CN105504038B - Turbot SmLTL recombinant proteins and its preparation and application method - Google Patents
Turbot SmLTL recombinant proteins and its preparation and application method Download PDFInfo
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- CN105504038B CN105504038B CN201610076867.4A CN201610076867A CN105504038B CN 105504038 B CN105504038 B CN 105504038B CN 201610076867 A CN201610076867 A CN 201610076867A CN 105504038 B CN105504038 B CN 105504038B
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
A kind of recombinant protein and its preparation and application method of turbot SmLTL, belong to molecular biology and genetic engineering field, it is made of 118 amino acid, molecular weight 13.47KDa, isoelectric point 8.52, amino acid sequence SEQ NO:1.The present invention is prepared for turbot SmLTL recombinant proteins, and turbot SmLTL recombinant proteins can inhibit infusorian vigor, and has fixation phenomenon with polypide, is that the research and development of the anti-infusorian drug of turbot play positive, important impetus.
Description
Technical field
The invention belongs to molecular biology and genetic engineering field, more particularly to a kind of turbot SmLTL recombinant proteins
And its preparation and application method.
Background technology
A large amount of mucus of fish skin secretion are covered in fish body surface and constitute first of the portal contacted with the external world extensively,
Protection fish body resists breeding environment variation and resists extraneous causal organism invasion and plays a crucial role.Fish body surface point
There are the multiple biological activities factors in secretion, and wherein mucus le ctin (lectin) is as important natural in fish epidermal mucus
Immune factor is gradually paid close attention to by domestic and foreign scholars.
Before the present invention makes, Lily-type lectin as one of main Types in mucus le ctin, be earliest from
(pufflectin) extracted in Fugu rubripes (Takifugu rubripes) epidermal mucus, is a kind of more special
Agglutinin, amino acid sequence have high homology with unifacial leaf mannose-binding lectin (MMB)；It is reported that MMB is a kind of heavy
The plant protection factor wanted, transfer-gen plant can resist the invasion of sucking insects and nematode etc.；At present for Lily-
Type lectin researchs are limited only to filefish T.rubripes, line murrel C.striata and Cynoglossus semilaevis C.semilaevis,
However, being rarely reported in application aspects researchs such as parasite inhibition for Lily-type lectin, the immune effect of insect resistace
It answers unclear.In conclusion turbot Lily-type lectin (SmLTL) recombinant protein is prepared and its is lived to shield infusorian
Power inhibits research, and at home and abroad there is no reports.
Invention content
The technical problem to be solved in the present invention is to provide a kind of preparation method and the egg of turbot SmLTL recombinant proteins
In vain to shield infusorian vigor inhibition application.
The present invention is completed according to following operational aspect：
A kind of recombinant protein of turbot SmLTL, it is made of 118 amino acid, and molecular weight 13.47KDa waits electricity
Point is 8.52, and amino acid sequence is as follows：
The preparation method of the recombinant protein of the turbot SmLTL, is as follows：
(1) design of gene order and excellent is carried out to turbot SmLTL (Genbank accession no.KU199003)
Change, and artificial synthesized nucleic acid fragment；
(2) nucleic acid fragment synthesized in step (1) is subcloned by table by NdeI/HindIII restriction enzyme sites
Up on carrier pET43.1a, recombinant expression plasmid pET-SmLTL is built；
(3) recombinant expression plasmid pET-SmLTL heat shock methods transformed competence colibacillus cell BL21 (DE3)；
(4) IPTG is added, 15 DEG C of expression overnight, and amplify culture；
(5) carrying out ultrasonic bacteria breaking, lysate are splined on nickel affinity column, purify destination protein, and dialyse；
(6) Western-blot methods detect recombinant expression protein.
Further, the artificial synthesized nucleic acid fragment of the step (1) is
Further, the amplification culture：Strain is inoculated into the culture mediums of LB containing ampicillin, in 37 DEG C of *
It is grown overnight in 200rpm shaking tables；It is inoculated within second day in the 3L TB culture mediums of the ampicillin containing 100ug/mL, is put into 37
When growing to OD=0.6-0.8 in DEG C * 200rpm shaking tables, shaking table temperature is down to 15 DEG C, final concentration 0.5mM is added after 1h
IPTG induced growths are stayed overnight；8000rpm*15min*4 DEG C is collected by centrifugation wet bacterium, and phosphate-buffered salt is used in combination to be resuspended once to clean bacterium
Body.
Further, with the ultrasonic wave of 500W power when the ultrasound cracks, ultrasound is carried out in ice bath, per ultrasound 3s,
Interval 8s amounts to 20min.
The present invention also provides one kind for inhibiting the ciliophoran composition of shield, including above-mentioned turbot SmLTL recombinant proteins.
The present invention also provides application of the turbot SmLTL recombinant proteins in inhibiting shield infusorian drug.
