARLKQYIYDVVGRYKGKVYAWDVVNEAIDENQPDSYRRSTWYEICGPEYIEKAFIWAHEADPNAKLFYNDYNTEISKKRDFIYNMVKNLKSKGIPIHGIGMQCHINVNWPSVSEIENSIKLFSSIPGIEIHITELDMSLYNYGSSENYSTPPQDLLQKQSQKYKEIFTMLKKYKNVVKSVTFWGLKDDYSWLRSFYGKNDWPLLFFEDYSAKPAYWAVIEASGVTTSSPTPTPTPTVTVTPTPTPTPTPTVTATPTPTPTPVSTPATGGQIKVLYANKETNSTTNTIRPWLKVVNSGSSSIDLSRVTIRYWYTVDGERAQSAVSDWAQIGASNVTFKFVKLSSSVSGADYYLEIGFKSGAGQLQPGKDTGEIQIRFNKSDWSNYNQGNDWSWLQSMTSYGENEKVTAYIDGVLVWGQEPSWDISELSISGEYVRSRIKGIPYQPIERTLKISQDQVACAPIGQPILPSDFEDGTRQGWDWDGPSGVKGALTIEEANGSNALSWEVEYPEKKLQDGWASAPRLILRNINTTRGDCKYLCFDFYLKPKQATKGELAIFLAFAPPSLNYWAQAEDSFNIDLTNLSTLKKTPDDLYSFKISFDLDKIKEGKIIGPDTHLRDIIIVVADVNSDFKGRMYLDNVRFTNMLFEDVTPQTTGYEAISKLYSKKIVNGISTNLFGPEKAVTRAEVAAMAVRLLDLQEESYNGEFVDVSKNSWYANEVSTAYKAGIILGDGKYIKPEKAVTREEMAVFAMRIYRILTDEKAEATEEIAISDKNSISSWARQDVNAAISLGLMDVFTDGSFGPKVKVTRAEATQIIYKILELTGKMHHHHHH, such as SEQ ID NO.2) at 37 ℃; a mutant enzyme with cellulase activity of 0.060U/mg at 37 ℃ is selected, and the transformed strain is numbered as SXT 2.
Sequencing of coding sequences and coding amino acid sequences
The strain SXT2 was inoculated in 6mL of LB medium (containing 50mg/L kanamycin sulfate) and the transformant was cultured overnight at 37 ℃ and 220rpm for 14 hours.
Extracting recombinant plasmid from proper amount of bacterial liquid according to the specification of the plasmid extraction kit, and sequencing the extracted plasmid by sequencing companyObtaining the coding sequence of the mutant enzyme is SQ2-1(GGATCCATGAAACGTAACCTGTTCCGCATCGTTTCTCGTGTTCTGCTGATTGCTTTCATCGCATCCATCTCTCTGGTGGGTGCTATGAGCTATTTCCCGGTAGAAACCCAGGCGGCCCCGGATTGGTCTATTCCGTCCCTGTGCGAATCTTATAAAGATGACTTTATGATCGGTGTTGCGATCCCTGCCCGTTGTCTGAGCAACGATACCGACAAACGCATGGTTCTGAAACATTTCAACTCTATCACCGCCGAAAACGAAATGAAGCCAGAAAGCCTGCTGGCCGGTCAGACTAGCACCGGCCTGTCTTACCGCTTTTCCACTGCAGACGCTTTTGTGGATTTCGCGAGCACTAACAAAATTGGCATCCGCGGCCATACCCTGGTTTGGCACAACCAGACCCCGGATTGGTTCTTCAAAGATTCTAACGGTCAGCGTCTGTCCAAGGACGCACTGCTGGCTCGTCTGCTGCAGTACATCTATGATGTTGTGGGCCGTTACAAGGGCAAAGTGTATGCGTGGGATGTAGTGAACGAGGCAATCGACGAAAACCAACCGGACAGCTACCGTCGCTCTACCTGGTACGAGATCTGTGGTCCGGAATACATTGAGAAAGCCTTCATCTGGGCCCACGAGGCTGACCCGAATGCGAAACTGTTTTACAACGATTATAATACCGAGATCAGCAAGAAACGTGACTTTATCTACAACATGGTTAAAAACCTGAAATCCAAGGGCATCCCTATTCACGGCATCGGTATGCAGTGTCATATTAATGTGAACTGGCCGTCCGTTAGCGAAATTGAAAACTCTATCAAACTGTTCTCCAGCATCCCGGGTATCGAAATTCATATCACTGAACTGGACATGTCTCTGTATAACTACGGCTCTAGCGAAAACTACTCCACCCCGCCGCAGGACCTGCTGCAGAAGCAAAGCCAGAAATACAAGGAAATTTTTACTATGCTGAAAAAATACAAAAACGTTGTTAAGAGCGTTACCTTCTGGGGTCTGAAAGACGACTACTCTTGGCTGCGCAGCTTCTACGGCAAAAACGACTGGCCGCTGCTGTTCTTCGAAGACTACTCTGCTAAACCGGCTTATTGGGCGGTCATCGAAGCTAGCGGCGTGACTACCTCCTCCCCGACCCCGACTCCGACCCCGACCGTAACCGTTACTCCGACCCCGACTCCGACTCCGACCCCAACTGTGACCGCGACTCCTACTCCGACCCCTACCCCGGTCTCCACTCCGGCAACTGGCGGCCAAATCAAAGTGCTGTACGCTAATAAGGAAACCAACTCCACCACGAATACCATCCGTCCGTGGCTGAAGGTTGTTAACAGCGGCTCCAGCAGCATCGACCTGTCTCGCGTTACTATCCGCTATTGGTATACCGTTGACGGTGAACGTGCCCAGTCCGCAGTCTCTGATTGGGCGCAGATCGGTGCTTCCAACGTCACTTTCAAATTTGTAAAACTGAGCTCTAGCGTGAGCGGTGCTTACTACTACCTGGAACTGGGTTTCAAGAGCGGTGCTGGTCAGCTGCAGCCGGGTAAAGACACTGGCGAGATCCAGATCCGTTTCAACAAATCTGACTGGTCTAACTATAACCAGGGCAACGACTGGTCTTGGCTGCAGTCCATGACCTCTTACGGTGAAAACGAGAAAGTTACTGCGTACATTGATGGTGTTCTGGTGTGGGGCCAGGAACCGTCTTGGGACATTAGCGAACTGTCTATTTCTGGTGAATACGTTCGCAGCCGTATCAAAGGTATCCCATACCAGCCGATCGAACGTACCCTGAAAATCTCCCAGGACCAGGTGGCATGTGCACCGATTGGCCAGCCGATCCTGCCGTCTGACTTCGAGGACGGTACCCGTCAGGGCTGGGATTGGGACGGCCCGTCCGGTGTTAAAGGTGCTCTGACCATTGAAGAAGCGAATGGCTCCAACGCTCTGAGCTGGGAAGTTGAGTACCCGGAAAAAAAACTGCAGGATGGCTGGGCATCTGCTCCTCGCCTGATCCTGCGTAATATTAACACCACGCGTGGTGATTGTAAATACCTGTGCTTCGACTTCTATCTGAAGCCGAAGCAGGCGACCAAAGGCGAACTGGCGATCTTCCTGGCGTTTGCTCCTCCGAGCCTGAACTACTGGGCTCAAGCTGAAGACTCCTTCAACATCGACCTGACTAATCTGTCTACTCTGAAGAAAACCCCGGATGATCTGTACTCTTTCGTTATTTCTTTCGATCTGAAAAAGATCAAAGAAGGCAAAATCATCGGCCCTGACACCCACCTGCGTGACATCATCATCGTTGTAGCAGACGTGAACTCTGACTTCAAAGGTCGTATGTATCTGGACAATGTGCGTTTCACGAACATGCTGTTCGAAGATGTAACGCCGCAGACTACGGGTTACGAAGCGATCTCTAAACTGTACTCTAAGAAAATTGTTAACGGCATCAGCACCAACCTGTTCGGCCCGGAGAAAGCTGTTACTCGCGCAGAAGTGGCGGCTATGGCAGTCCGTCTGCTGGACCTGCAAGAAGAATCTTACAACGGTGAGTTTGTAGACGTATCTAAAAACTCCTGGTACGCGAACGAAGTATCTACTGCGTACAAAGCAGGTATCATTCTGGGCGACGGTAAGTACATCAAACCGGAGAAAGCCGTTACTCGCGAGGAAATGGCGGTCTTCGCGATGCGTATCTACCGTATCCTGACTGACGAAAAAGCAGAAGCGACCGAAGAAATTGCAATTTCTGACAAAAACAGCATTTCTTCCTGGGCGCGTCAAGATGTAAACGCTGCTATCTCTCTGGGCCTGATGGACGTCTTCACTGACGGTTCTTTCGGCCCGAAGGTGAAAGTGACTCGTGCCGAAGCGACTCAGATTATTTATAAGATCCTGGAACTGACTGGTAAAATGCACCACCACCACCACCATTAAGAATTCE.g. SEQ ID NO.3), including restriction sites and a terminator TAA, the amino acid sequence of which is SQ2(MKRNLFRIVSRVLLIAFIASISLVGAMSYFPVETQAAPDWSIPSLCESYKDDFMIGVAIPARCLSNDTDKRMVLKHFNSITAENEMKPESLLAGQTSTGLSYRFSTADAFVDFASTNKIGIRGHTLVWHNQTPDWFFKDSNGQRLSKDALLARLLQYIYDVVGRYKGKVYAWDVVNEAIDENQPDSYRRSTWYEICGPEYIEKAFIWAHEADPNAKLFYNDYNTEISKKRDFIYNMVKNLKSKGIPIHGIGMQCHINVNWPSVSEIENSIKLFSSIPGIEIHITELDMSLYNYGSSENYSTPPQDLLQKQSQKYKEIFTMLKKYKNVVKSVTFWGLKDDYSWLRSFYGKNDWPLLFFEDYSAKPAYWAVIEASGVTTSSPTPTPTPTVTVTPTPTPTPTPTVTATPTPTPTPVSTPATGGQIKVLYANKETNSTTNTIRPWLKVVNSGSSSIDLSRVTIRYWYTVDGERAQSAVSDWAQIGASNVTFKFVKLSSSVSGAYYYLELGFKSGAGQLQPGKDTGEIQIRFNKSDWSNYNQGNDWSWLQSMTSYGENEKVTAYIDGVLVWGQEPSWDISELSISGEYVRSRIKGIPYQPIERTLKISQDQVACAPIGQPILPSDFEDGTRQGWDWDGPSGVKGALTIEEANGSNALSWEVEYPEKKLQDGWASAPRLILRNINTTRGDCKYLCFDFYLKPKQATKGELAIFLAFAPPSLNYWAQAEDSFNIDLTNLSTLKKTPDDLYSFVISFDLKKIKEGKIIGPDTHLRDIIIVVADVNSDFKGRMYLDNVRFTNMLFEDVTPQTTGYEAISKLYSKKIVNGISTNLFGPEKAVTRAEVAAMAVRLLDLQEESYNGEFVDVSKNSWYANEVSTAYKAGIILGDGKYIKPEKAVTREEMAVFAMRIYRILTDEKAEATEEIAISDKNSISSWARQDVNAAISLGLMDVFTDGSFGPKVKVTRAEATQIIYKILELTGKMHHHHHH, e.g. SEQ ID NO.3)SEQ ID NO.4)。
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Zhongnong Huawei biopharmaceutical (Hubei) Co., Ltd
<120> construction method of cellulase suitable for traditional Chinese medicine feed additive
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2961
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggatccatga aacgtaacct gttccgcatc gtttctcgtg ttgtactgat tgctttcatc 60
gcatccatct ctctggtggg tgctatgagc tatttcccgg tagaaaccca ggcggccccg 120
gattggtcta ttccgtccct gtgcgaatct tataaagatg actttatgat cggtgttgcg 180
atccctgccc gttgtctgag caacgatacc gacaaacgca tggttctgaa acatttcaac 240
tctatcaccg ccgaaaacga aatgaagcca gaaagcctgc tggccggtca gactagcacc 300
ggcctgtctt accgcttttc cactgcagac gcttttgtgg atttcgcgag cactaacaaa 360
attggcatcc gcggccatac cctggtttgg cacaaccaga ccccggattg gttcttcaaa 420
