CN114507682A - Gene for coding recombinant NlpC/P60 endopeptidase protein and application thereof - Google Patents
Gene for coding recombinant NlpC/P60 endopeptidase protein and application thereof Download PDFInfo
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- CN114507682A CN114507682A CN202210258133.3A CN202210258133A CN114507682A CN 114507682 A CN114507682 A CN 114507682A CN 202210258133 A CN202210258133 A CN 202210258133A CN 114507682 A CN114507682 A CN 114507682A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biology, and particularly relates to a gene for coding a recombinant NlpC/P60 endopeptidase protein and application thereof. The invention carries out codon optimization on NlpC/P60 endopeptidase gene by a genetic engineering method, then constructs a recombinant expression vector and a prokaryotic expression system, induces expression of recombinant fusion protein, obtains the recombinant NlpC/P60 endopeptidase protein for resisting escherichia coli, can achieve the killing effect of 2 orders of magnitude on escherichia coli BL21(DE3), provides a new method for resisting gram-negative bacteria such as escherichia coli, can solve the drug resistance problem of the gram-negative bacteria such as the escherichia coli, and has great significance for preventing and treating poultry colibacillosis and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a gene for coding a recombinant NlpC/P60 endopeptidase protein and application thereof.
Background
Escherichia coli (e.coli) is a diverse bacterial population that is a normal component of poultry micro-ecology. Coli is present throughout the gut, upper respiratory tract and on the skin and feathers of healthy birds. Although most strains of E.coli are not harmful to avian health, some strains are capable of causing disease parenterally. Those capable of causing avian diseases, or diseases when the host defense system is compromised, are known as Avian Pathogenic E.coli (APEC). APEC is a major cause of morbidity and mortality in poultry industry worldwide, and disease syndromes associated with APEC include hemorrhagic septicemia, air sacculitis, swollen head syndrome, hyperemia, enteritis, escherichia coli cellulitis, peritonitis, salpingitis, orchitis, osteomyelitis/coleitis (including turkey osteomyelitis syndrome), generalized ophthalmia and oophoritis. Coli can be the primary infectious pathogen, and can also cause secondary infections with other viruses or bacteria.
The prevention and control of colibacillosis is a great problem in the breeding industry, because colibacillosis is a part of normal intestinal flora of poultry, methods based on management strategies have limited success rate. The serious consequences of antibiotic abuse and the emergence of resistant strains, scientists have conducted many studies from the standpoint of reducing the source of infection, for example: the antagonistic effect of prebiotics, probiotics, bacteriophages, fatty acids, bacteriocins, antibiotic adjuvants, vaccines, etc. on escherichia coli is expected to advance new measures to reduce losses in the breeding industry while maintaining public health.
The NlpC/P60 endopeptidase is a highly evolved peptidoglycan hydrolase encoded by the bacterium itself and is a protein molecule in nature. Peptidases containing NlpC/P60 are involved in the catalysis of N-acetylurea-L-alanine or D-glutamyl-methanediamine phosphate linkages, and four major groups have been identified to date. (i) P60-like, (ii) AcmB/LytN-like, (iii) YaeF/poxvirus G6R, (iv) Lecithin Retinol Acylase (LRAT) -like. The NlpC/P60 structural domain is similar to papain, can exist alone or combine with other structural domains, and the catalytic function of the NlpC/P60 structural domain is changed along with the structural change, so that functionally diversified proteins can be formed. For example, the SH3 domain in the NlpC/P60 endopeptidase domain participates in the process of anchoring cell walls by hyaluronidase, regulates the concentration and anchoring position of hyaluronic acid, and forms an effective enzyme-substrate complex to play a role. Peptidoglycan (PG), one of the NlpC/P60 endopeptidases, is a major component of bacterial cell walls and is essential for maintaining structural integrity and internal osmotic pressure, shaping bacterial morphology, but is also highly dynamic, with glycosidic and amide bonds broken down or formed by different enzymes. The degradation products resulting from the catalytic activity of the NlpC/P60 endopeptidase can be recovered for re-biosynthesis of peptidoglycans, and can also act as signaling molecules, triggering the re-growth of antibiotic-resistant or dormant cells, or as effector molecules in an immune response. The NlpC/P60 endopeptidase can also be secreted into the environment or enter the periplasm of other bacteria through a VI type secretion system to influence the survival of the bacteria, so that the bacteria secreting the NlpC/P60 endopeptidase have competitive advantages.
