CN114214303B - Ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof - Google Patents

Ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof Download PDF

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CN114214303B
CN114214303B CN202210094887.XA CN202210094887A CN114214303B CN 114214303 B CN114214303 B CN 114214303B CN 202210094887 A CN202210094887 A CN 202210094887A CN 114214303 B CN114214303 B CN 114214303B
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lysozyme
oclyz1a
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姜雨
王香南
李宗军
张禹
王喜宏
田小倩
田�健
黄火清
罗会颖
姚斌
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Northwest A&F University
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Abstract

The invention discloses a ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof. The invention utilizes the goat rumen protozoan Ophryscolex caudatus genome data to obtain a specific lysozyme named as OCLyz1A of rumen protozoans through assembly, annotation analysis, protein expression and function experiments. The lysozyme enzyme from the novel source has high activity, has obvious inhibition effect on gram-positive bacteria represented by micrococcus luteus, and can be widely applied to livestock and poultry feed, food and bioengineering.

Description

Ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a secretion expression method of ruminant rumen protozoan' trichina cauda "(O.caudatus) specific lysozyme (lysozyme, lyz) in pichia pastoris and application thereof.
Background
Lysozyme, also known as muramidase or N-acetylmuramyl peptide glycan hydrolase; it is a natural protein, has no toxic side effect, and can be widely used in higher animal and plant tissues and secretions, protozoa, insects and various microorganisms. The lysozyme can be classified into egg white lysozyme, animal lysozyme, plant lysozyme, microbial lysozyme and bacteriophage lysozyme according to different sources; the microbial lysozyme is used as an effective antibacterial agent, and hydrolyzes beta-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine acetate on bacterial cell wall peptidoglycan through an active center, so that the peptidoglycan skeleton structure is broken, cell wall damage is caused, and finally, thalli cells are dissolved due to unbalanced osmotic pressure until the thalli cells die. Various lysozyme enzymes have been identified in ruminants (such as the ruminant rumen specific lysozyme LYZ1 disclosed in Chinese CN 111187765A), which is abundant in the digestive system. The lysozyme in a digestive system also has obvious effects on changing the intestinal microbiota of animals, increasing beneficial bacteria in the intestinal tract, enhancing the disease resistance of organisms and improving the production performance of the animals; by adding lysozyme into the feed to replace or partially replace antibiotics, a certain way is provided for solving the problems of drug resistance, drug residue and the like in the breeding industry.
Reports on lysozyme derived from ruminant rumen protozoan Ophryscolex caudatus have not been found, and only Ophryscolex caudatus has been reported to promote the digestion of fiber in the rumen.
Disclosure of Invention
The invention aims to provide a ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof. The lysozyme OCLyz1A can be used as a novel enzyme preparation and widely applied to livestock and poultry feed, food, medicine, bioengineering and enzyme engineering.
