CN117327706A - Composition for improving antiviral and immunity abilities of aquatic products and application thereof - Google Patents
Composition for improving antiviral and immunity abilities of aquatic products and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/178—Lectin superfamily, e.g. selectins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/7056—Lectin superfamily, e.g. CD23, CD72
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a composition for improving antiviral and immunity abilities of aquatic products and application thereof, wherein the composition comprises recombinant shrimp C-type lectin PcLT and astragalus membranaceus, and the nucleotide sequence of the recombinant shrimp C-type lectin PcLT is shown as SEQ ID No. 1. The nucleotide sequence of the main antigen structural functional region of the prawn C-type lectin PcLT is optimized, the optimized PcLT nucleotide sequence is recombined and expressed in escherichia coli, the expression efficiency of the PcLT is obviously improved, the expression efficiency of the protein after optimization is 3.2 times that of the protein after optimization, bacterial mud for expressing the protein and traditional Chinese medicine astragalus membranaceus are compatible according to a proper proportion by volume, and the prawn C-type lectin PcLT has sweet taste and mild property, and provides an important basis for resisting invasion of pathogenic microorganisms and enhancing nonspecific immunity of the prawns.
Description
Technical Field
The invention belongs to the field of genetic engineering and traditional Chinese medicine compatibility, and relates to a composition for improving antiviral and immunity abilities of aquatic products and application thereof.
Background
Shrimp belongs to crustaceans, which lack as strong an adaptive immune system as vertebrates, and rely mainly on innate immunity to combat the invasion of pathogenic microorganisms. Innate immunity, also called nonspecific immunity, is an immune response that is innate and not specific to a particular antigenic substance, has stability, and can be inherited to offspring, and mainly exhibits three functions: (1) The immune barrier includes skin mucosa barrier, blood brain barrier and placenta barrier. (2) Phagocytosis, including macrophages, monocytes, neutrophils, etc., is a powerful phagocytosis. The astragalus has the functions of stimulating macrophages to play phagocytic functions, enhancing the immunity of organisms, resisting viruses and bacteria, mainly contains various effective components such as astragaloside IV, astragalus polysaccharide, alkaloid and the like, particularly the astragalus polysaccharide belongs to an interferon inducer, can inhibit the activity of viruses, has the immunoregulatory activity, is beneficial to enhancing humoral immunity and nonspecific immunity, and further helps to avoid being infected by the viruses. (3) Body fluids, including blood, various secretions and interstitial fluids, contain killing substances such as complement, lysozyme, interferon, and the like. The shrimp C-type lectin PcLT is taken as an important humoral immunity factor, can participate in the recognition of pathogen related molecular modes, can regulate various immune responses, achieves the capability of resisting various diseases by improving the immunity of a host, and has broad-spectrum disease resistance. PcLT can help organism to clear bacteria rapidly, resist virus infection, and protect common attack of vibrio, white spot virus, etc. in aquaculture.
In summary, the existing shrimp C-type lectin PcLT has the problems of low protein expression quantity, weak antiviral and antibacterial abilities, and the like. How to provide a recombinant shrimp C-type lectin PcLT, greatly improving the protein expression efficiency, improving the antiviral and antibacterial capacities of shrimps, and making up the specific immunodeficiency of crustaceans, and is one of the problems to be solved in the technical field of genetic engineering at present.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a composition for improving the antiviral and immune abilities of aquatic products and application thereof, and the composition effectively improves the expression efficiency of the PcLT by optimizing the gene sequence of the PcLT and utilizing recombinant plasmids to express in an escherichia coli expression system, and combines the high-expression PcLT with traditional Chinese medicine astragalus membranaceus, thereby effectively improving the antiviral and antibacterial abilities of the shrimps and overcoming the defects of specific immunodeficiency and nonspecific immunodeficiency.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides recombinant shrimp C-type lectin PcLT, wherein the recombinant shrimp C-type lectin PcLT is obtained by gene optimization on a sequence shown in SEQ ID No.1, and the nucleotide sequence of the recombinant shrimp C-type lectin PcLT comprises a sequence shown in SEQ ID No. 2.