From the present research, NlpC/P60 endopeptidase has several advantages as a novel antibacterial agent: (1) has good antibacterial effect on gram-positive bacteria in vivo or in vitro; (2) the lytic activity to bacteria is high, and the problem of antibiotic resistance of the bacteria is not considered; (3) the possibility of resistance to the bacteria is low; (4) the safety is high, and the strong immunity antigenicity can not be caused; (5) it is easy to be transformed by genetic engineering so as to achieve better sterilization effect.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention provides a gene encoding a recombinant NlpC/P60 endopeptidase protein, which encodes a recombinant NlpC/P60 endopeptidase protein having good biological activity and stability.
Another object of the present invention is to provide the use of the above gene encoding a recombinant NlpC/P60 endopeptidase protein.
It is still another object of the present invention to provide a recombinant expression vector comprising the above gene encoding the recombinant NlpC/P60 endopeptidase protein.
The fourth purpose of the invention is to provide a recombinant expression strain, which comprises the recombinant expression vector.
The fifth purpose of the invention is to provide a preparation method of the recombinant NlpC/P60 endopeptidase protein.
The purpose of the invention is realized by the following technical scheme:
a gene encoding a recombinant NlpC/P60 endopeptidase protein, which has the nucleotide sequence shown below:
ATGTGCTCAACAAAATCTATTGATCCTTACGGTGAGCAGAATAGTTATGATAATATTGGGTCAAAATACCAGAAAAATGATACGACCTCAGGTTACAATTTCAATTATAACCAGGCGGCTGACGAATTCTACAATATTAATCTGAGCAAGTACCTTAATAAGAAACTCGGGAACGATTGCTCGGGGTTTGTAAGCTTAGTTAACGAAGACTCCAAGAGTCTGTATTTCGATGAGAACATTGTCAATAATTTTTACGACAAGAACGGTCGCAAGTCCCAAGCTATCTTCAACCTTTATAAGTCCCAAAACAAAATTAGCTATACCGACCCTAAACCGGGCGATTTAGTATTTTTTAACAATACCACAAGCCGGACGAACAAATCTAAAAATAAGGCAATTACTCACCTCGGTATTATCGACAAAGTTGAGTCTAATGGTACTATCACGTTCGTTCACAACATTAACGGGAAGAATATCAAAAGCGTAATGAACTTGAAGCACAAGAACACACATAAATTAAATGGCAAGAAAATCAACGCTTATATTATTCTCAAGTGTCAAACGCCTGTATGTTTGATTTCCAACAAATTCGCTGGCTTTGGGAAAGTGGACAAATTTGTGCAAATTGATGGTTCGCATCACCACCACCACCATTAA
the gene for coding the recombinant NlpC/P60 endopeptidase protein is applied to preparing an antibacterial product;
the antibacterial product is preferably used for resisting gram-negative bacteria;
the gram-negative bacteria resistance is preferably anti-Escherichia coli;
a recombinant expression vector, which is an expression vector containing the gene for coding the recombinant NlpC/P60 endopeptidase protein and is obtained by cloning the gene for coding the recombinant NlpC/P60 endopeptidase protein into the expression vector;
the expression vector is preferably pET-28a (+);
the gene for coding the recombinant NlpC/P60 endopeptidase protein is preferably inserted into pET-28a (+) through enzyme cutting sites SacI and BamHI;
a recombinant expression strain, which is a host strain containing the recombinant expression vector and is obtained by transferring the recombinant expression vector into the host strain;
the host strain is bacteria, yeast or fungi;
the host strain is preferably a bacterium, more preferably Escherichia coli (Escherichia coli), and most preferably Escherichia coli BL21(DE 3);
a method for preparing a recombinant NlpC/P60 endopeptidase protein, comprising the steps of:
(1) cloning the gene of the coded recombinant NlpC/P60 endopeptidase protein into an expression vector to obtain a recombinant expression vector;
(2) transferring the recombinant expression vector into a host strain to obtain a recombinant expression strain;
(3) inducing and expressing the recombinant expression strain, collecting bacterial liquid, crushing, separating and purifying to obtain recombinant NlpC/P60 endopeptidase protein;
the expression vector in the step (1) is pET-28a (+);
the host strain in the step (2) is bacteria, yeast or fungi;
the host strain described in step (2) is preferably a bacterium, more preferably Escherichia coli (Escherichia coli), and most preferably Escherichia coli BL21(DE 3);
the inducer for inducing expression in the step (3) is preferably IPTG;
the dosage of the IPTG is preferably 0.1mmol/L of final concentration;
the condition for inducing the expression in the step (3) is preferably inducing for 4 hours at 37 ℃;