gattctaacg gtcagcgtct gtccaaggac gcactgctgg ctcgtctgaa acagtacatc 480
tatgatgttg tgggccgtta caagggcaaa gtgtatgcgt gggatgtagt gaacgaggca 540
atcgacgaaa accaaccgga cagctaccgt cgctctacct ggtacgagat ctgtggtccg 600
gaatacattg agaaagcctt catctgggcc cacgaggctg acccgaatgc gaaactgttt 660
tacaacgatt ataataccga gatcagcaag aaacgtgact ttatctacaa catggttaaa 720
aacctgaaat ccaagggcat ccctattcac ggcatcggta tgcagtgtca tattaatgtg 780
aactggccgt ccgttagcga aattgaaaac tctatcaaac tgttctccag catcccgggt 840
atcgaaattc atatcactga actggacatg tctctgtata actacggctc tagcgaaaac 900
tactccaccc cgccgcagga cctgctgcag aagcaaagcc agaaatacaa ggaaattttt 960
actatgctga aaaaatacaa aaacgttgtt aagagcgtta ccttctgggg tctgaaagac 1020
gactactctt ggctgcgcag cttctacggc aaaaacgact ggccgctgct gttcttcgaa 1080
gactactctg ctaaaccggc ttattgggcg gtcatcgaag ctagcggcgt gactacctcc 1140
tccccgaccc cgactccgac cccgaccgta accgttactc cgaccccgac tccgactccg 1200
accccaactg tgaccgcgac tcctactccg acccctaccc cggtctccac tccggcaact 1260
ggcggccaaa tcaaagtgct gtacgctaat aaggaaacca actccaccac gaataccatc 1320
cgtccgtggc tgaaggttgt taacagcggc tccagcagca tcgacctgtc tcgcgttact 1380
atccgctatt ggtataccgt tgacggtgaa cgtgcccagt ccgcagtctc tgattgggcg 1440
cagatcggtg cttccaacgt cactttcaaa tttgtaaaac tgagctctag cgtgagcggt 1500
gctgactact acctggaaat cggtttcaag agcggtgctg gtcagctgca gccgggtaaa 1560
gacactggcg agatccagat ccgtttcaac aaatctgact ggtctaacta taaccagggc 1620
aacgactggt cttggctgca gtccatgacc tcttacggtg aaaacgagaa agttactgcg 1680
tacattgatg gtgttctggt gtggggccag gaaccgtctt gggacattag cgaactgtct 1740
atttctggtg aatacgttcg cagccgtatc aaaggtatcc cataccagcc gatcgaacgt 1800
accctgaaaa tctcccagga ccaggtggca tgtgcaccga ttggccagcc gatcctgccg 1860
tctgacttcg aggacggtac ccgtcagggc tgggattggg acggcccgtc cggtgttaaa 1920
ggtgctctga ccattgaaga agcgaatggc tccaacgctc tgagctggga agttgagtac 1980
ccggaaaaaa aactgcagga tggctgggca tctgctcctc gcctgatcct gcgtaatatt 2040
aacaccacgc gtggtgattg taaatacctg tgcttcgact tctatctgaa gccgaagcag 2100
gcgaccaaag gcgaactggc gatcttcctg gcgtttgctc ctccgagcct gaactactgg 2160
gctcaagctg aagactcctt caacatcgac ctgactaatc tgtctactct gaagaaaacc 2220
ccggatgatc tgtactcttt caaaatttct ttcgatctgg ataagatcaa agaaggcaaa 2280
atcatcggcc ctgacaccca cctgcgtgac atcatcatcg ttgtagcaga cgtgaactct 2340
gacttcaaag gtcgtatgta tctggacaat gtgcgtttca cgaacatgct gttcgaagat 2400
gtaacgccgc agactacggg ttacgaagcg atctctaaac tgtactctaa gaaaattgtt 2460
aacggcatca gcaccaacct gttcggcccg gagaaagctg ttactcgcgc agaagtggcg 2520
gctatggcag tccgtctgct ggacctgcaa gaagaatctt acaacggtga gtttgtagac 2580
gtatctaaaa actcctggta cgcgaacgaa gtatctactg cgtacaaagc aggtatcatt 2640
ctgggcgacg gtaagtacat caaaccggag aaagccgtta ctcgcgagga aatggcggtc 2700
ttcgcgatgc gtatctaccg tatcctgact gacgaaaaag cagaagcgac cgaagaaatt 2760
gcaatttctg acaaaaacag catttcttcc tgggcgcgtc aagatgtaaa cgctgctatc 2820
tctctgggcc tgatggacgt cttcactgac ggttctttcg gcccgaaggt gaaagtgact 2880
cgtgccgaag cgactcagat tatttataag atcctggaac tgactggtaa aatgcaccac 2940
caccaccacc attaagaatt c 2961
<210> 2
<211> 982
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Lys Arg Asn Leu Phe Arg Ile Val Ser Arg Val Val Leu Ile Ala
1 5 10 15
Phe Ile Ala Ser Ile Ser Leu Val Gly Ala Met Ser Tyr Phe Pro Val
