CN109897861B - Portunus trituberculatus chitinase gene, recombinant expression protein and application thereof - Google Patents
Portunus trituberculatus chitinase gene, recombinant expression protein and application thereof Download PDFInfo
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- CN109897861B CN109897861B CN201910135567.2A CN201910135567A CN109897861B CN 109897861 B CN109897861 B CN 109897861B CN 201910135567 A CN201910135567 A CN 201910135567A CN 109897861 B CN109897861 B CN 109897861B
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- protein
- gene
- ptcht4
- portunus trituberculatus
- chitinase
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Abstract
The invention discloses a portunus trituberculatus chitinase gene, recombinant expression protein and application thereof. The invention carries out the chitinase of the portunus trituberculatusPtCht4Cloning of genes and utilization ofPtCht4Constructing prokaryotic expression vector by nucleotide sequence of gene coding protein, and screening out a portunus trituberculatus capable of efficiently expressing portunus trituberculatusPtCht4The expression system of the gene recombinant protein can obtain the recombinant protein which can be correctly folded and has activity through protein purification and renaturation. The recombinant protein is used for in vitro bacteriostasis becausePtCht4The gene belongs to endogenous genes of the portunus trituberculatus, so that the drug prepared from the expressed protein can not cause rejection reaction of organisms, can excite the nonspecific immune system of the organisms, reduce the drug resistance of bacteria, and can provide a new idea for the research of novel drugs for the crabs due to the realization of in vitro antibacterial technology.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Portunus trituberculatus Miers chitinase gene and its recombinant expression
Albumen and application.
Background technique
Portunus trituberculatus Miers (Portunus trituberculatus) is under the jurisdiction of Arthropoda, Crustachia, Decapoda, shuttle
Zi Xie section, Portunus are the important sea-farming kinds in China, have fast growth, fine and tender taste, delicious flavour, nutrition rich
The advantages that rich is the high important marine product of economic value.But as the continuous deterioration of breeding environment increases the possibility of crab illness
Property, the development of crab is largely constrained, for the use for reducing external source drug, ecologic breeding is realized and pursues interests maximum
Change, the raising of Portunus trituberculatus Miers autoimmunity defence capability and the research of effective newtype drug are just particularly important.
Chitinase (Chitinase) is that one kind is widely present in arthropod, can be by cracking pathogen cell wall
Chitin (Chitin) is to play the crucial enzyme of immune function.By the structure and function of studying chitinase PtCht4 gene
Can, and then realize the In Vitro Bacteriostasis of recombinant protein.Have not yet to see the correlation of Portunus trituberculatus Miers chitinase gene In Vitro Bacteriostasis
Report.
Summary of the invention
The purpose of the present invention is to provide a kind of recombinant expression protein of Portunus trituberculatus Miers chitinase gene and its in body
It is outer it is antibacterial in application, the recombinant protein of the PtCht4 gene has significant antibacterial action, to enhance Portunus trituberculatus Miers pair
The resilience of pathogen provides experiment and supports.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a kind of Portunus trituberculatus Miers chitinase PtCht4 gene, nucleotides sequence list such as SEQ ID
Shown in NO:1.
The present invention also provides the Portunus trituberculatus Miers chitinase PtCht4 gene translation generate chitinase,
Amino acid sequence is as shown in SEQ ID NO:2.
The present invention provides the Portunus trituberculatus Miers chitinase PtCht4 gene generate recombinant expression protein, it
Preparation method the following steps are included:
(1) chitinase gene is cloned；
(2) building of recombinant expression carrier: carrying out the amplification of open reading frame to chitinase gene, to PCR product and
PET-28a plasmid carries out double digestion, and carries out PCR amplification, recovery purifying product, and linked system and competent cell are mixed,
Culture medium, coated plate culture, with T7, T7teminator primer screening purpose bacterial strain is added；
(3) inducing expression of recombinant protein: by purpose strain inoculated in culture medium culture, being transferred to competent cell, will
Obtained bacterial strain carries out inducing expression；
(4) recombinant protein isolates and purifies: the bacterial strain after inducing expression success being expanded culture, obtained thallus is added
Lysate is centrifuged after ultrasonication, obtains the destination protein present in expression bacterial strain in the form of inclusion body, inclusion body is washed
It washs, purify；
(5) renaturation of recombinant protein: recombinant protein after purification is packed into bag filter, is successively dialysed with dialyzate, finally
It is centrifuged, takes supernatant, obtain recombinant expression protein.