In order to achieve the purpose, the invention adopts the following technical scheme:
a ruminant rumen protozoan specific lysozyme OCLyz1A, the amino acid sequence of the lysozyme OCLyz1A is:
ADEYYTVQSGDYLGKIANRYGVSVSDLCNWNGISNPDLIYPGQRLLVKKSGSSSGSGSGSGSGSGKSGKIYSVTDSQMQKMGWKNYNLPDLNRCLKTFEINTVNRVRHFISQCSHESACGVYTQELGGQSYCSKYDGRRDLGNTQPGDGCRFKGAGYIQLTGRSNYQSFANYIGDSRVMEGVSYVSSKYPWTSAGYWWKSHGLNSLCDNGASVDVITKRVNGGYNGLAERKKYYQRACNIFN。
preferably, the gene coding sequence of lysozyme OCLyz1A comprises the following DNA sequence, or the gene coding sequence of lysozyme OCLyz1A is selected from sequences derived from ruminant rumen protozoan genome having 90% or more homology with the following DNA sequence:
5'-GCTGATGAATATTATACTGTTCAATCAGGTGATTATTTAGGTAAAATTGCAAACAGATATGGTGTATCAGTATCTGATTTATGTAATTGGAATGGTATTTCTAACCCTGATTTAATTTATCCAGGACAAAGACTTCTTGTTAAAAAATCAGGTTCTAGTTCAGGTTCAGGCTCAGGATCAGGTTCAGGTTCAGGTAAATCTGGAAAAATATATTCTGTAACAGATTCACAAATGCAAAAAATGGGTTGGAAAAATTACAATCTTCCTGATTTAAATAGATGTTTAAAAACATTTGAAATTAATACAGTTAATAGAGTCAGACATTTCATTTCTCAATGCTCACATGAATCTGCCTGTGGAGTTTATACTCAAGAATTAGGAGGTCAAAGCTATTGCTCAAAATATGATGGAAGAAGAGATTTAGGAAATACACAACCTGGTGATGGATGCAGATTTAAAGGGGCTGGATATATTCAATTAACTGGAAGATCTAATTACCAAAGTTTTGCAAATTATATTGGTGATTCAAGAGTAATGGAAGGTGTTTCTTATGTTTCTAGCAAATATCCTTGGACTTCTGCTGGTTATTGGTGGAAATCTCATGGTTTAAATTCTTTATGTGATAATGGAGCTTCTGTTGATGTAATTACTAAAAGAGTAAATGGAGGATATAATGGTCTTGCTGAAAGAAAAAAATATTATCAAAGAGCTTGTAATATCTTTAAT-3'。
preferably, the ruminant rumen protozoa is of the genus cephalococcum (Ophrysscolex), for example, there is a coccygeus caterpillar (Ophrysscolex caudatum).
The secretion expression method of the ruminant rumen protozoan specific lysozyme OCLyz1A in pichia pastoris comprises the following steps:
1) Construction of recombinant Pichia Strain
Constructing a methanol-induced recombinant vector according to the gene coding sequence of the lysozyme OCLyz1A, and transforming pichia pastoris competent cells into the recombinant vector to obtain a pichia pastoris recombinant strain;
2) Fermentation culture
Carrying out induction expression of recombinant lysozyme OCLyz1A on the pichia pastoris recombinant strain through fermentation to obtain fermentation liquor;
3) Collecting the supernatant of the fermentation liquor to obtain a recombinant lysozyme OCLyz1A protein sample.
Preferably, the target gene sequence expressed by the recombinant vector is shown in SEQ ID No.5, and the amino acid sequence of the recombinant lysozyme OCLyz1A is shown in SEQ ID No. 4.
The application of the ruminant rumen protozoan specific lysozyme OCLyz1A in preparing antibacterial drugs for inhibiting gram-positive bacteria.
The application of the ruminant rumen protozoan specific lysozyme OCLyz1A in preparing antibacterial drugs for inhibiting Micrococcus luteus (Micrococcus luteus).
The application of the ruminant rumen protozoan specific lysozyme OCLyz1A in preparing feed additives.
A feed additive comprises the specific lysozyme OCLyz1A of the ruminant rumen protozoan.
A food additive comprises the specific lysozyme OCLyz1A of rumen protozoan of ruminant.
The invention has the beneficial effects that:
the invention discovers a rumen protozoan specific lysozyme (named as OCLyz 1A) gene by assembling, annotating and analyzing ruminant rumen protozoan O.caudatus genome data, and establishes a secretion expression method of the lysozyme in pichia pastoris according to a gene coding sequence. The lysozyme has the function of inhibiting gram-positive bacteria (such as micrococcus luteus), can be used as a safe livestock and poultry feed and a food additive (such as an antibacterial agent), and has great application potential.
The invention constructs a methanol-induced pichia pastoris recombinant strain capable of efficiently secreting and expressing the lysozyme according to the sequence of the ruminant rumen protozoan O.caudatus specific lysozyme (i.e. lysozyme OCLyz 1A), so that the production of the lysozyme OCLyz1A has the advantages of simple operation and short period.
Drawings
FIG. 1 is the SDS-PAGE electrophoresis of the recombinant OCLyz1A gene expression product in the example, wherein Marker is the protein molecular weight.