preferably, the recombinant expression strain in the step (3) is cultured to a bacterial liquid OD600Adding an inducer for induction expression when the expression is 0.6-0.8;
the recombinant expression strain in the step (3) is preferably cultured to a bacterial liquid OD600When the expression level is 0.8, adding an inducer for induction expression;
the separation and purification method in the step (3) is preferably a nickel column affinity chromatography method;
compared with the prior art, the invention has the following advantages and effects:
(1) the invention carries out codon optimization on NlpC/P60 endopeptidase gene by a genetic engineering method, then constructs a recombinant expression vector and a prokaryotic expression system, induces expression of recombinant fusion protein, obtains the recombinant NlpC/P60 endopeptidase protein for resisting escherichia coli, can achieve the killing effect of 2 orders of magnitude on escherichia coli BL21(DE3), provides a new method for resisting gram-negative bacteria such as escherichia coli, can solve the drug resistance problem of the gram-negative bacteria such as the escherichia coli, and has great significance for preventing and treating poultry colibacillosis and the like.
(2) The production method of the recombinant NlpC/P60 endopeptidase protein provided by the invention is a prokaryotic expression method, is simple to operate and low in cost, and can realize large-scale production.
(3) The recombinant NlpC/P60 endopeptidase provided by the invention has high protein purity, good stability and biological activity, and great significance for controlling colibacillosis in poultry farms and reducing the probability of spreading the colibacillosis to human beings through a food production chain.
Drawings
FIG. 1 is a map of recombinant expression vector NlpC/P60 in pET-28a (+).
FIG. 2 is a graph showing the results of PCR identification of colonies in example 2, wherein lane 1: yeasen 2000Marker, lane 2: amplified fragment of the target gene, lane 3: sterile dd water-negative control.
FIG. 3 is a graph showing the results of protein purification by SDS-PAGE, wherein lane 1: SMOBIO PM2510 Marker, lane 2: the inducer IPTG is 0.1mmol/L, and the protein is purified at 37 ℃ for 4 h.
Fig. 4 is a graph showing the bactericidal effect of recombinant NlpC/P60 endopeptidase protein at pH 6, with the ordinate indicating the viable cell count in the 4 th well fluid.
FIG. 5 is a graph showing the effect of the recombinant NlpC/P60 endopeptidase protein in example 4 on the killing of E.coli strain BL21(DE3), A: blank control group to which PBS was added dropwise, B: the experimental group of recombinant NlpC/P60 endopeptidase protein was added dropwise.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Imidazole-free protein lysate Lysis buffer: NaH2PO4·H26.9g of O (MW 137.99g/mol) and 17.54g of NaCl (MW 58.44g/mol), adding about 900mL of deionized water, stirring and dissolving, adding NaOH to adjust the pH value of the solution to 8.0, and adding deionized water to reach the constant volume of 1000 mL;
lysis buffer: NaH2PO4·H26.9g of O (MW 137.99g/mol), 17.54g of NaCl (MW 58.44g/mol) and 0.68g of imidazole (MW 68.08g/mol) are added into about 900mL of deionized water, the mixture is stirred and dissolved, NaOH is added to adjust the pH value of the solution to 8.0, and the deionized water is added to the solution to reach the volume of 1000 mL;
elution buffer: NaH2PO4·H26.9g of O (MW 137.99g/mol), 17.54g of NaCl (MW 58.44g/mol) and 17.00g of imidazole (MW 68.08g/mol) are added into about 900mL of deionized water, the mixture is stirred and dissolved, NaOH is added to adjust the pH value of the solution to 8.0, and the deionized water is added to the solution to reach the volume of 1000 mL;
0.01mol PBS(pH＝7.4)：NaCl(MW 58.44g/mol)8g、KCl(MW l74.55g/mol)0.2g、Na2HPO4(MW 141.96g/mol)1.44g and KH2PO4(MW 136.09g/mol)0.24g dissolved in 800ml distilled water and the pH of the solution adjusted to 7.4 with HCl, maximumThen adding distilled water to a constant volume of 1L;
0.01mol PBS(pH＝6)：NaCl(MW 58.44g/mol)8g、KCl(MW l74.55g/mol)0.2g、Na2HPO4(MW 141.96g/mol)1.44g and KH2PO4(MW 136.09g/mol)0.24g, dissolved in 800ml of distilled water, the pH value of the solution is adjusted to 6 by HCl, and finally distilled water is added to make the volume to be 1L.
