CN103614399A - Eriocheir sinensis lysozyme gene, encoding protein and application thereof - Google Patents
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Abstract
The invention discloses an eriocheir sinensis lysozyme gene, wherein a nucleotide sequence is shown in SEQ ID NO.1. The invention provides a recombinant expression and purification method of the eriocheir sinensis lysozyme gene. The invention also provides a lysozyme polypeptide encoded by the eriocheir sinensis lysozyme gene and provides the use of a gene sequence and the use of the lysozyme; the amino acid sequence of the lysozyme polypeptide is shown in SEQ ID NO.2. The lysozyme disclosed by the invention has bacteriostatic activity to gram-positive bacteria, also has bacteriostatic activity to gram negative bacteria. The recombined lysozyme obtained by using the eriocheir sinensis lysozyme gene can be applied to development of antibacterial drugs and feed additives.
Description
Technical field:
The present invention relates to genetically engineered field, specifically relate to the N,O-Diacetylmuramidase of mitten crab lysozyme gene and coding thereof and described gene and there is the application in the recombinant protein of anti-microbial activity in preparation.
Background technology:
Invertebrates lacks acquired immunity, in animal body fluid, do not there is immunoglobulin (Ig), its body defensive raction has and is different from more vertebrate peculiar properties, its reaction mechanism is divided into cellular immunization and humoral immunization traditionally, mainly comprises the effect of some lytic enzymes, antimicrobial effect molecule and the conditioning factor in the engulfing of hemocyte, parcel and hemolymph etc.
Antibacterial peptide is that a class is extensively present in the amphiphilic small molecules basic polypeptide in whole organic sphere, is one of key factor of body non-specific immunity.Since Steiner finds cecropin cecropin, in various biologies, had been found that more than 1000 kind of antibacterial peptide.In recent years, in many invertebrates bodies, isolate the polypeptide with antimicrobial effect.N,O-Diacetylmuramidase claims again lysozyme, is extensively present in the various organs or secretory product of invertebrates, vertebrates, plant, bacterium and phage, and be the important molecule of inherent immunity system.As antibacterial peptide, the hydrolysis of the β-Isosorbide-5-Nitrae glycosidic link in N,O-Diacetylmuramidase energy catalysis bacteria cell wall between peptidoglycan N-Acetyl-D-glucosamine and-acetylmuramic acid, causes that bacteria cell wall breaks, content overflows and make bacterium dead.In addition N,O-Diacetylmuramidase also can be induced and be regulated the synthetic and secretion of other immune factor of body, collaborative other immune factor to carry out immune defense, and for the invertebrates that lacks specific immunity, N,O-Diacetylmuramidase is very important antibacterial peptide in its immunity system.
Sinensis huge legendary turtle crab (Eriocheir sinensis) is commonly called as river crab, belongs to Crustachia, Decapoda, and Grapsidae, Eriocheir, its meat flavour is delicious, has the important aquaculture kind of very high nutritive value and economic worth ，Shi China.Along with the deterioration of Sinensis huge legendary turtle crab breeding environment, disease frequently occurs, and Given this, the antibacterial peptide of research Sinensis huge legendary turtle crab has important effect for understanding crab immune defence mechanism.
In mitten crab, find that there is albumen or the peptide class of the extraneous pathogenic micro-organism of multiple opposing, as multiple lectin, coagulogen, but also do not have the report of lysozyme gene.
Summary of the invention:
The invention provides mitten crab lysozyme gene and proteins encoded thereof and application.
For achieving the above object, the technical solution used in the present invention is:
A mitten crab lysozyme gene Eslysozyme, is specially in sequence SEQIDNo.1 shown in base sequence.It is the cDNA sequence that is cloned into lysozyme gene Eslysozyme from mitten crab, and this sequence total length is 831bp, comprises open reading frame 426bp, and 5 ' the non-coding head of district is 25bp, and 3 ' the non-coding head of district is 383bp, a polyadenylic acid tail.Utilize pET32a expression vector recombinant expressed this albumen in e. coli bl21, expression product has bacteriostatic activity.
The proteins encoded of mitten crab lysozyme gene has the aminoacid sequence shown in SEQIDNo.2, and complete proteins encoded contains 141 amino acid, and wherein the 1-16 of an encoding sequence amino acid is signal peptide sequence.Mature peptide comprises 125 amino acid, predicts that this molecular weight of albumen is 14.08kDa.Mature peptide has typical Destabilase structural domain.
The present invention utilizes expressed sequence tagging method and cDNA end rapid amplifying technology from mitten crab, to be cloned into the full length sequence of the cDNA of lysozyme gene, pass through round pcr, the gene fragment of amplification coding N,O-Diacetylmuramidase mature peptide is also cloned in pET32a expression vector, realizes protokaryon in-vitro recombination expression in e. coli bl21.Recombinant products, through nickel sepharose purifying, after dialysis renaturation, has obtained restructuring antalzyme protein, and this recombinant protein has higher bacteriostatic activity after testing.The present invention provides theoretical basis for mitten crab immune defence mechanism, and is the disease control of mitten crab, and exploitation pharmaceutical prod and fodder additives are established application foundation.