FIG. 2 is a graph showing the bacteriostatic effects of the fermentation supernatants on Micrococcus luteus and Escherichia coli in examples.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples so as to facilitate the understanding of the present invention by those skilled in the art, but the scope of the present invention is not limited thereto.
Secretion expression method of ruminant rumen protozoan O.caudatus specific lysozyme OCLyz1A in pichia pastoris
1. Construction of Pichia pastoris GS115 recombinant Strain
1.1 the invention utilizes ruminant (including guanzhong milk goats, guanzhong milk goat rumen content collected in 5 months in 2019 from Yang Ling in Shanxi) rumen protozoan O.caudatus genome sequencing data to assemble, annotate and analyze genes thereof, and identifies and obtains a new goat rumen protozoan O.caudatus specific lysozyme (named as OCLyz 1A) gene (the coding sequence of which comprises a sequence shown in SEQ. ID. NO. 1). According to the corresponding amino acid sequence of the lysozyme OCLyz1A gene obtained by identification, a recombinant rumen protozoan O.caudatus lysozyme OCLyz1A gene sequence (synthesized by general organisms (Anhui) GmbH, the sequence design completion time is 2021 years and 5 months, as shown in SEQ. ID. NO. 5) with signal peptide removed is obtained according to the codon preference of escherichia coli (used for cloning). The amino acid sequence of the rumen protozoan O.caudatus lysozyme OCLyz1A recombinant protein coded by the gene sequence is shown as follows:
ADEYYTVQSGDYLGKIANRYGVSVSDLCNWNGISNPDLIYPGQRLLVKKSGSSSGSGSGSGSGSGKSGKIYSVTDSQMQKMGWKNYNLPDLNRCLKTFEINTVNRVRHFISQCSHESACGVYTQELGGQSYCSKYDGRRDLGNTQPGDGCRFKGAGYIQLTGRSNYQSFANYIGDSRVMEGVSYVSSKYPWTSAGYWWKSHGLNSLCDNGASVDVITKRVNGGYNGLAERKKYYQRACNIFN (see seq. Id. No. 4).
1.2 amplifying recombinant rumen protozoan O.caudatus lysozyme OCLyz1A gene sequence from a cloning vector by using a designed primer LYZ-F, LYZ-R (shown as SEQ. ID. NO.2 and SEQ. ID. NO. 3), performing double enzyme digestion (EcoR I and Not I) on an expression vector pPIC9 (purchased from Invitrogen company) and the recombinant rumen protozoan O.caudaus lysozyme OCLyz1A gene sequence respectively, cutting out a gene fragment (shown as SEQ. ID. NO. 5) for coding mature lysozyme, connecting the gene fragment to the pPIC9 expression vector to obtain a recombinant plasmid pPIC9-OCLyz1A, and extracting the plasmid by using a small root plasmid medium-amount kit.
LYZ-F：
5'-GGAATTCCATCATCATCATCACCATGCTGATGAATATTACACCGTTCAGAG-3'LYZ-R：
5'-TAAAGCGGCCGCATTGAAGATATTACATGCACGCTG-3'
1.3 preparation of Pichia pastoris GS115 competent cell
Pichiapastoris GS115 (preserved by Beijing animal veterinary institute, national academy of agricultural sciences) on YPD solid plates was selected for monoclonal cloning, inoculated into 3mL of YPD liquid medium, and subjected to shake culture at 30 ℃ and 200 rmp. The thallus grows to OD 600-1.0, is transferred to a 100mL YPD shake flask and is cultured by a shaking table at 30 ℃ and 200 rmp. When the thallus grows to OD 600-1.0, centrifuging for 5min at 4 ℃ at 5000rmp, removing supernatant, adding 200mL of ice water, and resuspending; centrifuging at 4 ℃ for 5min at 5000rmp, removing supernatant, adding 100mL of ice water, and resuspending; centrifuging at 4 deg.C at 5000rmp for 5min, removing supernatant, adding 50mL ice water, and resuspending; centrifuging at 4 deg.C at 1500g for 5min, removing supernatant, adding 4mL of sorbitol (1M), and resuspending; centrifuging at 4 deg.C for 5min at 1500g, removing supernatant, adding 2mL sorbitol (1M), resuspending, and collecting.