The invention utilizes bioinformatics means to determine the gene sequence information of the main structural functional region of the shrimp C-type lectin PcLT and optimize the sequence, and provides a preparation method of the shrimp C-type lectin PcLT, which expresses in an escherichia coli expression system by means of optimized recombinant plasmid, is simple and easy to operate and has high expression efficiency.
SEQ ID No.1：
ATGAATGTGGAAGCTTTTCTTCTGTTGTTAGGTTCCGTCTTGGTCTATGGCGATTCTCCAATAAACAAGGCCAGATATCCTGCTGCGGATATAACCTGCCCTTTGCCGTACAAAGCAGTTGGAGGTCGCTGCATTCTGTTCGCGGTGGCCGAGGCTGGCGCCTGGCACGACATGGGATACTTCTGCGAGACACTCGGGGGCAAACTGGTCGTTATTGACGACTCCACCTTCATGGAGGCTCTTACGGACTA CATCTTGGATGGTGAGTTCTCAACCACGGAATTCTGGATTGGAGCGACGGACGAGGTGACGGAAGGGGTGTGGAAGTGGGTCAATGGGAAGACGGTGAGGATGCGCATGCCTCTCTGGCTCACGTGTTCGGACGACCAGGAACCCAACGGTGGCAGCGCCGAGAACTGTCTGGCCATCACGAGCGACCATAACTACTACTTCGTCGACGCCGATTGCGCTCTAGCAATGAATCCCATTTGTGAATACACAGTACTTTAG。
SEQ ID No.2：
ATGAACGTTGAAGCTTTCCTGCTGCTGCTGGGTTCTGTTCTGGTTTACGGTGACTCTCCGATCAACAAAGCTCGTTACCCGGCTGCTGACATCACCTGCCCGCTGCCGTACAAAGCTGTTGGTGGTCGTTGCATCCTGTTCGCTGTTGCTGAAGCTGGTGCTTGGCACGACATGGGTTACTTCTGCGAAACCCTGGGTGGTAAACTGGTTGTTATCGACGACTCTACCTTCATGGAAGCTCTGACCGACTACATCCTGGACGGTGAATTCTCTACCACCGAATTCTGGATCGGTGCTACCGACGAAGTTACCGAAGGTGTTTGGAAATGGGTTAACGGTAAAACCGTTCGTATGCGTATGCCGCTGTGGCTGACCTGCTCTGACGACCAGGAACCGAACGGTGGTTCTGCTGAAAACTGCCTGGCTATCACCTCTGACCACAACTACTACTTCGTTGACGCTGACTGCGCTCTGGCTATGAACCCGATCTGCGAATACACCGTTCTGTAA。
Preferably, the method of gene optimization comprises the steps of:
(1) Determining the gene sequence of the main structural functional region of the shrimp C-type lectin PcLT by using a bioinformatics means;
(2) Adjusting the codon usage preference to increase the codon adaptation index to 0.9-0.96;
(3) Removing repeated areas in the original sequence and removing bad peaks;
(4) Modification of the restriction sites and negative cis-acting sites for subcloning;
(5) The whole sequence is finely adjusted, so that the translation efficiency is improved, and the half life of mRNA is prolonged.
The dot values of 0.9 and 0.96 are specifically selected from 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, and the like.
In a second aspect, the present invention provides a recombinant vector for expressing recombinant shrimp C-type lectin PcLT, said recombinant vector comprising the nucleotide sequence of recombinant shrimp C-type lectin PcLT according to the first aspect.
Preferably, the recombinant vector further comprises a strong promoter, a tag protein, a multiple cloning site, a restriction enzyme site and an ampicillin resistance sequence.
Preferably, the recombinant vector comprises recombinant E.coli expression vector pET32a.
Preferably, the nucleotide sequence of the recombinant vector is shown as SEQ ID No. 3.