20 25 30
Glu Thr Gln Ala Ala Pro Asp Trp Ser Ile Pro Ser Leu Cys Glu Ser
35 40 45
Tyr Lys Asp Asp Phe Met Ile Gly Val Ala Ile Pro Ala Arg Cys Leu
50 55 60
Ser Asn Asp Thr Asp Lys Arg Met Val Leu Lys His Phe Asn Ser Ile
65 70 75 80
Thr Ala Glu Asn Glu Met Lys Pro Glu Ser Leu Leu Ala Gly Gln Thr
85 90 95
Ser Thr Gly Leu Ser Tyr Arg Phe Ser Thr Ala Asp Ala Phe Val Asp
100 105 110
Phe Ala Ser Thr Asn Lys Ile Gly Ile Arg Gly His Thr Leu Val Trp
115 120 125
His Asn Gln Thr Pro Asp Trp Phe Phe Lys Asp Ser Asn Gly Gln Arg
130 135 140
Leu Ser Lys Asp Ala Leu Leu Ala Arg Leu Lys Gln Tyr Ile Tyr Asp
145 150 155 160
Val Val Gly Arg Tyr Lys Gly Lys Val Tyr Ala Trp Asp Val Val Asn
165 170 175
Glu Ala Ile Asp Glu Asn Gln Pro Asp Ser Tyr Arg Arg Ser Thr Trp
180 185 190
Tyr Glu Ile Cys Gly Pro Glu Tyr Ile Glu Lys Ala Phe Ile Trp Ala
195 200 205
His Glu Ala Asp Pro Asn Ala Lys Leu Phe Tyr Asn Asp Tyr Asn Thr
210 215 220
Glu Ile Ser Lys Lys Arg Asp Phe Ile Tyr Asn Met Val Lys Asn Leu
225 230 235 240
Lys Ser Lys Gly Ile Pro Ile His Gly Ile Gly Met Gln Cys His Ile
245 250 255
Asn Val Asn Trp Pro Ser Val Ser Glu Ile Glu Asn Ser Ile Lys Leu
260 265 270
Phe Ser Ser Ile Pro Gly Ile Glu Ile His Ile Thr Glu Leu Asp Met
275 280 285
Ser Leu Tyr Asn Tyr Gly Ser Ser Glu Asn Tyr Ser Thr Pro Pro Gln
290 295 300
Asp Leu Leu Gln Lys Gln Ser Gln Lys Tyr Lys Glu Ile Phe Thr Met
305 310 315 320
Leu Lys Lys Tyr Lys Asn Val Val Lys Ser Val Thr Phe Trp Gly Leu
325 330 335
Lys Asp Asp Tyr Ser Trp Leu Arg Ser Phe Tyr Gly Lys Asn Asp Trp
340 345 350
Pro Leu Leu Phe Phe Glu Asp Tyr Ser Ala Lys Pro Ala Tyr Trp Ala
355 360 365
Val Ile Glu Ala Ser Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro
370 375 380
Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
385 390 395 400
Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro
405 410 415
Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn
420 425 430
Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly
435 440 445
Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr
450 455 460
Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile
465 470 475 480
Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val
485 490 495
Ser Gly Ala Asp Tyr Tyr Leu Glu Ile Gly Phe Lys Ser Gly Ala Gly
500 505 510
Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn
515 520 525
Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu
530 535 540
Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile
545 550 555 560
Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Trp Asp Ile Ser Glu
565 570 575
Leu Ser Ile Ser Gly Glu Tyr Val Arg Ser Arg Ile Lys Gly Ile Pro
580 585 590
Tyr Gln Pro Ile Glu Arg Thr Leu Lys Ile Ser Gln Asp Gln Val Ala
595 600 605
Cys Ala Pro Ile Gly Gln Pro Ile Leu Pro Ser Asp Phe Glu Asp Gly
610 615 620
Thr Arg Gln Gly Trp Asp Trp Asp Gly Pro Ser Gly Val Lys Gly Ala
625 630 635 640
Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Val
645 650 655
Glu Tyr Pro Glu Lys Lys Leu Gln Asp Gly Trp Ala Ser Ala Pro Arg
660 665 670
Leu Ile Leu Arg Asn Ile Asn Thr Thr Arg Gly Asp Cys Lys Tyr Leu
675 680 685
Cys Phe Asp Phe Tyr Leu Lys Pro Lys Gln Ala Thr Lys Gly Glu Leu
690 695 700