1.4, carrying out linearization on the pPIC9-OCLyz1A plasmid to be transformed by using Bgl II restriction enzyme, and electrically transforming to a pichia pastoris GS115 competent cell; uniformly coating the transformed bacteria liquid on an MD solid plate for culture, selecting a positive monoclonal, namely the pichia pastoris GS115 recombinant strain, and placing the recombinant strain in a glycerol tube for freeze-drying and preservation.
2. Fermentation culture
2.1 streaking and activating an expression strain (pichia GS115 recombinant strain) on an MD solid plate, picking a single clone into 50mL YPD liquid culture medium after a colony grows out, and carrying out shake culture at 30 ℃ and 200 rpm.
2.2 the thallus grows to OD 600-1.0, absorb 500 mul bacterial liquid and transfer to 200mL BMGY liquid medium for shake flask culture, 30 ℃ shake bed incubation for 2 days.
2.3 Centrifuging at 5000rpm for 5min, collecting thallus, transferring to 100mL BMMY liquid culture medium, shake-flask culturing at 30 deg.C for 4 days, and adding 500 μ L methanol as inducer every 12 h.
2.4 after the induction is finished, centrifuging at 12000rpm for 10min, and collecting the supernatant of the fermentation liquid.
The formula of the culture medium involved in the steps (20 g/L agar powder is added into the corresponding solid culture medium):
1) YPD liquid medium (1L): 10g of yeast extract, 20g of tryptone and 20g of glucose.
2) MD solid plate (1L): agarose 20g and glucose 20g.
3) BMGY liquid Medium (1L): yeast extract 10g, tryptone 20g and glycerol 10mL, after 15min of sterilization at 121 ℃ was added YNB 100mL and 500 Xbiotin 2mL on a sterile console.
4) BMMY liquid medium (1L): yeast extract 10g and tryptone 20g, sterilized at 121 ℃ for 15min, then 100mL YNB, 500 Xbiotin 2mL and methanol 5mL were added to the sterile console.
Among them, 500 Xbiotin contained 0.02g of biotin in 100 mL.
3. Identification of recombinant proteins in fermentation broths
3.1 Collection of protein samples
The supernatant of the fermentation liquid is a protein sample.
3.2 protein electrophoresis
And (3) identifying the rumen protozoan O.caudatus lysozyme OCLyz1A recombinant protein in the supernatant of the fermentation liquor by SDS-PAGE electrophoresis.
3.2.1 reagents
Glycerol, tetramethylethylenediamine (TEMED), ammonium Persulfate (APS), methanol, glacial acetic acid, ethanol, sodium Dodecyl Sulfate (SDS);
phosphate buffer (0.1 mol/L, pH 6.2): 11.70g of sodium dihydrogen phosphate (NaH) 2 PO 4 ·2H 2 O), 7.86g disodium hydrogen phosphate (Na) 2 HPO 4 ·12H 2 O) and 0.372g disodium ethylenediaminetetraacetate (EDTA-2 Na) were metered into 1000mL sterile water.
Preparation of denatured protein electrophoresis buffer (1 ×): 1.25mol/L glycine, 0.125mol/L Tris and 5g/L SDS.
3.2.2 preparation of the separation gel and the concentrated gel
Gel formulation time was about 2h, concentrated and split gels were formulated according to the following formulation (table 1):
TABLE 1 separation and concentrate formulations
Figure BDA0003490445710000061
Note: SDS, APS and TEMED were added sequentially in succession at the end.
After the separation gel is solidified (about 30 min), 4% concentrated gel is added on the separation gel, and after the separation gel is completely solidified, the separation gel is protein gel, and is taken down and placed at 4 ℃ for standby.