SEQ ID No.3：
atccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatctcagtggtggtggtggtggtgctcgagtgcggccgcaagcttgtcgacggagctcgaattcggatccgatatcagccatggccttgtcgtcgtcgtcggtacccagatctgggctgtccatgtgctggcgttcgaatttagcagcagcggtttctttcataccagaaccgcgtggcaccagaccagaagaatgatgatgatgatggtgcatatggccagaaccagaaccggccaggttagcgtcgaggaactctttcaactgacctttagacagtgcacccactttggttgccgccacttcaccgtttttgaacagcagcagagtcgggataccacggatgccatatttcggcgcagtgccagggttttgatcgatgttcagttttgcaacggtcagtttgccctgatattcgtcagcgatttcatccagaatcggggcgatcattttgcacggaccgcaccactctgcccagaaatcgacgaggatcgccccgtccgctttgagtacatccgtgtcaaaactgtcgtcagtcaggtgaataattttatcgctcatatgtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatcgatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgagatcccggacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggacatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtaagttagctcactcattaggcaccgggatctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgcca。
Preferably, the construction method of the recombinant vector comprises the following steps:
(1) Double enzyme digestion is carried out on the gene fragment of the optimized PcLT sequence and the pET32a plasmid, and after the enzyme digestion products are separated by agarose gel electrophoresis, the target strip and the carrier strip are recovered by gel digestion;
(2) And (3) connecting by using T4 ligase, transforming into competent cells, and screening by using ampicillin to obtain a recombinant plasmid pET32a-PcLT.
In a third aspect, the present invention provides a method for producing recombinant shrimp C-type lectin PcLT, said method comprising: expressing the recombinant vector of the second aspect in escherichia coli to obtain recombinant PcLT, centrifuging the escherichia coli, filtering bacterial sludge by a stainless steel screen, and homogenizing under high pressure.
Preferably, the rotational speed of the centrifugation is 500-2000rpm for 5-20min.
Specific values of 500-2000rpm may be selected from 500rpm, 600rpm, 800rpm, 1000rpm, 1200rpm, 1400rpm, 1600rpm, 1800rpm, 2000rpm.
The specific point value of the above 5-20min can be selected from 5min, 6min, 8min, 10min, 12min, 14min, 16min, 18min, and 20min.
Preferably, the E.coli comprises E.coli BL21 (DE 3).
Preferably, the stainless steel screen has a pore size of 60-100 mesh.
The specific point values in the 60-100 mesh can be 60 mesh, 70 mesh, 75 mesh, 80 mesh, 85 mesh, 90 mesh, 95 mesh, 100 mesh, etc.
Preferably, the preparation method comprises the following steps:
(1) Transferring the recombinant vector of claim 3 or 4 into competent cells of escherichia coli by a chemical conversion method, coating the escherichia coli with the recombinant plasmid on an LB plate containing 0.5-2 mug/mL of ampicillin resistance after the conversion is finished, and culturing at a constant temperature of 35-37 ℃;
(2) Picking single colony grown on a flat plate, pure culturing, extracting plasmids, and sequencing and identifying the plasmids to obtain recombinant bacteria;
(3) Shaking and culturing the recombinant strain in LB liquid medium containing 0.5-2 mug/mL of ampicillin at 35-37 ℃ for overnight;
(4) Inoculating the cultured strain solution to LB medium to make OD 600 The initial concentration is 0.1-0.3, and the culture is carried out at constant temperature of 35-37 ℃ for 0.5-3h by shaking until OD 600 Adding IPTG inducer at 0.5-0.7, and adding IPTG to final concentration of 0.5-2mM;
(5) After the induction is carried out overnight, the fermentation is stopped, and the recombinant shrimp C-type lectin PcLT is obtained by centrifugally collecting thalli.
Specific spot values among the above 0.5-2. Mu.g/mL may be selected from 0.5. Mu.g/mL, 0.6. Mu.g/mL, 0.8. Mu.g/mL, 1.0. Mu.g/mL, 1.2. Mu.g/mL, 1.4. Mu.g/mL, 1.6. Mu.g/mL, 1.8. Mu.g/mL, 2. Mu.g/mL, etc.
Specific point values of 0.1 to 0.3 may be selected from 0.1, 0.2, 0.3, etc.
The specific values of 35-37deg.C can be 35 deg.C, 36 deg.C, 37 deg.C, etc.
Specific point values in the above 0.5-3h may be selected from 0.5h, 0.6h, 0.8h, 1.2h, 1.4h, 1.6h, 2.8h, 3h, etc.
Specific values among the above 0.5 to 2mM may be selected from 0.5mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM, 2mM, etc.