Accompanying drawing explanation
Be illustrated as the restraining effect of mitten crab N,O-Diacetylmuramidase recombinant protein of the present invention to streptococcus aureus (Staphylococcus aureus), subtilis (Bacillus subtilis) and intestinal bacteria (E.coli) growth.
Embodiment:
In the following examples, the invention will be further elaborated, but the invention is not restricted to this.
Embodiment 1:
The mitten crab lysozyme gene that clone obtains, has the sequence shown in SEQ ID NO.1.
From
http:// www.ncbi.nlm.nih.govthe est sequence of mitten crab cDNA library (Taxonomy ID:95602) is downloaded in website, these sequences are spliced, wherein splicing aminoacid sequence of deriving of sequence and the similarity of Penaeus monodon N,O-Diacetylmuramidase are 60%, with the similarity of Litopenaeus vannamei N,O-Diacetylmuramidase be 58.6%, through SMART software, predict, this sequence has a typical Destabilase structural domain.According to this splicing sequences Design forward primer P1 (5 '-AGACAGGGGACGGAGAGTACGA-3 ') and and adaptor dT primer (5 '-GGCCACGCGTCGACTAGTACT17-3 '), the cDNA of mitten crab is template, carry out pcr amplification, pcr amplification product is diluted to 100 times as template, utilize forward primer P2 (5 '-TCACGTATTTGGTTCAGGTAAG-3 ') and adaptor dT primer to carry out pcr amplification for the second time, agarose gel electrophoresis detects and reclaims object fragment, be connected to pMD-18T carrier, transform in intestinal bacteria TOP10 competent cell, choosing positive colony delivers to biotech firm and checks order.Sequencing result is lysozyme gene with analyzing this amplified fragments of demonstration through BLAST.By est sequence and lysozyme gene 3 ' the terminal sequence splicing of increasing, obtain the cDNA full length sequence of lysozyme gene, as shown in SEQ ID NO.1.Design primer P3 (5 '-CTTCGAATTCCGACACCAA-3 ') and P4 (5 '-TTTTTGTGCTTAAGAGCCCTAATA-3 ') to verify lysozyme gene sequence.
Embodiment 2:
Structure, expression and the purifying of mitten crab lysozyme gene coding region RT-PCR expression vector
1, the structure of recombinant expression vector
According to mitten crab lysozyme gene sequence and expression vector pET32a(Novagen company) cloning site, the present invention adopts EcoRI and the Xho I restriction enzyme site in pET32a, during design primer, at upstream primer, introduce EcoRI restriction enzyme site, downstream primer is introduced Xho I restriction enzyme site.Design and synthesize the primer for the mitten crab lysozyme gene coding region fragment that increases, P5 (5 '-GAATTCGACCCGCTGGAGGAC-3 ') and P6(5 '-CTCGAGACGGAGAGGCATTGTGT-3 ').Using the cDNA of mitten crab as template amplification Eslysozyme fragment, at 16 ℃, connect pMD18-T and Eslysozyme fragment, to connect product and transform intestinal bacteria TOP10 competent cell, with penbritin and round pcr screening positive clone, obtain pMD18-T-Eslysozyme recombinant plasmid.Under 37 ℃ of water bath condition, with EcoRI and Xho I respectively enzyme cut pET32a and pMD18-T-Eslysozyme.Enzyme is cut end, with 1% agarose gel electrophoresis enzyme, cuts product, reclaims pET32a carrier large fragment and with the Eslysozyme fragment of EcoRI and Xho I restriction enzyme site, then, under the effect of T4DNA ligase enzyme, 16 ℃ of connections, spends the night.Connect product and transform intestinal bacteria TOP10, with penbritin screening and bacterium colony PCR, screen and check order.
2, the expression of recombinant protein and purifying
The recombinant strains using in this research is BL21, its with plysS plasmid can express T7 N,O-Diacetylmuramidase, can suppress the leakage that inductor inserts gene before adding and express, therefore can improve the tolerance of Host Strains to toxicity representation.
The abduction delivering step of recombinant protein is as follows:
(1) picking mono-clonal, in the LB substratum with penbritin, 220rpm, 37 ℃ are cultured to OD
600be about 0.5.
(2) adding final concentration is 0.8mmol L
-1iPTG, 220rpm, continue to cultivate 4-6h for 37 ℃.
(3) 4 ℃, 5000rpm, centrifugal 10min, collects bacterial sediment.Add 100 μ l sample-loading buffers, centrifugal after cracking 10min in boiling water bath, get supernatant, in 15% SDS-PAGE electrophoretic analysis, coomassie brilliant blue R_250 dyeing, records result with gel imaging system after decolouring.
Do not do induction contrast simultaneously, get 1ml bacterium liquid centrifugal, after supernatant discarded, add 80 μ l water and 20 μ l5X albumen sample-loading buffers, 100 ℃ of boiling water boil 10 minutes, slightly centrifugal, collect supernatant liquor and and precipitation, SDS-PAGE detects expression product.