3.2.3 protein sampling
Mixing 40 μ L of fermentation broth supernatant with 10 μ L of 5 xSDS-PAGE Sample Buffer (Kang Run Bio) and boiling for 5min, centrifuging for 1min at 12000rmp, and collecting the above solution as much as possible to obtain a protein Sample-Buffer solution mixture.
3.2.4 electrophoresis
The protein gel was mounted on the electrophoresis tank and 1 × denatured protein electrophoresis buffer was added to the tank.
Loading: to a 1.5X 5mm loading well, 20. Mu.L of the protein sample-buffer mixture was added.
Setting electrophoresis conditions: the gel was run at 80V for 30min, and the voltage was adjusted to 120V after the protein sample was run to the separation gel.
3.2.5 dyeing and decolouring
The protein gel was incubated in Coomassie brilliant blue G-250 stain (Kang Run organisms) for 15-30min. After incubation, the gel is washed twice with water, and the protein gel is placed in a destaining solution for about 3 hours, and the destaining solution is replaced for 2 times.
The formula of the destaining solution is as follows: 10% ethanol and 10% glacial acetic acid.
3.2.6 observations
The bands were observed in a gel imager. The results show that: a clear protein band (the size is about 26.6 KD) with the target molecular weight and almost no other impurity band appears, namely, the rumen protozoan O.caudatus lysozyme OCLyz1A recombinant protein can be very easily obtained through the secretory expression of the Pichia pastoris GS115 recombinant strain, and particularly, the detailed formula is shown in figure 1.
(II) rumen protozoan O.caudatus specific lysozyme OCLyz1A bacteriostatic activity of ruminant
Bacteriostatic tests were performed using LB agar medium with micrococcus luteus (ATCC 4698) and escherichia coli (CVCC 3367).
Sterilizing an LB agar culture medium, respectively mixing Micrococcus luteus and Escherichia coli, pouring the plates, after the culture medium is cooled and solidified, firstly placing an Oxford cup with the diameter of 0.5cm on each plate, respectively adding 200 mu L of fermentation supernatant and yeast (a strain transformed with pPIC 9) culture solution supernatant (negative control) containing empty carriers into each Oxford cup, placing the oxford cups in an incubator at 37 ℃, observing the growth condition of a bacteriostatic ring after 24 hours, and taking a picture.
The LB agar medium formula (1L): 10g of sodium chloride, 5g of yeast extract, 10g of tryptone and 20g of agar powder, and autoclaving at 121 ℃ for 21min.
As can be seen from FIG. 2, the secretion expression product of the Pichia pastoris GS115 recombinant strain constructed by the invention has bacteriostatic activity against Micrococcus luteus, and has an obvious bacteriostatic zone, but has no inhibitory effect on Escherichia coli, and the detail is shown in FIG. 2.
Lysozyme activity was measured for lysozyme (lysozyme, i.e., lysozyme OCLyz 1A) specific to rumen protozoa o.caudatus, according to the requirements of food safety national standard food additive lysozyme (GB 1886.257-2016). One lysozyme activity unit is defined as the amount of lysozyme required to cause a change in absorbance at 450nm per minute to 0.001 using a suspension of Micrococcus luteus at 25 ℃ and pH 6.2. Finally, the enzyme activity of OCLyz1A is 4302U/mL.