Ala Ile Phe Leu Ala Phe Ala Pro Pro Ser Leu Asn Tyr Trp Ala Gln
705 710 715 720
Ala Glu Asp Ser Phe Asn Ile Asp Leu Thr Asn Leu Ser Thr Leu Lys
725 730 735
Lys Thr Pro Asp Asp Leu Tyr Ser Phe Lys Ile Ser Phe Asp Leu Asp
740 745 750
Lys Ile Lys Glu Gly Lys Ile Ile Gly Pro Asp Thr His Leu Arg Asp
755 760 765
Ile Ile Ile Val Val Ala Asp Val Asn Ser Asp Phe Lys Gly Arg Met
770 775 780
Tyr Leu Asp Asn Val Arg Phe Thr Asn Met Leu Phe Glu Asp Val Thr
785 790 795 800
Pro Gln Thr Thr Gly Tyr Glu Ala Ile Ser Lys Leu Tyr Ser Lys Lys
805 810 815
Ile Val Asn Gly Ile Ser Thr Asn Leu Phe Gly Pro Glu Lys Ala Val
820 825 830
Thr Arg Ala Glu Val Ala Ala Met Ala Val Arg Leu Leu Asp Leu Gln
835 840 845
Glu Glu Ser Tyr Asn Gly Glu Phe Val Asp Val Ser Lys Asn Ser Trp
850 855 860
Tyr Ala Asn Glu Val Ser Thr Ala Tyr Lys Ala Gly Ile Ile Leu Gly
865 870 875 880
Asp Gly Lys Tyr Ile Lys Pro Glu Lys Ala Val Thr Arg Glu Glu Met
885 890 895
Ala Val Phe Ala Met Arg Ile Tyr Arg Ile Leu Thr Asp Glu Lys Ala
900 905 910
Glu Ala Thr Glu Glu Ile Ala Ile Ser Asp Lys Asn Ser Ile Ser Ser
915 920 925
Trp Ala Arg Gln Asp Val Asn Ala Ala Ile Ser Leu Gly Leu Met Asp
930 935 940
Val Phe Thr Asp Gly Ser Phe Gly Pro Lys Val Lys Val Thr Arg Ala
945 950 955 960
Glu Ala Thr Gln Ile Ile Tyr Lys Ile Leu Glu Leu Thr Gly Lys Met
965 970 975
His His His His His His
980
<210> 3
<211> 2961
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggatccatga aacgtaacct gttccgcatc gtttctcgtg ttctgctgat tgctttcatc 60
gcatccatct ctctggtggg tgctatgagc tatttcccgg tagaaaccca ggcggccccg 120
gattggtcta ttccgtccct gtgcgaatct tataaagatg actttatgat cggtgttgcg 180
atccctgccc gttgtctgag caacgatacc gacaaacgca tggttctgaa acatttcaac 240
tctatcaccg ccgaaaacga aatgaagcca gaaagcctgc tggccggtca gactagcacc 300
ggcctgtctt accgcttttc cactgcagac gcttttgtgg atttcgcgag cactaacaaa 360
attggcatcc gcggccatac cctggtttgg cacaaccaga ccccggattg gttcttcaaa 420
gattctaacg gtcagcgtct gtccaaggac gcactgctgg ctcgtctgct gcagtacatc 480
tatgatgttg tgggccgtta caagggcaaa gtgtatgcgt gggatgtagt gaacgaggca 540
atcgacgaaa accaaccgga cagctaccgt cgctctacct ggtacgagat ctgtggtccg 600
gaatacattg agaaagcctt catctgggcc cacgaggctg acccgaatgc gaaactgttt 660
tacaacgatt ataataccga gatcagcaag aaacgtgact ttatctacaa catggttaaa 720
aacctgaaat ccaagggcat ccctattcac ggcatcggta tgcagtgtca tattaatgtg 780
aactggccgt ccgttagcga aattgaaaac tctatcaaac tgttctccag catcccgggt 840
atcgaaattc atatcactga actggacatg tctctgtata actacggctc tagcgaaaac 900
tactccaccc cgccgcagga cctgctgcag aagcaaagcc agaaatacaa ggaaattttt 960
actatgctga aaaaatacaa aaacgttgtt aagagcgtta ccttctgggg tctgaaagac 1020
gactactctt ggctgcgcag cttctacggc aaaaacgact ggccgctgct gttcttcgaa 1080
gactactctg ctaaaccggc ttattgggcg gtcatcgaag ctagcggcgt gactacctcc 1140
tccccgaccc cgactccgac cccgaccgta accgttactc cgaccccgac tccgactccg 1200
accccaactg tgaccgcgac tcctactccg acccctaccc cggtctccac tccggcaact 1260
ggcggccaaa tcaaagtgct gtacgctaat aaggaaacca actccaccac gaataccatc 1320
cgtccgtggc tgaaggttgt taacagcggc tccagcagca tcgacctgtc tcgcgttact 1380
atccgctatt ggtataccgt tgacggtgaa cgtgcccagt ccgcagtctc tgattgggcg 1440
cagatcggtg cttccaacgt cactttcaaa tttgtaaaac tgagctctag cgtgagcggt 1500
gcttactact acctggaact gggtttcaag agcggtgctg gtcagctgca gccgggtaaa 1560
