CN104212717A - Application of antifreeze protein of paralichthys olivaceus - Google Patents

Abstract

The invention relates to the technical field of screening and application of functional genes in the aquatic biotechnology and particularly relates to an application of antifreeze protein of paralichthys olivaceus. The IV-type antifreeze protein of the paralichthys olivaceus is used as an application for preparing a low-temperature protective agent. The obtained IV-type antifreeze protein of the paralichthys olivaceus is cloned and expressed and the low-temperature protective effect of the IV-type antifreeze protein is analyzed by a low-temperature protection experiment of bacteria in vitro. The protein is easily and massively expressed by the bacteria, can be naturally folded under the conditions of expression and purification, has the characteristics of natural protein, and avoids the denaturation and renaturation processes of in-vitro protein expression.

Description

A kind of application of lefteye flounder antifreeze protein

Technical field

The present invention relates to the functional gene screening and application technical field in aquatic wholesale market, specifically a kind of application of lefteye flounder antifreeze protein.

Background technology

Under extreme cold conditions, fish in order to avoid freezing to the damage of body, antifreeze protein of evolving out (Antifreeze protein, AFP).AFP is found in the blood of a kind of Nototheniidae fish Antarctic Nototheniids (Trematomus nicolai) in Mcmurdo straits, the South Pole by DeVries.Its biological function is be combined with ice crystal specifically, is adsorbed on its surface and suppresses it to grow, changing its form, thus slow down ice-crystal growth speed.In addition, also there is recrystalization inhabition effect, make the ice crystal in body mainly little with volume and uniform state existence, thus avoid temperature to reduce the injury caused organism.AFP is divided into antifreeze glycoprotein (AFGPs) and not sugary antifreeze protein according to whether sugary albumen is or be called antifreeze peptide (AFP), the difference that not sugary antifreeze protein forms according to its amino acid, is divided into again I type, II type, III type familial combined hyperlipidemia.

AFP-IV is a kind of novel antifreeze proteins that first Deng etc. is separated to from the serum of long angle sculpin (Myoxocephalus octodecimspinosis).This antifreeze protein is usually containing 108 amino acid, and molecular weight is about 12.3KDa, and contains the Glu up to 17%, and sequence and film lipophorin have similarity (being about 22%), are therefore considered to be evolved by film lipophorin.This protein three-dimensional structure is the helical bundle that 4 left hand alpha-helix antiparallel arrangements are folded into, and wherein hydrophobic grouping is inside, and hydrophilic radical is outside, and the surface of hydrophilic group can be combined with ice crystal, stops ice crystal to infiltrate from skin surface.In addition, AFP-IV is have also discovered in the fish such as side line Antarctic Fish (Pleuragramma antarcticum) and the first Antarctic Fish of leather (Notothenia coriiceps), and carried out RT-PCR expression respectively, find that AFP-IV heat stagnation value is 0.08 DEG C when concentration is 0.5mg/mL, there is Activity of Antifreeze.

Lefteye flounder (Paralichthys olivaceus), be one of important marine fish of China, its strong adaptability, can tolerate the low temperature of 2 DEG C.Therefore, clone and identify lefteye flounder AFP-IV albumen, can be the functional study of this albumen in low-temperature protection and basis is provided.

Summary of the invention

The object of the invention is the application providing a kind of lefteye flounder antifreeze protein.

The technical solution used in the present invention is for achieving the above object:

An application for lefteye flounder antifreeze protein, lefteye flounder IV type antifreeze protein is as the application preparing cryoprotective agent.

Described lefteye flounder AFP IV albumen is for the preparation of the application of the cryoprotective agent outside bacterial body.

Described cryoprotective agent can maintain biological normal existence or reduce biological injury, dead material under referring to low temperature.Minimum temperature wherein in low temperature can reach 0 DEG C.

Described lefteye flounder AFP IV is by shown in sequence table SEQ ID No.1 nucleotide sequence.Described sequence clones the Gene A FP IV of the fish IV type Antifreeze protein gene homology obtained from lefteye flounder cDNA, and its cDNA total length 652bp, comprises 375bp open reading frame, 124 amino acid of encoding.

The advantage that the present invention has:

The present invention clones and expresses the lefteye flounder IV type antifreeze protein obtained, and analyzes its low-temperature protection effect by external bacterium low-temperature protection Protection.This albumen easily by bacterium great expression, naturally can fold, have the characteristic of native protein, avoid the sex change renaturation process of protein expression in vitro under expression of the present invention, purification condition.