The advantageous effect of the present invention compared with prior art：
First, by the method for codon optimization, the shortcomings that overcoming the codon-bias of Escherichia coli so that albumen obtains
Obtain high expression；
Second, the present invention is by being prepared by recombinant turbot SmLTL recombinant proteins, and turbot SmLTL recombinant protein energy
Enough inhibit infusorian vigor, and there is fixation phenomenon with polypide, is that the research and development performance of the anti-infusorian drug of turbot is positive, important
Impetus.
Description of the drawings
Fig. 1 is the SDS-PAGE results of turbot SmLTL recombinant protein induced expression products；Wherein Lane M:SDS-
PAGE Protein marker；Lane 0:Control；Lane 1:15 DEG C of overnight inductions；Lane 2:37 DEG C of induction 4h.
Fig. 2 is the SDS-PAGE results of SmLTL recombinant protein purification products；Wherein Lane M:SDS-PAGE Protein
marker；Lane 1:Full bacterium breaks supernatant after bacterium centrifugation；Lane 2:Efflux after supernatant is incubated with Ni-IDA；Lane 3-6:
The Buffer A eluents of 50mM imidazoles；Lane 7-10:The Buffer A eluents of 100mM imidazoles；Lane 11-22:300mM
The Buffer A eluents of imidazoles.
Fig. 3 is the Western-blot analysis results of SmLTL recombinant proteins；LaneM2:Western Blot Marker；
Lane 1：Recombinant protein specific band.
Specific implementation mode
Technical scheme of the present invention is further explained below by embodiment and attached drawing, but the protection model of the present invention
It encloses and is not limited in any form by embodiment.
Embodiment 1, the optimization of the nucleic acid sequence of turbot SmLTL
1. the nucleic acid sequence optimization and synthesis of turbot SmLTL
Using the codon optimization software (MaxCodonTM of moral Thailand biotechnology (Nanjing) Co., Ltd recent development
Optimization Program (V13)), carry out SmLTL (Genbank access ion no.KU199003) gene order
Design and optimization, the nucleic acid sequence of optimization is：
2. on subclone to expression vector pET43.1a, building recombinant expression plasmid pET-SmLTL
Nucleic acid sequence after being optimized step 1 by NdeI/HindIII restriction enzyme sites is subcloned into expression vector
On pET43.1a, recombinant expression plasmid pET-SmLTL is built；
The expression and purification of embodiment two, SmLTL albumen in E. coli system
1.SmLTL protein expressions and identification
(1) purifying of expression vector：
Competent cell BL21 (DE3) is taken out from -80 DEG C of refrigerators, and is immediately placed in ice water, is melted on ice
2-5 minutes；The expression vector plasmid pET-SmLTL DNA of about 100ng are directly added into competent cell, gentle agitation mixes
It is subsequently placed at 30min on ice-water bath；Centrifuge tube is placed into 42 DEG C of water-baths, heat shock 90s；Quickly centrifuge tube is transferred in ice-water bath
3min；The LB liquid medium of 200ul is taken to be added in the competent cell converted after taking out；It is put into shaking table (37 DEG C of *
1h is grown in 195rpm)；50ul-80ul suspension even spreads are drawn to contain in 100ug/mL ampicillins (Amp) LB tablets；
Tablet is put on workbench several minutes so that the liquid of coating is absorbed, is inverted, 37 DEG C of overnight incubations.
(2) induced expression of bacterial strain：
It is inoculated with monoclonal from conversion tablet, is added in the LB culture mediums of 3 4ml Amp containing 100ug/mL, number is mark
It is denoted as " 0 " " 1 " " 2 "；It is 0.5-0.8 (it is generally necessary to 2-3 hours) that 37 DEG C of * 200rpm, which are cultivated to OD600,；Nothing in growth course
Sample is taken out under the conditions of bacterium measures OD600 values；Final concentration 0.5mM IPTG are added into Tube propagation liquid, and (" 0 " control group is not added with
IPTG), usually " 0 " " 1 " places 15 DEG C of overnight inductions, and " 2 " place 37 DEG C of induction 4h.
(3) SDS-PAGE identifies induced expression result：
12000rpm*10min*4 DEG C of centrifugation of each 400-600uL culture solutions in " 0 " " 1 " " 2 " test tube is taken respectively, removes supernatant
500ul dd H are added in liquid2Thalline, and 12000rpm*10min*4 DEG C of centrifugation again is resuspended in O, removes supernatant；50 μ l are added
Phosphate-buffered salt (PBS) mix be resuspended precipitation；2 × SDS sample-loading buffers that 50 μ l are added heat rapidly sample at 100 DEG C
15min makes albuminous degeneration, then 12000rpm*5min*4 DEG C of centrifuging and taking supernatant electrophoresis；10min 100V voltage stabilizing electrophoresis before electrophoresis,
200V voltage stabilizings electrophoresis to bromophenol blue band is migrated to from gel bottom 1cm after bromophenol blue indicator enters separation gel, takes out gel
It is dyed, is then continued in destainer with Coomassie brilliant blue dyeing liquor, decoloration to clear background.