It is further: in the step (2) double digestion use substep digestion, the restriction enzyme used be BamHI and
Not I。
It is further: substep digestion method described in the step (2) are as follows: first by the PCR amplification body containing BamHI
30 DEG C of reaction 10min in PCR instrument are tied up to, then the Not I of 2ul is added into reaction solution, after mixing centrifugation, 37 DEG C in PCR instrument
React 10min.
It is further: the PCR amplification system are as follows: 10 × QuickCut Buffer, 10 μ L；Purified pcr product/pET-
28a, 10 μ l/45 μ L；QuickCut BamH I, 2 μ L；Mend sterilizing ddH2O to 100 μ L.
It is further: T7 in the step (2), T7teminator primer are as follows:
T7:5 '-TAATACGACTCACTATAGGG-3 '；
T7teminator:5 '-GCTAGTTATTGCTCAGCGG-3 '.
Further: inductive condition is to add the IPTG of final concentration of 1.0mM to induce at 37 DEG C in the step (3)
6h。
The present invention also provides the recombinant expression proteins of the Portunus trituberculatus Miers chitinase gene to prepare bacteriostatic agent
In application.
Further: the concentration of recombinant expression protein is 5mg/mL-25mg/mL in the bacteriostatic agent, and the bacteriostatic agent is used
In inhibition vibrio parahaemolytious and staphylococcus aureus.
Compared with prior art, advantages of the present invention and technical effect are as follows: the present invention is by carrying out Portunus trituberculatus Miers chitin
Matter enzyme PtCht4 gene cloning constructs prokaryotic expression carrier using the nucleotide sequence of PtCht4 gene coded protein, filters out
A kind of expression system for capableing of high efficient expression Portunus trituberculatus Miers PtCht4 gene recombinant protein is obtained by protein purification and renaturation
It can correctly fold and active recombinant protein.
Recombinant protein is used for In Vitro Bacteriostasis by the present invention, constantly gropes and optimize antibacterial condition, finally determines various concentration
PtCht4 recombinant protein to the inhibitory effects of different bacterium, PtCht4 gene belongs to the endogenous gene of Portunus trituberculatus Miers, thus table
Drug made of albumen up to output makes body not generate rejection, and can excite the nospecific immunity system of body
System, reduces the drug resistance of bacterium, realizations of In Vitro Bacteriostasis technology can be provided for the research of crab class newtype drug theoretical basis with
Experiment is supported.
Detailed description of the invention
Fig. 1 is PtCht4 prokaryotic expression recombinant protein and purification result, and A. does not induce control；B. the experimental group induced；C.
Broken recombinant protein；D. supernatant after being crushed；E. (inclusion body) is precipitated after being crushed；F. unpurified recombination after solubilization of inclusion bodies
Albumen；G. recombinant protein after purification；
Fig. 2 is the PtCht4 recombinant protein renaturation solution of various concentration to different bacterium bacteriostasis rate.
Specific implementation method
Technical solution of the present invention is described in detail below by specific embodiment and result figure.
Embodiment 1
One, the clone of Portunus trituberculatus Miers chitinase PtCht4 gene
According to the est sequence of chitinase gene in Portunus trituberculatus Miers transcript profile database, Primer Premier is utilized
5.0 RACE of software design 3 ' and 5 ' specific primers and universal primer UPM/NUP clone PtCht4 gene using RACE technology.
The primer information that the present embodiment is used is shown in Table 1.
1 primer information of table
It is (as shown in SEQ ID NO:1) that the cDNA of PtCht4 gene, which is held from 5 ' to 3 ' terminal sequences:
Sequence signature: PtCht4 gene is made of 1549 nucleotide, is doublestranded cDNA molecule, linear topology structure.