Preferably, the chemical conversion method of step (1) includes the steps of:
(1) Uniformly mixing competent cells with the recombinant vector, and standing on ice for 15-60min;
(2) Heat shock in water bath at 40-50deg.C for 30-60s, rapidly placing back into ice, and standing for 2-10min;
(3) Mixing with sterile LB liquid medium without antibiotics, homogenizing, shake culturing at 35-37deg.C for 15-60min.
The specific point value of 15-60min can be selected from 15min, 16min, 18min, 20min, 30min, 40min, 50min, 55min, and 60min.
The specific point value of the above 2-10min can be selected from 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, and 10min.
In a fourth aspect, the present invention provides an antiviral composition for aquatic products comprising a bacterial mud comprising recombinant shrimp C-type lectin PcLT of the first aspect and astragalus root; the volume ratio of the bacterial mud to the astragalus is 1 (2-6), and the aquatic product comprises shrimps.
The specific point values in the above 2-6 can be selected from 2, 3, 4, 5 and 6.
In a fifth aspect, the invention provides the use of the recombinant shrimp C-type lectin PcLT of the first aspect, the recombinant vector of the second aspect or the composition for shrimp antiviral of the fourth aspect in the preparation of a medicament for treating shrimp immune diseases.
Compared with the prior art, the invention has the following beneficial effects:
(1) The nucleotide sequence of the main antigen structural functional region of the prawn C-type lectin PcLT is optimized, the optimized PcLT nucleotide sequence is recombined and expressed in escherichia coli, the expression efficiency of the PcLT is obviously improved, and the optimized protein expression efficiency is 3.2 times of that of the protein which is not optimized;
(2) According to the invention, the high-expression PcLT is compatible with the traditional Chinese medicine astragalus membranaceus, so that the antiviral and antibacterial capacities of the shrimps are effectively improved, the defects of specific immunodeficiency and nonspecific immunodeficiency of crustaceans are overcome, and the phagocytosis and humoral effect in the nonspecific immunity of the shrimps are enhanced.
Drawings
FIG. 1 is a diagram showing the dual cleavage assay of pET32a-PcLT plasmids;
FIG. 2 is a graph showing comparison of protein expression before and after optimization.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
The gene sequence of shrimp C-type lectin PcLT was determined.
(1) The nucleotide sequence and the coded amino acid sequence are searched in NCBI, the nucleotide sequence of the shrimp C-type lectin PcLT used for synthesis and construction is obtained by screening, and the full-length sequence, the open reading frame and the antigen structural functional region of the shrimp C-type lectin PcLT gene are determined by consulting the literature;
(2) Adjusting the codon usage preference to meet the highest expression profile of the target host, increasing the codon adaptation index from 0.5 to 0.96 (CAI values 0.8-1.0 are considered good high expression);
(3) Removing the repeated region in the original sequence, removing the bad peak and avoiding the stem-loop structure in mRNA;
(4) Modification of the restriction sites and negative cis-acting sites for subcloning;
(5) And fine tuning the whole sequence, improving translation efficiency, prolonging the half life of mRNA, obtaining an optimized shrimp C-type lectin PcLT gene sequence, and sending the sequence to a biological engineering (Shanghai) stock company for synthesis, wherein the sequence is shown as SEQ ID No. 2.
Example 2
Construction of recombinant vector and expression of target protein.
(1) The synthetic PcLT gene sequence (shown as SEQ ID NO. 2) and pET32a plasmid (shown as SEQ ID NO. 3) are subjected to BamHI and Xho I double enzyme digestion, enzyme digestion products are separated by agarose gel electrophoresis, target bands and carrier bands are recovered by gel digestion, and are connected by T4 ligase, transformed into BL21 (DE 3) competent cells, and screened by using 1 mug/mL ampicillin to obtain a new plasmid pET32a-PcLT;
(2) Picking single colony growing on a flat plate, pure culturing, extracting plasmids, carrying out double enzyme digestion identification on the plasmids, and sending the plasmids with correct identification to Shanghai workers for sequencing identification to obtain recombinant bacteria;
(3) Shaking culture of recombinant bacteria in LB liquid medium containing 1 mug/mL of ampicillin at 37 ℃ overnight;
(4) Inoculating the cultured strain overnight into LB medium containing 1 μg/mL of ampicillin to make OD 600 The initial concentration is 0.2, and the culture is carried out for about 2 hours at the constant temperature of 37 ℃ and the OD is reached 600 Adding IPTG inducer when the concentration reaches about 0.6, wherein the final concentration of the added IPTG is 1mM;
(5) Stopping fermentation after induction overnight, respectively taking bacterial liquid before induction and bacterial liquid after induction, concentrating bacterial liquid after quantification, adding 5 xSDS (sodium dodecyl sulfate) loading buffer solution to ensure that the final concentration of the buffer solution is 1 xSDS, putting into boiling water, boiling for 10min, and detecting protein expression condition by SDS-PAGE gel electrophoresis, wherein the protein expression rate after optimization is higher and is 3.2 times that of non-optimized protein.