The pET-28a (+) vector, E.coli DH 5. alpha., E.coli BL21(DE3) and the like in the examples are commercially available.
EXAMPLE 1 construction of NlpC/P60 endopeptidase protein plasmid NlpC/P60 in pET-28a (+)
(1) Optimizing the nucleotide sequence of the NlpC/P60 endopeptidase protein according to the amino acid sequence of the NlpC/P60 endopeptidase protein to obtain the optimized nucleotide sequence of the NlpC/P60 endopeptidase protein, wherein the original nucleotide sequence of the NlpC/P60 endopeptidase protein and the optimized nucleotide sequence of the NlpC/P60 endopeptidase protein are shown as follows:
original nucleotide sequence of NlpC/P60 endopeptidase protein:
ATGTGGTTCAAGGGGGTACTCACGATCTCTGCCCTACTACTAATCCTGTCAGGGTGTTCAACCAAATCCATAGACCCATACGGCGAACAGAATTCCTATGACAACATTGGCTCCAAGTACCAAAAGAATGATACCACGAGTGGATATAACTTCAATTACAACCAAGCTGCCGACGAGTTTTATAACATAAATCTCTCCAAATATCTTAACAAAAAGTTAGGTAATGACTGCAGTGGATTCGTGTCTTTAGTTAATGAGGATTCCAAATCGTTATATTTCGACGAAAATATTGTCAATAATTTCTACGACAAAAACGGCCGTAAGAGCCAAGCCATCTTTAACCTCTACAAGTCACAGAACAAGATCTCATATACCGATCCTAAACCTGGTGATCTGGTGTTCTTCAACAACACGACATCTCGCACAAACAAGTCTAAGAATAAAGCCATAACTCATTTAGGTATAATCGATAAGGTCGAGAGCAATGGTACGATAACTTTTGTTCATAACATCAACGGCAAAAACATCAAATCGGTGATGAACTTAAAACATAAAAACACCCATAAGCTAAATGGTAAAAAAATAAATGCCTACATCATCCTAAAGTGTCAGACGCCTGTGTGTTTGATATCAAACAAGTTCGCAGGTTTTGGCAAGGTCGACAAATTCGTACAAATAGAT
nucleotide sequence of optimized NlpC/P60 endopeptidase protein:
ATGTGCTCAACAAAATCTATTGATCCTTACGGTGAGCAGAATAGTTATGATAATATTGGGTCAAAATACCAGAAAAATGATACGACCTCAGGTTACAATTTCAATTATAACCAGGCGGCTGACGAATTCTACAATATTAATCTGAGCAAGTACCTTAATAAGAAACTCGGGAACGATTGCTCGGGGTTTGTAAGCTTAGTTAACGAAGACTCCAAGAGTCTGTATTTCGATGAGAACATTGTCAATAATTTTTACGACAAGAACGGTCGCAAGTCCCAAGCTATCTTCAACCTTTATAAGTCCCAAAACAAAATTAGCTATACCGACCCTAAACCGGGCGATTTAGTATTTTTTAACAATACCACAAGCCGGACGAACAAATCTAAAAATAAGGCAATTACTCACCTCGGTATTATCGACAAAGTTGAGTCTAATGGTACTATCACGTTCGTTCACAACATTAACGGGAAGAATATCAAAAGCGTAATGAACTTGAAGCACAAGAACACACATAAATTAAATGGCAAGAAAATCAACGCTTATATTATTCTCAAGTGTCAAACGCCTGTATGTTTGATTTCCAACAAATTCGCTGGCTTTGGGAAAGTGGACAAATTTGTGCAAATTGATGGTTCGCATCACCACCACCACCATTAA
(2) through gene synthesis, the optimized nucleotide sequence is synthesized, Sac I and BamH I restriction sites (synthesized by Shanghai general Co., Ltd.) are respectively added at two ends of the gene, then the obtained target gene fragment and pET-28a (+) vector are subjected to Sac I and BamH I double restriction and are connected to obtain a recombinant expression vector, named NlpC/P60 in pET-28a (+), and the size of the recombinant expression vector is about 6020bp (figure 1).