The nucleotide sequence coded amino acid sequence of PtCht4 gene is (as shown in SEQ ID NO:2):
Sequence signature: the cDNA of PtCht4 gene encodes 462 amino acid, Theoretical pi 5.03, molecular weight altogether
Mw51.171Da。
Two, the preparation of the recombinant protein of Portunus trituberculatus Miers chitinase PtCht4 gene
1, the building of recombinant expression carrier
1) amplification of open reading frame
According to the nucleotide sequence of PtCht4 gene coded protein, the polyclonal position of pET-28a expression vector is comprehensively considered
The restriction endonuclease sites (Bam HI, Not I) of point and gene, using 5.0 software Design primers of Primer Premier,
Restriction enzyme site and protection base, synthetic primer are introduced at 5 ' ends of upstream primer and downstream primer
PCR amplification target gene open reading frame (ORF).Utilize ultraviolet specrophotometer and 1% agarose gel electrophoresis
Detect the quality and concentration of PCR product, gel extraction.
2) double digestion
Double digestion is carried out to PCR product and pET-28a plasmid with restriction enzyme BamHI and Not I, using substep enzyme
It cuts, carries out PCR amplification, system is shown in Table 2.
2 PCR amplification system of table
30 DEG C of reaction 10min in PCR instrument.After reaction, the Not I of 2ul is added into reaction solution, after of short duration centrifugation,
PCR instrument, reacts 10min by 37 DEG C.1% agarose gel electrophoresis gel extraction, PCR after UV spectrophotometer measuring digestion
Product and pET-28a plasmid concentration are respectively 74.5ng/ μ L, 57.3ng/ μ L.
3) recovery product connects, and linked system is shown in Table 3, mixes, of short duration centrifugation, 22 DEG C of reaction 20min of PCR instrument.
3 PCR product linked system of table
4) 20 μ L connection products are mixed with 100 μ LDH5 α competent cells, stands 30min, 42 DEG C of metal bath heat on ice
After swashing 90s, ice bath 2min, the LB culture medium of the addition sterile nonreactive of 900uL into centrifuge tube, 37 DEG C of 180rpm shaken cultivation 1h,
Make relevant resistant maker gene expression, thallus recovery on plasmid.Coated plate in containing 100 μ g/mL kanamycins LB plate on, 37
DEG C, it is incubated overnight.Picking positive monoclonal, with T7, T7teminator primer screens purpose bacterium solution after carrying out bacterium colony PCR identification
It is sequenced.
T7:5 '-TAATACGACTCACTATAGGG-3 ' (SEQ ID NO:12)；
T7teminator:5 '-GCTAGTTATTGCTCAGCGG-3 ' (SEQ ID NO:13).
2, the inducing expression of recombinant protein
1) building of bacterial strain is expressed
The plasmid-bearing strains correctly built to sequencing are inoculated in the LB liquid containing 100 μ g/mL kanamycins by 1:100
Plasmid is extracted in culture medium, after 37 DEG C of culture 12h and recombinant plasmid is transferred to Rosetta (DE3) impression according to the method described above
It in state cell, and picks out positive colony and is sequenced, the correct strain of obtained sequencing is saved as kind of daughter bacteria.
2) inducing expression of recombinant bacterial strain
Kind daughter bacteria 1: 100 is inoculated in the 100mLLB fluid nutrient medium containing 100 μ g/mL kanamycins, and 37 DEG C,
200rpm is cultivated to OD600=0.5~0.6, and bacteria suspension is averagely assigned in 10mL centrifuge tube, final concentration is separately added into
0.1mM, 0.5mM, 0.8mM, 1.0mM IPTG inducer, respectively at 37 DEG C, 25 DEG C, 20 DEG C, 16 DEG C, 200rpm continues to cultivate,
0h, 2h, 4h, 6h, 12h after induction, for 24 hours each time point respectively take 1mL bacterium solution, and 4 DEG C of 12000rpm centrifugation 20min collect thallus
Precipitating, and thallus is resuspended with the PBS (0.1M, pH 7.4) of pre-cooling.Thallus is mixed according to 4: 1 ratio and 4 × albumen loading is slow
Fliud flushing, after boiling water boiling 10min, SDS-PAGE polyacrylamide gel detection induction situation is so that it is determined that optimal inductive condition
Are as follows: the IPTG for adding final concentration of 1.0mM induces 6h at 37 DEG C.