As shown in Figure 1：The SmLTL recombinant proteins of codon optimization electrophoresis in SDS-PAGE, the results showed that through codon
Optimization design, can be in BL21 (DE3) strain high level expression under conditions of 15 DEG C of overnight inductions.
(4) SmLTL albumen amplification culture
It takes in the LB culture mediums of strain 20ul to the 4mL of ampicillin containing 100ug/mL of " 1 " conservation, in shaking table (37 DEG C of *
Growth is stayed overnight in 200rpm)；It is inoculated within second day in the 3L TB culture mediums of the ampicillin containing 100ug/mL, is put into shaking table (37
DEG C * 200rpm) in when growing to OD=0.6-0.8, shaking table temperature is down to 15 DEG C, final concentration 0.5mM IPTG are added after about 1h
Induced growth is overnight (about 16h)；
8000rpm*15min*4 DEG C is collected by centrifugation wet bacterium, and phosphate-buffered salt (PBS) is used in combination to be resuspended once to clean thalline.
2.SmLTL protein purifications
(1) BufferA is used:50mM Tris, 150mM NaCl, 1mM DTT, 1%Triton X-100,1ug/mL
Pepstatin A, 1ug/mL Leupeptin, 20mM imidazoles, bacterium mud is resuspended in pH8.0, and (usual lysate dosage is 10-15mL/g
Bacterium mud)；Ultrasound cracking carries out ultrasound with 500W power in ice bath, per ultrasound 3s, interval 8s, amounts to 20min, terminates aobvious
Micro- microscopic observation broken results；13000rpm*30min*4 DEG C of centrifugation retains supernatant, and 0.45 μm of membrane filtration is used in combination；Use Buffer
A balances 3mL Ni-IDA columns, and balance about 5-10CV is until ultraviolet registration reaches baseline；After the supernatant homostasis of the 3rd step filtering
Ni-IDA columns mixing be placed on rotary mixer in 4 DEG C be incubated 60-80min；With chromatographic column retention contain Lily
The Ni-IDA columns of Lectin albumen are used in combination Buffer A to rinse 5-10CV until ultraviolet registration reaches baseline；With containing respectively
Buffer A (being free of Triton X-100) elution target protein of 50mM, 100mM and 300mM imidazoles, flow control exist
1.5mL/min, and collect the eluant component of each imidazole gradient；Each 50 μ l of collection component are taken to be added on 2 × SDS of 50 μ l
Sample buffer solution, sample 10min is heated at 100 DEG C rapidly makes albuminous degeneration, then 12000rpm*5min*4 DEG C of centrifuging and taking supernatant electricity
Swimming.
(2) the SDS-PAGE electrophoresis of SmLTL expression products, 10min 100V voltage stabilizing electrophoresis before electrophoresis, later bromophenol blue refer to
Show that agent enters separation gel 200V voltage stabilizings electrophoresis to bromophenol blue band and migrates to from gel bottom 1cm, takes out gel Coomassie brilliant blue
Dyeing liquor dyes, and then continues in destainer, decoloration to clear background；The relatively high Lane 3-8 of purity are collected, for saturating
Analysis experiment.
As shown in Figure 2：Nickel column affinity purification SmLTL recombinant proteins, column is washed with the imidazoles of various concentration, according to SmLTL weights
It is balanced between histone yield and purity, the relatively high Lane 3-8 of purity is collected, for experiment of dialysing.
3.SmLTL albumen is dialysed
Target protein is carried out dialysis into 500mL protein storage liquid Buffer B：50mM Tris-HCl, 150mM NaCl, 10%
In Glycerol, pH8.0, with 4 DEG C of rotations of magnetic stirring apparatus, liquid is changed every 2h, total changes liquid 3 times, and dialyse about 8h；Dialysis terminates
Afterwards, with 0.45 μm of membrane filtration, and jelly is dispensed in -80 DEG C.
4.Western blot identifications
Sample transfers 3h after SDS-PAGE, by protein band on gel at 50V, until on nitrocellulose membrane；With 1%
BSA room temperatures close 1h, and TBST washes film 3 times (5min/ times), the diluted Anti-His antibody of 1.5mlTBST, room temperature is added to incubate
2h is educated, TBST washes 3 addition ELIAS secondary antibody sheep anti-mouse igg-HRP of film, is incubated at room temperature 2h；TBST washes 3 addition substrate solutions of film, shake
15min is swung, after abundant colour developing, tap water, which rinses, terminates reaction.