(3) Plasmid extraction, double digestion confirmation of plasmid and sequencing
Transforming the recombinant expression vector NlpC/P60 in pET-28a (+) obtained in the step (1) into recipient bacterium DH5 alpha, coating LB (kanamycin (Kan) containing 30 mg/L)) plate containing kanamycin with bacterium liquid for resuscitation and activation, culturing for 16h at 37 ℃, picking a single colony and replanting the plate; selecting a part of the transferred bacterial colonies, transferring the selected bacterial colonies into a 100mL liquid LB culture medium, shaking the bacterial colonies on a shaker at 37 ℃ and 200rpm for 16h, and temporarily storing a part of bacterial liquid at 4 ℃; collecting a part of bacterial liquid by using a 50mL centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, and performing plasmid extraction; then carrying out SacI and BamHI double enzyme digestion on the plasmid, confirming the size of the enzyme digestion fragment by nucleic acid electrophoresis, and displaying the enzyme digestion result: the recombinant expression plasmid is successfully transferred into a target gene fragment with the size of about 657 bp; the plasmid was further sequenced. And performing amplification culture on the positive clones with correct sequencing, extracting plasmids, storing the plasmids in a refrigerator at the temperature of-20 ℃, and placing the bacterial liquid in LB solution containing 15-20% of glycerol by volume fraction at the temperature of-80 ℃ for seed preservation.
(4) The recombinant expression vector NlpC/P60' in pET-28a (+): connecting an original nucleotide sequence (not optimized) of NlpC/P60 endopeptidase protein with a pET-28a (+) vector, transforming escherichia coli DH5 alpha, extracting a plasmid, carrying out double enzyme digestion identification, sequencing, carrying out amplification culture on a positive clone with correct sequencing, extracting the plasmid, storing the plasmid into a refrigerator at the temperature of minus 20 ℃, and placing a bacterial liquid at the temperature of minus 80 ℃ by using an LB solution containing 15-20% of glycerol by volume fraction for seed preservation.
Example 2 inducible expression of recombinant NlpC/P60 endopeptidase protein
(1) The recombinant expression plasmid NlpC/P60 in pET-28a (+) (with NlpC/P60' in pET-28a (+) as a control) for expressing the recombinant NlpC/P60 endopeptidase protein, which was preserved in example 1, was transformed into recipient E.coli BL21(DE3) according to a conventional method, which specifically comprises: mixing 25 μ g recombinant expression plasmid and 100 μ L BL21(DE3) competent cell, standing on ice for 30min, and hot shocking in 42 deg.C water bath for 90 s; then adding 1ml LB broth culture medium, resuscitating and activating for 1h on a shaker at 37 ℃ and 200rpm, centrifuging for 1min at 3000rpm, discarding 90% of supernatant, coating the bacterial liquid on LB (kanamycin-containing kanamycin (Kan)) plate containing kanamycin for resuscitating and activating, and culturing for 16h at 37 ℃; selecting a single colony, carrying out colony PCR confirmation by using a T7 primer, and transferring a clone colony of a target band with the size of about 657bp to a coating plate again and storing the clone colony; the specific PCR identification procedure is as follows:
forward primer T7-F: 5'-TAATACGACTCACTATAGG-3', respectively;
reverse primer T7-R: 5'-TGCTAGTTATTGCTCAGCGG-3', respectively;
an amplification system:
max Master Mix (Dye Pl. mu.s) 25. mu.L of enzyme, 2. mu.L of forward primer (10 pmol/. mu.L), 2. mu.L of reverse primer (10 pmol/. mu.L), 3. mu.L of gene template, ddH2O make up to 50. mu.L.
And (3) amplification reaction conditions: 3min at 95 ℃; at 95 ℃ for 15s, 56 ℃ for 15s and 72 ℃ for 60s, and performing 40 cycles; 5min at 72 ℃.
After completion of the PCR reaction, the target band of about 657bp was visualized by electrophoresis on a 1% mass fraction agarose gel (FIG. 2).
(2) Selecting a part of the transferred bacterial colonies to be transferred into 100mL liquid LB culture medium at 37℃,Shaking the bacteria on a shaking table at 200 rpm; OD of bacterial liquid to be treated600When the value is 0.8, the induction is carried out by IPTG, and the final concentration of the IPTG is 0.1 mmol/L; after inducing for 4h at 37 ℃, collecting the bacterial liquid by using a 50mL centrifuge tube, centrifuging for 10min at 8000rpm, discarding the supernatant, washing the bacterial liquid twice by using PBS, and storing at-20 ℃.