3, it isolates and purifies
1) separation of bacterium solution is broken
Bacterial strain after inducing expression success is expanded into culture to 100mL, OD600=0.5~0.6 is added final concentration of
After the IPTG of 1.0mM induces 6h at 37 DEG C, 6000rpm is centrifuged 10min, collects thallus, according to thallus weight, every 1g thallus adds
Enter 10mL lysate (20mM Na3PO4, 0.5M NaCl, 20mM imidazoles, 10U DNase I, 50mM PMSF, 0.3mg/mL bacteriolyze
Enzyme), thallus is resuspended.After 4 DEG C of placement 1h in -80 DEG C refrigerator multigelation 3 times, ultrasonication is carried out under condition of ice bath, ultrasound
Power is 200W, and 8s ultrasound, 8s interval, being crushed the time is 40min.By broken liquid 12000rpm, 4 DEG C of centrifugation 10min,
Supernatant precipitating is collected respectively, and SDS-PAGE polyacrylamide gel, which detects, determines PtCht4 destination protein in the form of inclusion body
Exist in expression bacterial strain.
The inclusion body of collection is used into buffer solution A (50mM Tris-HCl, 100mM NaCl, 0.5mM EDTA-Na respectively2,
2M Urea, 1%Triton X-100) and buffer solution B (50mM Tris-HCl, 100mM NaCl, 0.5mM EDTA-Na2, 2M
Urea 2 times, 12000rpm, 4 DEG C centrifugation 20min) are washed respectively, and 20mL lysate (100mM Tris- is added after collecting precipitating
HCl, 500mM NaCl, 8M Urea, 10mM imidazoles), ice bath stirring to solubilization of inclusion bodies, 12000rpm, 4 DEG C of centrifugation 30min,
Undissolved precipitating is removed, is PtCht4 protein solution to be purified after supernatant 0.22um filter membrane suction filtration degerming.
2) protein purification
The ddH of 3 times of columnar volumes is added into ProteinIsoTM Ni-NTA Resin cylinder2O is rinsed, in column
After ddH2O drains off, addition Binding buffer (100mM Tris, 500mM Nacl, 8M Urea, 10mM imidazoles, pH 7.4,
0.3mg/mL lysozyme and 2%Triton-X 100 is added in used time) to balance the pH of resin；By destination protein solution to be purified
Sample is added slowly in pillar, uses Binding buffer and Wash buffer (100mM Tris, 500mM Nacl, 8M respectively
Urea, 20mM imidazoles, pH7.4) cleaning pillar, remove impurity；Elution buffer (100mM Tris, 500mM is added
Nacl, 8M Urea, 250mM imidazoles, pH 7.4) after be placed at room temperature for 20min after collect eluent.SDS-PAGE polyacrylamide
Gel detection purification effect, as shown in Figure 1.
4, the renaturation of recombinant protein
By bag filter in treatment fluid (10mM NaHCO3, 1mM EDTA) in boil about 20min, then washed 3 times with ddH2O,
4 DEG C of preservations in 1mM EDTA solution.Recombinant protein after purification is fitted into the bag filter handled well, dialyzate I is successively used
(25mM Tris, 200mM Nacl, 1mM EDTA, 5mM DTT, 6M Urea, pH 7.4), II (25mM Tris, 50mM
1mM GSSG and 1mM GSH, pH 7.4 is added in Nacl, 1mM EDTA, 4M Urea, used time) and III (25mM Tris, 20mM
1mM GSSG and 1mM GSH, pH 7.4 is added in Nacl, 1mM EDTA, 2M Urea, used time) dialysis 12h is respectively stirred at 4 DEG C,
Successively with dialyzate IV, (25mM Tris, 10mM Nacl, 1mM EDTA, 1M Urea, 1mM DTT, 1% glycine are used later
When be added 1mM GSSG and 1mM GSH, pH 7.4), V (25mM Tris, 10mM Nacl, 1mM EDTA, 0.5M Urea, 1mM
DTT, 1% glycine, the used time be added 1mM GSSG and 1mM GSH, pH 7.4) and VI (25mM Tris, 10mM NaCl, 5% is sweet
Oil, pH 7.4) dialysis 3h is respectively stirred at 4 DEG C, the liquid after dialysis is gone in new centrifuge tube, 4 DEG C, 12000rpm, from
Heart 20min takes supernatant, the as recombinant protein after renaturation.Destination protein concentration is 52mg/mL after measuring renaturation using BCA method.