gacactggcg agatccagat ccgtttcaac aaatctgact ggtctaacta taaccagggc 1620
aacgactggt cttggctgca gtccatgacc tcttacggtg aaaacgagaa agttactgcg 1680
tacattgatg gtgttctggt gtggggccag gaaccgtctt gggacattag cgaactgtct 1740
atttctggtg aatacgttcg cagccgtatc aaaggtatcc cataccagcc gatcgaacgt 1800
accctgaaaa tctcccagga ccaggtggca tgtgcaccga ttggccagcc gatcctgccg 1860
tctgacttcg aggacggtac ccgtcagggc tgggattggg acggcccgtc cggtgttaaa 1920
ggtgctctga ccattgaaga agcgaatggc tccaacgctc tgagctggga agttgagtac 1980
ccggaaaaaa aactgcagga tggctgggca tctgctcctc gcctgatcct gcgtaatatt 2040
aacaccacgc gtggtgattg taaatacctg tgcttcgact tctatctgaa gccgaagcag 2100
gcgaccaaag gcgaactggc gatcttcctg gcgtttgctc ctccgagcct gaactactgg 2160
gctcaagctg aagactcctt caacatcgac ctgactaatc tgtctactct gaagaaaacc 2220
ccggatgatc tgtactcttt cgttatttct ttcgatctga aaaagatcaa agaaggcaaa 2280
atcatcggcc ctgacaccca cctgcgtgac atcatcatcg ttgtagcaga cgtgaactct 2340
gacttcaaag gtcgtatgta tctggacaat gtgcgtttca cgaacatgct gttcgaagat 2400
gtaacgccgc agactacggg ttacgaagcg atctctaaac tgtactctaa gaaaattgtt 2460
aacggcatca gcaccaacct gttcggcccg gagaaagctg ttactcgcgc agaagtggcg 2520
gctatggcag tccgtctgct ggacctgcaa gaagaatctt acaacggtga gtttgtagac 2580
gtatctaaaa actcctggta cgcgaacgaa gtatctactg cgtacaaagc aggtatcatt 2640
ctgggcgacg gtaagtacat caaaccggag aaagccgtta ctcgcgagga aatggcggtc 2700
ttcgcgatgc gtatctaccg tatcctgact gacgaaaaag cagaagcgac cgaagaaatt 2760
gcaatttctg acaaaaacag catttcttcc tgggcgcgtc aagatgtaaa cgctgctatc 2820
tctctgggcc tgatggacgt cttcactgac ggttctttcg gcccgaaggt gaaagtgact 2880
cgtgccgaag cgactcagat tatttataag atcctggaac tgactggtaa aatgcaccac 2940
caccaccacc attaagaatt c 2961
<210> 4
<211> 982
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Lys Arg Asn Leu Phe Arg Ile Val Ser Arg Val Leu Leu Ile Ala
1 5 10 15
Phe Ile Ala Ser Ile Ser Leu Val Gly Ala Met Ser Tyr Phe Pro Val
20 25 30
Glu Thr Gln Ala Ala Pro Asp Trp Ser Ile Pro Ser Leu Cys Glu Ser
35 40 45
Tyr Lys Asp Asp Phe Met Ile Gly Val Ala Ile Pro Ala Arg Cys Leu
50 55 60
Ser Asn Asp Thr Asp Lys Arg Met Val Leu Lys His Phe Asn Ser Ile
65 70 75 80
Thr Ala Glu Asn Glu Met Lys Pro Glu Ser Leu Leu Ala Gly Gln Thr
85 90 95
Ser Thr Gly Leu Ser Tyr Arg Phe Ser Thr Ala Asp Ala Phe Val Asp
100 105 110
Phe Ala Ser Thr Asn Lys Ile Gly Ile Arg Gly His Thr Leu Val Trp
115 120 125
His Asn Gln Thr Pro Asp Trp Phe Phe Lys Asp Ser Asn Gly Gln Arg
130 135 140
Leu Ser Lys Asp Ala Leu Leu Ala Arg Leu Leu Gln Tyr Ile Tyr Asp
145 150 155 160
Val Val Gly Arg Tyr Lys Gly Lys Val Tyr Ala Trp Asp Val Val Asn
165 170 175
Glu Ala Ile Asp Glu Asn Gln Pro Asp Ser Tyr Arg Arg Ser Thr Trp
180 185 190
Tyr Glu Ile Cys Gly Pro Glu Tyr Ile Glu Lys Ala Phe Ile Trp Ala
195 200 205
His Glu Ala Asp Pro Asn Ala Lys Leu Phe Tyr Asn Asp Tyr Asn Thr
210 215 220
Glu Ile Ser Lys Lys Arg Asp Phe Ile Tyr Asn Met Val Lys Asn Leu
225 230 235 240
Lys Ser Lys Gly Ile Pro Ile His Gly Ile Gly Met Gln Cys His Ile
245 250 255
Asn Val Asn Trp Pro Ser Val Ser Glu Ile Glu Asn Ser Ile Lys Leu
260 265 270
Phe Ser Ser Ile Pro Gly Ile Glu Ile His Ile Thr Glu Leu Asp Met
275 280 285
Ser Leu Tyr Asn Tyr Gly Ser Ser Glu Asn Tyr Ser Thr Pro Pro Gln