(3) And (3) taking 30mL of imidazole-free protein lysate Lysis buffer to resuspend the thallus in the step (2), and crushing the thallus by using an ultrasonication instrument, wherein the ultrasonication procedure comprises 3 seconds of crushing, 5 seconds of separation and 30min of ultrasonication.
(4) Centrifuging the ultrasonically-crushed product at 8000rpm for 15min, collecting the supernatant, taking a small amount of the supernatant, adding 4 xSDS (sodium dodecyl sulfate) loading buffer solution, uniformly mixing, boiling in boiling water for 10min, if the heated sample has a viscous product, instantly centrifuging the sample, taking the supernatant, loading the supernatant into a glue hole of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) prefabricated glue purchased from Kismei, simultaneously adding an equivalent amount of protein SMOBIO PM2510 Maker, adjusting to 100V for glue running by electrophoretic voltage, and carrying out glue running for 120 min. Taking out the gel block from the glass plate, and slightly placing in a staining tank containing Coomassie brilliant blue solution on a shaking table for 30 min; then pouring out the Coomassie brilliant blue solution from the dyeing tank, adding clear water to wash the gel block, putting the gel block into the dyeing tank in a shaking table, and decoloring the gel block with clear water for 30min to obtain a visible cleaning strip.
After an inducer IPTG is added into a strain of escherichia coli BL21(DE3) containing a recombinant expression vector NlpC/P60' in pET-28a (+) for induction expression according to the method, the bacterial liquid OD600 is reduced from 0.8 to 0.4, which shows that the NlpC/P60 endopeptidase coded by the unoptimized NlpC/P60 endopeptidase gene can kill the recipient bacterium BL21(DE3) after being expressed in the recipient bacterium BL21(DE3), further the recipient bacterium BL21(DE3) is killed, and the subsequent expression cannot be carried out.
After the escherichia coli BL21(DE3) strain containing the recombinant expression vector NlpC/P60 in pET-28a (+) is added with an inducer IPTG to perform induction expression according to the method, the bacterial liquid OD600 is increased from 0.8 to 2.0, which shows that the recombinant NlpC/P60 endopeptidase coded by the codon-optimized NlpC/P60 endopeptidase gene can be normally expressed in a recipient bacterium BL21(DE3) and cannot generate killing effect on a recipient BL21(DE3), and the protein purification result of SDS-PAGE electrophoresis detection shows that: the recombinant NlpC/P60 endopeptidase protein is successfully expressed in a soluble way.
EXAMPLE 3 purification, dialysis and Sterilization of recombinant NlpC/P60 endopeptidase protein
(1) Column equilibration: first use ddH2O removing ethanol from the Ni column, ddH2O was greater than 5 column volumes and column equilibration was performed with lysine buffer.
(2) Loading: the supernatant obtained in step (4) of example 2 was slowly poured into a nickel column.
(3) And (3) elution: the column was eluted with 500. mu.l of Elution buffer solution, and the liquid dropped from the nickel column was collected by a 1.5mL ep tube and eluted 4 times to give 4 tubes of purified protein.
(4) And (3) dialysis: and (4) putting the 2 nd and 3 rd tube proteins in the step (3) into a 3000Da protein dialysis bag, adding 1L PBS (pH 7.4), placing in a refrigerator at 4 ℃, and dialyzing the protein sample until the next day.
(5) And (3) degerming: placing dialyzed protein in 1.5ml ep tube, adding 20% glycerol, centrifuging at 12000g for 5min, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane for sterilization to obtain target protein, and storing at-20 deg.C.
(6) And (3) identification: adding a small amount of target protein into 4 xSDS loading buffer solution, mixing uniformly, boiling in boiling water for 10min, performing instant centrifugation on the heated sample if the sample has a viscous product, loading the supernatant into a glue hole of SDS-PAGE 15% prefabricated glue purchased from Kinry, simultaneously adding an equal amount of protein SMOBIO PM2510 Maker, adjusting to 100V glue running by electrophoretic voltage, and carrying out glue running for 120 min. Taking out the gel block from the glass plate, and slightly placing in a staining tank containing Coomassie brilliant blue solution on a shaking table for 30 min; then pouring out the Coomassie brilliant blue solution from the dyeing tank, adding clear water to wash the gel block, putting the gel block into the dyeing tank in a shaking table, and decoloring the gel block with clear water for 30min to obtain a visible cleaning strip.