As shown in Figure 3：With the antibody of anti-His labels, Western blot identifications are carried out to SmLTL recombinant proteins, pre-
There is specific band at the molecular size range (about 13KD) of meter.
Embodiment three, turbot SmLTL recombinant proteins are to shield infusorian vigor inhibiting effect
Gently shake shield cilium polypide collection liquid (a concentration of 3.1 × 102/ ml), 1ml collection liquids are drawn, EP pipes are transferred to
In, into EP pipes be added various concentration SmLTL recombinant proteins (200,100,50,25,12.5ug/ml) be incubated under 28 °.It is right
According to group experiment without SmLTL recombinant proteins.Under the microscope, shield is ciliophoran under the conditions of observation of different time points various concentration deposits
Quantity living and dead, (3,6,12,24,48h).Under each concentration conditions, there are three repeating groups, the death rate takes it average
Number.
As shown in table 1：Increase with the concentration of SmLTL recombinant proteins, has significant impact, concentration to shield infusorian vigor
Bigger, the death rate is higher.
1 SmTL recombinant proteins of table are to infusorian vigor inhibition analysis
Claims (6)
1. a kind of recombinant protein of turbot SmLTL, it is characterised in that it is made of 118 amino acid, and molecular weight is
13.47KDa, isoelectric point 8.52, amino acid sequence are SEQ ID NO:1.
2. the preparation method of the recombinant protein of turbot SmLTL described in claim 1, it is characterised in that its specific steps are such as
Under：
(1) turbot SmLTL gene orders are designed and are optimized, and artificial synthesized nucleic acid fragment SEQ ID NO:2, institute
The turbot SmLTL gene order acquisition patterns stated are Genbank accession no. KU199003；
(2) by NdeI/HindIII restriction enzyme sites by step（1）The nucleic acid fragment of middle synthesis is subcloned into expression and carries
On body pET43.1a, recombinant expression plasmid pET- SmLTL are built;
(3) recombinant expression plasmid pET- SmLTL heat shock methods transformed competence colibacillus cell BL21 (DE3)；
(4) IPTG is added, 15 DEG C of expression overnight, and amplify culture；
(5) carrying out ultrasonic bacteria breaking, lysate are splined on nickel affinity column, purify destination protein, and dialyse；
(6) Western-blot methods detect recombinant expression protein.
3. the preparation method of the recombinant protein of the turbot SmLTL described in claim 2, it is characterised in that the amplification training
It supports：Strain is inoculated into the culture mediums of LB containing ampicillin, is grown overnight in 200rpm shaking tables at 37 DEG C of shaking table temperature；The
It is inoculated within two days in the 3L TB culture mediums of the ampicillin containing 100ug/mL, is put at 37 DEG C of shaking table temperature in 200rpm shaking tables
When growing to OD=0.6-0.8, shaking table temperature is down to 15 DEG C, final concentration 0.5mM IPTG induced growths are added after 1h and stay overnight；
The total 15min of 8000rpm are centrifuged at 4 DEG C, wet bacterium is collected after centrifugation, and phosphate-buffered salt is used in combination to be resuspended once to clean thalline.
4. the preparation method of the recombinant protein of the turbot SmLTL described in claim 2, it is characterised in that the ultrasound cracking
When with the ultrasonic wave of 500W power, ultrasound is carried out in ice bath, per ultrasound 3s, interval 8s, total 20min.
5. one kind including the weight of turbot SmLTL described in claim 1-4 any one for inhibiting the ciliophoran composition of shield
Histone.
6. application of the recombinant protein of turbot SmLTL described in claim 1 in inhibiting shield infusorian drug.
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Citations (3)
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|---|---|---|---|---|
| CN101434955A (en) * | 2008-12-18 | 2009-05-20 | 山东大学 | Recombinant expression method of insect C-type lectin and use |
| CN103833839A (en) * | 2012-11-27 | 2014-06-04 | 沈阳药科大学 | C-type lectin as well as preparation method and application thereof |
| CN104302769A (en) * | 2011-12-28 | 2015-01-21 | 科学与工业研究会 | Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof |
-
2016
- 2016-02-04 CN CN201610076867.4A patent/CN105504038B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434955A (en) * | 2008-12-18 | 2009-05-20 | 山东大学 | Recombinant expression method of insect C-type lectin and use |
| CN104302769A (en) * | 2011-12-28 | 2015-01-21 | 科学与工业研究会 | Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof |
| CN103833839A (en) * | 2012-11-27 | 2014-06-04 | 沈阳药科大学 | C-type lectin as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus;S Tsutsui等;《Comparative Biochemistry & Physiology Part B Biochemistry & Molecular Biology》;20060430;第143卷(第4期);第514-519页 * |
| 鱼类体表粘液凝集素研究进展;黄智慧等;《动物学研究》;20131231;第34卷(第6期);第674-678页 * |
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