290 295 300
Asp Leu Leu Gln Lys Gln Ser Gln Lys Tyr Lys Glu Ile Phe Thr Met
305 310 315 320
Leu Lys Lys Tyr Lys Asn Val Val Lys Ser Val Thr Phe Trp Gly Leu
325 330 335
Lys Asp Asp Tyr Ser Trp Leu Arg Ser Phe Tyr Gly Lys Asn Asp Trp
340 345 350
Pro Leu Leu Phe Phe Glu Asp Tyr Ser Ala Lys Pro Ala Tyr Trp Ala
355 360 365
Val Ile Glu Ala Ser Gly Val Thr Thr Ser Ser Pro Thr Pro Thr Pro
370 375 380
Thr Pro Thr Val Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro
385 390 395 400
Thr Val Thr Ala Thr Pro Thr Pro Thr Pro Thr Pro Val Ser Thr Pro
405 410 415
Ala Thr Gly Gly Gln Ile Lys Val Leu Tyr Ala Asn Lys Glu Thr Asn
420 425 430
Ser Thr Thr Asn Thr Ile Arg Pro Trp Leu Lys Val Val Asn Ser Gly
435 440 445
Ser Ser Ser Ile Asp Leu Ser Arg Val Thr Ile Arg Tyr Trp Tyr Thr
450 455 460
Val Asp Gly Glu Arg Ala Gln Ser Ala Val Ser Asp Trp Ala Gln Ile
465 470 475 480
Gly Ala Ser Asn Val Thr Phe Lys Phe Val Lys Leu Ser Ser Ser Val
485 490 495
Ser Gly Ala Tyr Tyr Tyr Leu Glu Leu Gly Phe Lys Ser Gly Ala Gly
500 505 510
Gln Leu Gln Pro Gly Lys Asp Thr Gly Glu Ile Gln Ile Arg Phe Asn
515 520 525
Lys Ser Asp Trp Ser Asn Tyr Asn Gln Gly Asn Asp Trp Ser Trp Leu
530 535 540
Gln Ser Met Thr Ser Tyr Gly Glu Asn Glu Lys Val Thr Ala Tyr Ile
545 550 555 560
Asp Gly Val Leu Val Trp Gly Gln Glu Pro Ser Trp Asp Ile Ser Glu
565 570 575
Leu Ser Ile Ser Gly Glu Tyr Val Arg Ser Arg Ile Lys Gly Ile Pro
580 585 590
Tyr Gln Pro Ile Glu Arg Thr Leu Lys Ile Ser Gln Asp Gln Val Ala
595 600 605
Cys Ala Pro Ile Gly Gln Pro Ile Leu Pro Ser Asp Phe Glu Asp Gly
610 615 620
Thr Arg Gln Gly Trp Asp Trp Asp Gly Pro Ser Gly Val Lys Gly Ala
625 630 635 640
Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Val
645 650 655
Glu Tyr Pro Glu Lys Lys Leu Gln Asp Gly Trp Ala Ser Ala Pro Arg
660 665 670
Leu Ile Leu Arg Asn Ile Asn Thr Thr Arg Gly Asp Cys Lys Tyr Leu
675 680 685
Cys Phe Asp Phe Tyr Leu Lys Pro Lys Gln Ala Thr Lys Gly Glu Leu
690 695 700
Ala Ile Phe Leu Ala Phe Ala Pro Pro Ser Leu Asn Tyr Trp Ala Gln
705 710 715 720
Ala Glu Asp Ser Phe Asn Ile Asp Leu Thr Asn Leu Ser Thr Leu Lys
725 730 735
Lys Thr Pro Asp Asp Leu Tyr Ser Phe Val Ile Ser Phe Asp Leu Lys
740 745 750
Lys Ile Lys Glu Gly Lys Ile Ile Gly Pro Asp Thr His Leu Arg Asp
755 760 765
Ile Ile Ile Val Val Ala Asp Val Asn Ser Asp Phe Lys Gly Arg Met
770 775 780
Tyr Leu Asp Asn Val Arg Phe Thr Asn Met Leu Phe Glu Asp Val Thr
785 790 795 800
Pro Gln Thr Thr Gly Tyr Glu Ala Ile Ser Lys Leu Tyr Ser Lys Lys
805 810 815
Ile Val Asn Gly Ile Ser Thr Asn Leu Phe Gly Pro Glu Lys Ala Val
820 825 830
Thr Arg Ala Glu Val Ala Ala Met Ala Val Arg Leu Leu Asp Leu Gln
835 840 845
Glu Glu Ser Tyr Asn Gly Glu Phe Val Asp Val Ser Lys Asn Ser Trp
850 855 860
Tyr Ala Asn Glu Val Ser Thr Ala Tyr Lys Ala Gly Ile Ile Leu Gly
865 870 875 880
Asp Gly Lys Tyr Ile Lys Pro Glu Lys Ala Val Thr Arg Glu Glu Met
885 890 895
Ala Val Phe Ala Met Arg Ile Tyr Arg Ile Leu Thr Asp Glu Lys Ala
900 905 910
Glu Ala Thr Glu Glu Ile Ala Ile Ser Asp Lys Asn Ser Ile Ser Ser
915 920 925
Trp Ala Arg Gln Asp Val Asn Ala Ala Ile Ser Leu Gly Leu Met Asp
930 935 940
Val Phe Thr Asp Gly Ser Phe Gly Pro Lys Val Lys Val Thr Arg Ala
945 950 955 960