(7) As a result, as shown in FIG. 3, the soluble expression of the recombinant NlpC/P60 endopeptidase protein was successful, wherein the concentration of the purified, dialyzed and sterilized recombinant NlpC/P60 endopeptidase protein was 1.934 mg/ml.
Example 4 verification of the antibacterial function of the recombinant NlpC/P60 endopeptidase protein
(1) Escherichia coli strain BL21(DE3) was transferred to LB solid medium and cultured overnight at 37 ℃.
(2) Selecting part of the bacterial colonies in the step (1) to be placed in LB liquid culture medium, and culturing the bacterial colonies to OD on a shaker at 37 ℃ and 200rpm600＝1.0。
(3) Taking 10 mu L of the bacterial liquid obtained in the step (2), diluting 10 mu L of the bacterial liquid with sterile 0.01mol of PBS (pH 6) in a multiple ratio on a 96-well plate4And (4) doubling. That is, 90. mu.L of sterile 0.01mol PBS (pH 6) was added to 1-4 wells, 10. mu.L of the bacterial suspension obtained in step (2) was added to the 1 st well, and the pipetting was repeated 5 times, and 10. mu.L of the liquid in the 1 st well was added to the 2 nd well, and the pipetting was repeated 5 times, and this was continued until the addition to the 4 th well.
(4) mu.L of the 4 th well liquid from step (3) was added to a blank well, and 5. mu.L of the recombinant NlpC/P60 endopeptidase protein purified and sterile filtered from step (5) of example 3 (blank plus 5. mu.L of sterile 0.01mol PBS pH 7.4) was added thereto, blown up and mixed 5 times, covered, and incubated in a 37 ℃ incubator for 16 hours.
(5) After the incubation is finished, all the liquid of the blank group and the experimental group is taken out, the liquid is dripped on an LB solid culture medium, the culture medium is uniformly distributed with the liquid by rotating the culture medium, after the liquid is dried in the air, the liquid is placed in a 37 ℃ incubator for inverted culture overnight, and whether the protein has antibacterial activity or not is judged by counting the number of bacterial colonies on the second day.
(6) As shown in FIGS. 4 and 5, after the data are processed, the recombinant NlpC/P60 endopeptidase protein achieves a killing effect of 2 orders of magnitude on the Escherichia coli strain BL21(DE 3).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> gene encoding recombinant NlpC/P60 endopeptidase protein and use thereof
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 657
<212> DNA
<213> Artificial
<220>
<223> Gene encoding recombinant NlpC/P60 endopeptidase protein
<400> 1
atgtgctcaa caaaatctat tgatccttac ggtgagcaga atagttatga taatattggg 60
tcaaaatacc agaaaaatga tacgacctca ggttacaatt tcaattataa ccaggcggct 120
gacgaattct acaatattaa tctgagcaag taccttaata agaaactcgg gaacgattgc 180
tcggggtttg taagcttagt taacgaagac tccaagagtc tgtatttcga tgagaacatt 240
gtcaataatt tttacgacaa gaacggtcgc aagtcccaag ctatcttcaa cctttataag 300
tcccaaaaca aaattagcta taccgaccct aaaccgggcg atttagtatt ttttaacaat 360
accacaagcc ggacgaacaa atctaaaaat aaggcaatta ctcacctcgg tattatcgac 420
aaagttgagt ctaatggtac tatcacgttc gttcacaaca ttaacgggaa gaatatcaaa 480
agcgtaatga acttgaagca caagaacaca cataaattaa atggcaagaa aatcaacgct 540
tatattattc tcaagtgtca aacgcctgta tgtttgattt ccaacaaatt cgctggcttt 600
gggaaagtgg acaaatttgt gcaaattgat ggttcgcatc accaccacca ccattaa 657
<210> 2
<211> 681
<212> DNA
<213> Artificial
<220>
<223> original nucleotide sequence of NlpC/P60 endopeptidase protein
<400> 2
atgtggttca agggggtact cacgatctct gccctactac taatcctgtc agggtgttca 60
accaaatcca tagacccata cggcgaacag aattcctatg acaacattgg ctccaagtac 120
caaaagaatg ataccacgag tggatataac ttcaattaca accaagctgc cgacgagttt 180