Example 3
Centrifuging recombinant bacteria of the shrimp lectin PcLT expressed in the example 2, filtering the obtained bacterial sludge by using a 80-mesh stainless steel screen, homogenizing the obtained bacterial sludge by using a sterile high-pressure homogenizer, and mixing the homogenized protein solution with astragalus solution according to the ratio of 1:4, mixing the materials according to the volume ratio, and sub-packaging the materials to 250 mL/bottle for aquatic animal experiments.
Example 4
Centrifuging recombinant bacteria of the shrimp lectin PcLT expressed in the example 2, filtering the obtained bacterial sludge by using a 80-mesh stainless steel screen, homogenizing the obtained bacterial sludge by using a sterile high-pressure homogenizer, and mixing the homogenized protein solution with astragalus solution according to the ratio of 1:2, mixing the materials according to the volume ratio, and sub-packaging the materials to 250 mL/bottle for aquatic animal experiments.
Example 5
Centrifuging recombinant bacteria of the shrimp lectin PcLT expressed in the example 2, filtering the obtained bacterial sludge by using a 80-mesh stainless steel screen, homogenizing the obtained bacterial sludge by using a sterile high-pressure homogenizer, and mixing the homogenized protein solution with astragalus solution according to the ratio of 1:6, mixing the materials according to the volume ratio, and sub-packaging the materials to 250 mL/bottle for aquatic animal experiments.
Example 6
Centrifuging recombinant bacteria of the shrimp lectin PcLT expressed in the example 2, filtering the obtained bacterial sludge by using a 80-mesh stainless steel screen, homogenizing the obtained bacterial sludge by using a sterile high-pressure homogenizer, and mixing the homogenized protein solution with astragalus solution according to the ratio of 1: mixing at a volume ratio of 1, and sub-packaging to 250 mL/bottle for aquatic animal experiment.
Example 7
Centrifuging recombinant bacteria of the shrimp lectin PcLT expressed in the example 2, filtering the obtained bacterial sludge by using a 80-mesh stainless steel screen, homogenizing the obtained bacterial sludge by using a sterile high-pressure homogenizer, and mixing the homogenized protein solution with astragalus solution according to the ratio of 1:8, mixing the materials according to the volume ratio, and sub-packaging the materials to 250 mL/bottle for aquatic animal experiments.
Test example 1
And (5) testing viral diseases of the prawns.
The recombinant shrimp lectin PcLT and astragalus root composition product is used for the shrimp pond of Guangxi farmers with viral erythrozoonosis, the disease is obviously improved, and the specific use condition and the result are shown in the following table 1.
TABLE 1
In conclusion, the nucleotide sequence of the main antigen structural functional region of the prawn C-type lectin PcLT is optimized, the optimized PcLT nucleotide sequence is recombined and expressed in escherichia coli, the expression efficiency of the PcLT is obviously improved, the protein expression efficiency after optimization is 3.2 times that of the protein expression efficiency after optimization, bacterial mud for expressing the protein and traditional Chinese medicine astragalus membranaceus are compatible according to a proper proportion by volume, and the prawn C-type lectin PcLT has sweet taste and mild property, and provides an important basis for resisting invasion of pathogenic microorganisms and enhancing nonspecific immunity of the prawns.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. The recombinant shrimp C-type lectin PcLT is characterized in that the recombinant shrimp C-type lectin PcLT is obtained by gene optimization on a sequence shown in SEQ ID No.1, and the nucleotide sequence of the recombinant shrimp C-type lectin PcLT comprises a sequence shown in SEQ ID No. 2.