Embodiment 2
1, the In Vitro Bacteriostasis of Portunus trituberculatus Miers chitinase PtCht4 gene recombinant protein
Strain to be tested Gram-negative bacteria vibrio parahaemolytious (Vibrio parahaemolyticus) and gram-positive bacteria
Staphylococcus aureus (Staphylococcus aureus) is inoculated into fresh after 28 DEG C and 37 DEG C are incubated overnight
In 2216E fluid nutrient medium, 180rpm/min cultivates 5-6h to OD600 ≈ 1.PtCht4 recombinant protein renaturation solution is diluted to 5,
10,15,20,25mg/mL, take 50uL103-104The PtCht4 recombinant protein renaturation of each concentration of CFU/mL bacterial suspension and equivalent
Liquid is uniformly mixed, and the sterile 1 × PBS of same volume is added in control group, is sufficiently shaken up, respectively at 28 DEG C and 37 DEG C culture 2-3h,
It is spread evenly across on 2216E solid medium, is incubated overnight, record plate thallus number, calculate bacteriostasis rate.
The PtCht4 recombinant protein of 4 various concentration of table is to different bacterium bacteriostasis rate
As shown in table 4 and Fig. 2,5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL PtCht4 recombinant protein
Renaturation solution is respectively 0.236842 to the bacteriostasis rate of vibrio parahaemolytious and staphylococcus aureus, 0.479757,0.552632,
0.61336,0.710526 and 0.19039,0.286491,0.349048,0.43699,0.478694.Illustrate the PtCht4
Recombinant protein has significant inhibitory effect to the proliferation of above-mentioned pathogen, and with increasing for protein concentration, bacteriostasis rate is dramatically increased,
And the albumen of same concentrations is significantly higher than staphylococcus aureus to the inhibiting rate of vibrio parahaemolytious.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Sequence table
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Claims (1)
1. Portunus trituberculatus Miers chitinasePtCht4The recombinant expression protein of gene exists preparing the application in bacteriostatic agent, feature
In: the concentration of recombinant expression protein is 5mg/mL-25mg/mL in the bacteriostatic agent, and the bacteriostatic agent is for inhibiting secondary haemolysis arc
Bacterium and staphylococcus aureus；
The Portunus trituberculatus Miers chitinasePtCht4The nucleotides sequence list of gene is as shown in SEQ ID NO:1, three wart
Swimming crab chitinasePtCht4The preparation method of the recombinant expression protein of gene the following steps are included:
(1) chitinase gene is cloned；
(2) building of recombinant expression carrier: the amplification of open reading frame is carried out to chitinase gene, to PCR product and pET-
28a plasmid carries out double digestion, and carries out PCR amplification, recovery purifying product, and linked system and competent cell are mixed, and is added
Culture medium, coated plate culture, with T7, T7teminator primer screening purpose bacterial strain；
(3) inducing expression of recombinant protein: by purpose strain inoculated in culture medium culture, being transferred to competent cell, will obtain
Bacterial strain carry out inducing expression；
(4) recombinant protein isolates and purifies: the bacterial strain after inducing expression success being expanded culture, obtained thallus is added and is cracked
Liquid is centrifuged after ultrasonication, obtains the destination protein present in expression bacterial strain in the form of inclusion body, inclusion body is washed,
Purifying；
(5) renaturation of recombinant protein: being packed into bag filter for recombinant protein after purification, successively dialysed with dialyzate, finally centrifugation,
Supernatant is taken, recombinant expression protein is obtained.
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Non-Patent Citations (3)
| Title |
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| Portunus trituberculatus chitinase 4 mRNA, complete cds;Song,L. and Wang,L;《GenBank: MH137202.1》;20190112;全文 * |
| Song,L. and Wang,L.Portunus trituberculatus chitinase 4 mRNA, complete cds.《GenBank: MH137202.1》.2019, * |
| 三疣梭子蟹(Portunus trituberculatus)几丁质酶PtCht3 基因克隆鉴定及表达分析;张凤等;《渔业科学进展》;20170430;第38卷(第2期);第167-174页 * |
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