tataacataa atctctccaa atatcttaac aaaaagttag gtaatgactg cagtggattc 240
gtgtctttag ttaatgagga ttccaaatcg ttatatttcg acgaaaatat tgtcaataat 300
ttctacgaca aaaacggccg taagagccaa gccatcttta acctctacaa gtcacagaac 360
aagatctcat ataccgatcc taaacctggt gatctggtgt tcttcaacaa cacgacatct 420
cgcacaaaca agtctaagaa taaagccata actcatttag gtataatcga taaggtcgag 480
agcaatggta cgataacttt tgttcataac atcaacggca aaaacatcaa atcggtgatg 540
aacttaaaac ataaaaacac ccataagcta aatggtaaaa aaataaatgc ctacatcatc 600
ctaaagtgtc agacgcctgt gtgtttgata tcaaacaagt tcgcaggttt tggcaaggtc 660
gacaaattcg tacaaataga t 681
<210> 3
<211> 19
<212> DNA
<213> Artificial
<220>
<223> Forward primer T7-F
<400> 3
taatacgact cactatagg 19
<210> 4
<211> 20
<212> DNA
<213> Artificial
<220>
<223> reverse primer T7-R
<400> 4
tgctagttat tgctcagcgg 20
Claims (10)
1. A gene for coding recombinant NlpC/P60 endopeptidase protein is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. Use of the gene encoding the recombinant NlpC/P60 endopeptidase protein of claim 1 in the preparation of an antibacterial product.
3. A recombinant expression vector comprising the gene encoding the recombinant NlpC/P60 endopeptidase protein according to claim 1, wherein the gene encoding the recombinant NlpC/P60 endopeptidase protein according to claim 1 is cloned into the expression vector.
4. The recombinant expression vector of claim 3, wherein:
the expression vector is pET-28a (+).
5. The recombinant expression vector of claim 4, wherein:
the gene for coding the recombinant NlpC/P60 endopeptidase protein is inserted into pET-28a (+) through enzyme cutting sites SacI and BamHI.
6. A recombinant expression strain characterized by comprising the recombinant expression vector according to any one of claims 3 to 5, which is obtained by transferring the recombinant expression vector according to any one of claims 3 to 5 into a host strain.
7. The recombinant expression strain of claim 6, wherein:
the host strain is bacteria, yeast or fungi.
8. A method for preparing a recombinant NlpC/P60 endopeptidase protein, comprising the steps of:
(1) cloning the gene encoding the recombinant NlpC/P60 endopeptidase protein of claim 1 into an expression vector to obtain a recombinant expression vector;
(2) transferring the recombinant expression vector into a host strain to obtain a recombinant expression strain;
(3) and (3) inducing and expressing the recombinant expression strain, collecting bacterial liquid, crushing, separating and purifying to obtain the recombinant NlpC/P60 endopeptidase protein.
9. The method of producing the recombinant NlpC/P60 endopeptidase protein according to claim 8, wherein:
the inducer for inducing expression in the step (3) is IPTG.
10. The method of producing the recombinant NlpC/P60 endopeptidase protein according to claim 8, wherein:
the separation and purification method in the step (3) is a nickel column affinity chromatography method.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245937A (en) * | 2012-04-17 | 2014-12-24 | 弗·哈夫曼-拉罗切有限公司 | Method for the expression of polypeptides using modified nucleic acids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104245937A (en) * | 2012-04-17 | 2014-12-24 | 弗·哈夫曼-拉罗切有限公司 | Method for the expression of polypeptides using modified nucleic acids |
Non-Patent Citations (5)
| Title |
|---|
| 俞敏锋: "单核细胞增生李斯特菌p60结构域功能鉴定及其底物结合特性研究", 《中国优秀硕士学位论文全文数据库（基础科学辑）》 * |
| 孙坦: "单核细胞增生李斯特菌p60异源表达条件的优化及其抑菌效果的研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 * |
| 无: "无", 《GENBANK》 * |
| 胡凯等: "炭疽芽孢杆菌新抗原NlpC的重组表达与免疫评价", 《生物技术通讯》 * |
| 顾浩: "产单核细胞李斯特菌p60蛋白结构域的剖析", 《中国优秀硕士学位论文全文数据库（生物学辑）》 * |
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