2. The recombinant shrimp C-type lectin PcLT of claim 1, wherein the method of gene optimization comprises the steps of:
(1) Determining the gene sequence of the main structural functional region of the shrimp C-type lectin PcLT by using a bioinformatics means;
(2) Adjusting the codon usage preference to increase the codon adaptation index to 0.9-0.96;
(3) Removing repeated areas in the original sequence and removing bad peaks;
(4) Modification of the restriction sites and negative cis-acting sites for subcloning;
(5) The whole sequence is finely adjusted, so that the translation efficiency is improved, and the half life of mRNA is prolonged.
3. A recombinant vector for expressing recombinant shrimp C-type lectin PcLT, characterized in that the recombinant vector comprises the nucleotide sequence of recombinant shrimp C-type lectin PcLT of claim 1;
preferably, the recombinant vector further comprises a strong promoter, a tag protein, a multiple cloning site, a restriction enzyme site and an ampicillin resistance sequence;
preferably, the recombinant vector comprises a recombinant escherichia coli expression vector pET32a;
preferably, the nucleotide sequence of the recombinant vector is shown as SEQ ID No. 3.
4. The recombinant vector according to claim 3, wherein the construction method of the recombinant vector comprises the steps of:
(1) Double enzyme digestion is carried out on the gene fragment of the optimized PcLT sequence and the pET32a plasmid, and after the enzyme digestion products are separated by agarose gel electrophoresis, the target strip and the carrier strip are recovered by gel digestion;
(2) And (3) connecting by using T4 ligase, transforming into competent cells, and screening by using ampicillin to obtain a recombinant plasmid pET32a-PcLT.
5. A method for preparing recombinant shrimp C-type lectin PcLT, the method comprising: expressing the recombinant vector of claim 3 or 4 in escherichia coli to obtain recombinant PcLT, centrifuging the escherichia coli, filtering bacterial sludge by a stainless steel screen, and homogenizing under high pressure.
6. The method according to claim 5, wherein the rotational speed of the centrifugation is 500-2000rpm for 5-20min;
preferably, the escherichia coli comprises escherichia coli BL21 (DE 3);
preferably, the stainless steel screen has a pore size of 60-100 mesh.
7. The preparation method according to claim 5 or 6, characterized in that the preparation method comprises the steps of:
(1) Transferring the recombinant vector of claim 3 or 4 into competent cells of escherichia coli by a chemical conversion method, coating the escherichia coli with the recombinant plasmid on an LB plate containing 0.5-2 mug/mL of ampicillin resistance after the conversion is finished, and culturing at a constant temperature of 35-37 ℃;
(2) Picking single colony grown on a flat plate, pure culturing, extracting plasmids, and sequencing and identifying the plasmids to obtain recombinant bacteria;
(3) Shaking and culturing the recombinant strain in LB liquid medium containing 0.5-2 mug/mL of ampicillin at 35-37 ℃ for overnight;
(4) Inoculating the cultured strain solution to LB medium to make OD 600 The initial concentration is 0.1-0.3, and the culture is carried out at constant temperature of 35-37 ℃ for 0.5-3h by shaking until OD 600 Adding IPTG inducer at 0.5-0.7, and adding IPTG to final concentration of 0.5-2mM;
(5) After the induction is carried out overnight, the fermentation is stopped, and the recombinant shrimp C-type lectin PcLT is obtained by centrifugally collecting thalli.
8. The method of claim 7, wherein the chemical conversion process of step (1) comprises the steps of:
(1) Uniformly mixing competent cells with the recombinant vector, and standing on ice for 15-60min;
(2) Heat shock in water bath at 40-50deg.C for 30-60s, rapidly placing back into ice, and standing for 2-10min;
(3) Mixing with sterile LB liquid medium without antibiotics, homogenizing, shake culturing at 35-37deg.C for 15-60min.
9. An antiviral composition for aquatic products, characterized in that it comprises a bacterial sludge containing recombinant shrimp C-type lectin PcLT according to claim 1 or 2 and astragalus root; the volume ratio of the bacterial mud to the astragalus root is 1 (2-6); the aquatic product comprises shrimp.
10. Use of the recombinant shrimp C-type lectin PcLT of claim 1 or 2, the recombinant vector of claim 3 or 4 or the composition for shrimp antiviral of claim 9 for the preparation of a medicament for the treatment of shrimp immune diseases.
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