The purification step of recombinant protein is as follows:
Adopt metal chelate chromatography method, utilize imidazolyl and metal ion (Ni on Histidine side chain
2+) etc. the characteristic of chelating, the TRX albumen that makes His-Tag prepared by gene recombination technology merge recombinant protein and pET32a empty carrier abduction delivering obtains purifying.Main experimental procedure operation is as follows:
(1) the required buffer formulation of purifying His label recombinant protein under denatured state:
A. damping fluid I: preparation: 0.5mol L
-1naH
2pO
419ml, 0.5mol L
-1na
2hPO
481ml, NaCl29.3g and urea 480g, after dissolving, constant volume is to 1L.
B. damping fluid II: preparation: 0.5mol L
-1naH
2pO
419ml, 0.5mol L
-1na
2hPO
481ml, NaCl29.3g, imidazoles 34g and urea 480g, after dissolving, constant volume is to 1L.
C. damping fluid III: imidazole concentration is 50mmol L
-1buffer B: damping fluid I90ml, with damping fluid II10ml; Imidazole concentration is 400mmol L
-1buffer B: damping fluid I20ml, with damping fluid II80ml.
(2) purification step
A. centrifugal collection thalline, adds 20-30ml damping fluid I.Centrifuging and taking supernatant after ultrasonic disruption thalline.0.45 μ m membrane filtration for bacteria breaking liquid.
B. use 10 bed volumes of damping fluid I balance, coutroi velocity is 1ml min
-1.
C. bacteria breaking liquid be take to flow velocity as 1ml min
-1loading.
D. with damping fluid I, wash 10 column volumes, flow velocity is 1ml min
-1.
E. with containing respectively 50mmol L
-1, 400mmol L
-1the damping fluid III of imidazoles carries out stepwise elution, and flow velocity is 2ml min
-1, collect each stepwise elution peak, 5 column volumes of each concentration wash-out, by molecular size range and the purity of SDS-PAGE detection fusion rotein.
F. with pure water stream, wash 5 column volumes, then wash 5 column volumes by 20% ethanol stream, pillar is preserved in 4～8 ℃ of environment.Recombinant protein exists with inclusion body form, with ordinary method, through inclusion body purification, renaturation, obtains mitten crab N,O-Diacetylmuramidase recombinant protein.
Embodiment 3:
The Antibacterial Activity of mitten crab N,O-Diacetylmuramidase recombinant protein
The bacteriostatic activity of recombinant protein is slightly to change according to the method for Rathinakumar etc.Measure the impact of recombinant protein Eslysozyme on growth curve of bacteria.
(1) test bacterium is streptococcus aureus (Staphylococcus aureus), subtilis (Bacillus subtilis) and intestinal bacteria (E.coli).When tested bacterium grows to logarithmic phase, centrifugal, Tris-HCl (50mmol l for precipitation
-1, pH=8.0) dissolve, making concentration reach every milliliter is 10
3colony-forming unit.
(2) in flat 96 orifice plates (Costar, Fisher), every hole adds 50 μ l bacteria suspensions.Albumen, with after Tris-HCl gradient dilution, is added in 96 orifice plates, and each concentration is established 5 repetitions, and reference protein TRX is made as control group.
(3) incubated at room is 3 hours, then adds the substratum of 150 μ l.
(4) mix.Put into 28 ℃ of incubator incubated overnight.
(5) by microplate reader, in 600nm, detect each hole OD value.
Bacteriostatic experiment result: restructuring antalzyme protein has bacteriostatic activity to intestinal bacteria, subtilis and streptococcus aureus, as shown in the figure.
Claims (3)
1. mitten crab lysozyme gene, is characterized in that: the cDNA sequence of mitten crab lysozyme gene is the nucleotide sequence shown in sequence table SEQ ID No1.
2. by a proteins encoded for mitten crab lysozyme gene claimed in claim 1, it is characterized in that: described proteins encoded aminoacid sequence is as shown in SEQ ID No2.
3. an application as sterilant by the recombinant protein of mitten crab lysozyme gene claimed in claim 1, is characterized in that: in intestinal bacteria, express, obtain the recombinant protein with bacteriostatic activity.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955523A (en) * | 2010-03-10 | 2011-01-26 | 中国科学院海洋研究所 | Preparation and application of Eriocheir sinensis whey acidic protein (WAP) with bacteriostatic activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955523A (en) * | 2010-03-10 | 2011-01-26 | 中国科学院海洋研究所 | Preparation and application of Eriocheir sinensis whey acidic protein (WAP) with bacteriostatic activity |
Non-Patent Citations (4)
| Title |
|---|
| LI，F: "Eriocheir sinensis lysozyme mRNA,complete cds;GenBank:JN416111.1", 《GENBANK》 * |
| LI，F: "Lysozyme;GenBank:AEU04535.1", 《GENBANK》 * |
| UNIPROT: "UniProtKB-G9HZ47", 《UNIPROT》 * |
| 马贵华等: "中华鳌蟹免疫因子-溶菌酶的初步研究", 《淡水渔业》 * |
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