<110> northwest university of agriculture and forestry science and technology; beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> ruminant rumen protozoan specific lysozyme OCLyz1A and application thereof
<160> 5
<210> 1
<211> 726
<212> DNA
<213> O.caudatus
<400> 1
gctgatgaat attatactgt tcaatcaggt gattatttag gtaaaattgc aaacagatat 60
ggtgtatcag tatctgattt atgtaattgg aatggtattt ctaaccctga tttaatttat 120
ccaggacaaa gacttcttgt taaaaaatca ggttctagtt caggttcagg ctcaggatca 180
ggttcaggtt caggtaaatc tggaaaaata tattctgtaa cagattcaca aatgcaaaaa 240
atgggttgga aaaattacaa tcttcctgat ttaaatagat gtttaaaaac atttgaaatt 300
aatacagtta atagagtcag acatttcatt tctcaatgct cacatgaatc tgcctgtgga 360
gtttatactc aagaattagg aggtcaaagc tattgctcaa aatatgatgg aagaagagat 420
ttaggaaata cacaacctgg tgatggatgc agatttaaag gggctggata tattcaatta 480
actggaagat ctaattacca aagttttgca aattatattg gtgattcaag agtaatggaa 540
ggtgtttctt atgtttctag caaatatcct tggacttctg ctggttattg gtggaaatct 600
catggtttaa attctttatg tgataatgga gcttctgttg atgtaattac taaaagagta 660
aatggaggat ataatggtct tgctgaaaga aaaaaatatt atcaaagagc ttgtaatatc 720
tttaat 726
<200> 2
<200> 51
<202> DNA
<203> LYZ-F
<400> 2
ggaattccat catcatcatc accatgctga tgaatattac accgttcaga g 51
<200> 3
<200> 36
<202>DNA
<203> LYZ-R
<400> 3
taaagcggcc gcattgaaga tattacatgc acgctg 36
<200> 4
<200> 242
<202> PRT
<203> O.caudatus
<400> 4
Ala Asp Glu Tyr Tyr Thr Val Gln Ser Gly Asp Tyr Leu Gly Lys Ile
1 5 10 15
Ala Asn Arg Tyr Gly Val Ser Val Ser Asp Leu Cys Asn Trp Asn Gly
20 25 30
Ile Ser Asn Pro Asp Leu Ile Tyr Pro Gly Gln Arg Leu Leu Val Lys
35 40 45
Lys Ser Gly Ser Ser Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
50 55 60
Gly Lys Ser Gly Lys Ile Tyr Ser Val Thr Asp Ser Gln Met Gln Lys
65 70 75 80
Met Gly Trp Lys Asn Tyr Asn Leu Pro Asp Leu Asn Arg Cys Leu Lys
85 90 95
Thr Phe Glu Ile Asn Thr Val Asn Arg Val Arg His Phe Ile Ser Gln
100 105 110
Cys Ser His Glu Ser Ala Cys Gly Val Tyr Thr Gln Glu Leu Gly Gly
115 120 125
Gln Ser Tyr Cys Ser Lys Tyr Asp Gly Arg Arg Asp Leu Gly Asn Thr
130 135 140
Gln Pro Gly Asp Gly Cys Arg Phe Lys Gly Ala Gly Tyr Ile Gln Leu
145 150 155 160
Thr Gly Arg Ser Asn Tyr Gln Ser Phe Ala Asn Tyr Ile Gly Asp Ser
165 170 175
Arg Val Met Glu Gly Val Ser Tyr Val Ser Ser Lys Tyr Pro Trp Thr
180 185 190
Ser Ala Gly Tyr Trp Trp Lys Ser His Gly Leu Asn Ser Leu Cys Asp
195 200 205
Asn Gly Ala Ser Val Asp Val Ile Thr Lys Arg Val Asn Gly Gly Tyr
210 215 220
Asn Gly Leu Ala Glu Arg Lys Lys Tyr Tyr Gln Arg Ala Cys Asn Ile
225 230 235 240
Phe Asn
<210> 5
<211> 726
<212> DNA
<213> Artificial sequence
<400> 5
gctgatgaat attacaccgt tcagagtggc gattatctgg gcaaaattgc caatcgttat 60
ggtgttagtg ttagcgatct gtgcaattgg aatggcatta gcaatccgga tctgatctat 120
ccgggtcagc gtctgctggt taaaaaatca ggtagtagca gtggcagtgg tagcggcagt 180
ggcagtggca gcggcaaaag tggtaaaatc tatagtgtga ccgatagcca gatgcagaaa 240
atgggctgga aaaattataa tctgccggat ctgaatcgtt gtctgaaaac ctttgaaatt 300
aacaccgtta atcgcgttcg tcattttatt agtcagtgca gtcatgaaag cgcatgtggt 360
gtgtataccc aggaactggg tggtcagagc tattgtagta aatatgatgg ccgtcgcgat 420
ctgggcaata cccagccggg tgacggttgc cgttttaaag gcgcaggcta tattcagctg 480
accggtcgta gtaattatca gagctttgca aattatatcg gcgatagtcg tgttatggaa 540