Glu Ala Thr Gln Ile Ile Tyr Lys Ile Leu Glu Leu Thr Gly Lys Met
965 970 975
His His His His His His
980
Claims (6)
1. The cellulase suitable for the traditional Chinese medicine feed additive is characterized in that the nucleotide sequence of the coding enzyme is shown as SEQ ID NO. 3.
2. The cellulase suitable for the Chinese medicinal feed additive according to claim 1, wherein the amino acid sequence is shown as SEQ ID No. 4.
3. The method for constructing cellulase suitable for the traditional Chinese medicine feed additive according to claim 1 or 2, which is characterized by comprising the following steps:
(1) carrying out error-prone PCR amplification by using a synthetic sequence with a nucleotide sequence shown as SEQ ID NO.1 as a template;
(2) carrying out double enzyme digestion on the amplified product by using restriction enzymes BamHI and EcoRI, and recovering a gene fragment;
(3) carrying out double digestion on the expression vector pRSFduet-1 by using the same restriction enzyme as the restriction enzyme in the step (2);
(4) connecting the gene fragment recovered in the step (2) with the vector subjected to double enzyme digestion in the step (3) to obtain a recombinant expression vector pRSFduet-1-SQ, then transforming the recombinant expression vector pRSFduet-1-SQ into an Escherichia coli E.coli DH5 alpha competent cell, extracting an expression vector plasmid, identifying and sequentially numbering the transformed strains;
(5) and the transformed strain is subjected to protein expression, purification and enzyme activity analysis to screen target cellulase.
4. The method of claim 3, wherein the error-prone PCR reaction system of step (1): error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, 5mM MnCl23.0 μ L, 10 μ g/μ L DNA template 1 μ L, 10 μ M PCR primer 2 μ L, Taq DNA polymerase 1 μ L, sterilized double distilled water 17 μ L; the procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1 min; annealing at 45 deg.C for 1 min; extending at 72 ℃ for 4 min; after 32 cycles, extension was carried out for 10min at 72 ℃.
5. The method according to claim 4, wherein the enzyme digestion system in steps (2) and (3): 11 μ L of ultrapure water, 1 μ L of BamHI, 1 μ L of EcoRI, 3 μ L of Buffer, and 14 μ L of target gene.
6. Use of a cellulase adapted to a traditional Chinese medicine feed additive according to claim 1 or 2 in fermentation.
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| CN114196695A (en) * | 2021-11-26 | 2022-03-18 | 中农华威生物制药(湖北)有限公司 | Construction method of high-activity traditional Chinese medicine feed additive trypsin |
| CN114958809A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Construction method of endo-beta-glucanase suitable for high-temperature granulation of feed |
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| CN114196695A (en) * | 2021-11-26 | 2022-03-18 | 中农华威生物制药(湖北)有限公司 | Construction method of high-activity traditional Chinese medicine feed additive trypsin |
| CN114958809A (en) * | 2022-06-14 | 2022-08-30 | 中农华威生物制药(湖北)有限公司 | Construction method of endo-beta-glucanase suitable for high-temperature granulation of feed |
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