ggtgttagtt atgttagcag taaatatccg tggaccagcg caggttattg gtggaaaagt 600
catggcctga atagtctgtg cgataatggc gcaagcgtgg atgttattac caaacgcgtg 660
aatggtggtt ataatggtct ggcagaacgt aaaaaatatt atcagcgtgc atgtaatatc 720
ttcaat 726

Claims (8)

1. A ruminant rumen protozoan specific lysozyme OCLyz1A is characterized in that: the amino acid sequence of the lysozyme OCLyz1A is as follows:
ADEYYTVQSGDYLGKIANRYGVSVSDLCNWNGISNPDLIYPGQRLLVKKSGSSSGSGSGSGSGSGKSGKIYSVTDSQMQKMGWKNYNLPDLNRCLKTFEINTVNRVRHFISQCSHESACGVYTQELGGQSYCSKYDGRRDLGNTQPGDGCRFKGAGYIQLTGRSNYQSFANYIGDSRVMEGVSYVSSKYPWTSAGYWWKSHGLNSLCDNGASVDVITKRVNGGYNGLAERKKYYQRACNIFN。
2. a method for the secretory expression of the ruminant rumen protozoan specific lysozyme OCLyz1A in pichia pastoris, according to claim 1, wherein: the method comprises the following steps:
1) Construction of recombinant Pichia Strain
Constructing a recombinant vector according to the coding sequence of the lysozyme OCLyz1A, and transforming the recombinant vector into pichia pastoris competent cells to obtain a pichia pastoris recombinant strain;
2) Fermentation culture
Carrying out induction expression of recombinant lysozyme OCLyz1A on the pichia pastoris recombinant strain through fermentation to obtain fermentation liquor;
3) Collecting the supernatant of the fermentation liquor to obtain a recombinant lysozyme OCLyz1A protein sample.
3. The method of claim 2, wherein: the sequence of a target gene expressed by the recombinant vector is shown in SEQ.ID.NO. 5.
4. Use of the ruminant rumen protozoan specific lysozyme, OCLyz1A, as defined in claim 1, for the preparation of an antibacterial medicament for inhibiting gram-positive bacteria.
5. Use of the ruminant rumen protozoan specific lysozyme OCLyz1A of claim 1 for the preparation of a medicament for inhibiting micrococcus luteus (micrococcus luteus)Micrococcus luteus) The use in antibacterial agents.
6. Use of the ruminant rumen protozoan specific lysozyme, OCLyz1A, as defined in claim 1, for the preparation of a feed additive.
7. A feed additive, characterized in that: comprising the ruminant rumen protozoan specific lysozyme OCLyz1A of claim 1.
8. A food additive characterized by: comprising the ruminant rumen protozoan specific lysozyme OCLyz1A of claim 1.
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US5041236A (en) * 1989-10-27 1991-08-20 The Procter & Gamble Company Antimicrobial methods and compositions employing certain lysozymes and endoglycosidases
CN111187765A (en) * 2020-02-17 2020-05-22 西北农林科技大学 Ruminant rumen specific lysozyme LYZ1 and application thereof
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CN111187765A (en) * 2020-02-17 2020-05-22 西北农林科技大学 Ruminant rumen specific lysozyme LYZ1 and application thereof
CN113584004A (en) * 2020-12-24 2021-11-02 甘肃农业大学 Application of bovine rumen microbial cellulase eg gene in lactobacillus expression

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Use of Lysozyme as a Feed Additive on In vitro Rumen Fermentation and Methane Emission;Ashraf A. Biswas et al.;《 Asian Australas. J. Anim. Sci.》;20161130;第29卷(第11期);第1601-1607页 *
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