Accompanying drawing explanation

PET-30a (+) the carrier composition schematic diagram that Fig. 1 provides for the embodiment of the present invention.

The recombinant large-tooth flounder IV type antifreeze protein purified product schematic diagram that Fig. 2 provides for the embodiment of the present invention.

The recombinant large-tooth flounder IV type antifreeze protein that Fig. 3 provides for the embodiment of the present invention is to the anti-low-temperature protection effect curves of bacterium.

Embodiment

Embodiment 1: the acquisition of lefteye flounder IV type Antifreeze protein gene

1. adopt Trizol to extract lefteye flounder RNA

A) get lefteye flounder zygote about 100 μ L, first add 200 μ L Trizol and fully grind, then add 800 μ L Trizol rifle head piping and druming evenly, homogenised sample is placed 5min at 15-30 DEG C;

B) leave standstill rear 4 DEG C of centrifugal 10min of 12000 × g, get supernatant in new centrifuge tube;

C) then in new test tube, add 200 μ L chloroforms again, thermal agitation 15s, leave standstill 2-3min;

D) at 4 DEG C after leaving standstill, 12000 × g, centrifugal 15min.Centrifugal rear sample divides three layers, and RNA is mainly at aqueous phase;

E) 600 μ L aqueous phases are transferred to new centrifuge tube, add 600 μ L Virahols, put upside down mixing, room temperature leaves standstill 10min, precipitated rna;

F) with 4 DEG C after staticly settling, 12000 × g, centrifugal 10min, RNA are deposited at the bottom of pipe in a gluey lamella;

G) add 1mL 75% ethanol (preparation of DEPC water) in pipe after centrifugation, vibrate latter 4 DEG C, the centrifugal 5min of 7500 × g, then removes supernatant;

H) dry air RNA is about 5min, adds 20 μ L DNase, RNase free water dissolution precipitation, can put into 58 DEG C of water-bath 10min and RNA is dissolved completely, preserve with-80 DEG C for subsequent use after RNA sample liquid nitrogen flash freezer.

The structure in 2.cDNA library

Completed by precious biotechnology (Dalian) company limited.Be specially lefteye flounder Total RNA, first extract PolyA+RNA, then reverse transcription synthetic double chain cDNA, through end smoothing, connect Adaptor, enzyme cut, remove short-movie section after be connected on pBluescript II SK (+) carrier and obtain elementary cDNA library.By connecting fluid Partial Conversion, be coated with the dull and stereotyped cDNA library obtaining 1,000,000 clonal expansions and amplify.

3. lefteye flounder IV type Antifreeze protein gene sequence

By 1,000,000 amplification library glycerol bacterium conserving liquid dilutions 10 ⁵coated plate doubly, chooses Colony Culture, extracts plasmid, carries out double digestion with EcoR I, Xho I, screens the fragment varied in size, and then corresponding mono-clonal checks order.Sequencing primer: T3Primer:5 '-ATTAACCCTCACTAAAGGGAA-3 ', T7Primer:5 '-TAATACGACTCACTATAGGG-3 '.Acquisition sequence is carried out Blast comparison at ncbi database, obtains lefteye flounder IV type Antifreeze protein gene sequence (Genebank:AF384676) as shown in SEQ ID No.1.

SEQ ID No.1

This cDNA total length is 781bp, and its open reading frame is 465bp, originates in 34bp, ends at 498bp, 154 amino-acid residues of encoding.In addition, the 3`UTR of 5`UTR and 283bp of this gene also containing 33bp, has the typical tailing signal AATAAA of vertebrates and PolyA tail.The various physico-chemical properties of ExPASy ProtParam online tool to lefteye flounder AFP IV protein are used to carry out forecast analysis, result shows that this protein relative molecular weight is 17.2KDa, theoretical iso-electric point pI is 7.95, be made up of 154 amino-acid residues, wherein Gln (Q) is 12, account for 7.8% of total content, its N holds the signal peptide shearing site for prediction between 20-21 amino acid.In ncbi database, Homology search is carried out to lefteye flounder AFP IV aminoacid sequence, find that itself and piebald Xiphophorus helleri, blue or green Medaka, spot garpike and atlantic salmon AFP-IV albumen consistence are respectively 65%, 62%, 42% and 44%.

Embodiment 2: the in-vitro recombination expression of lefteye flounder IV type antifreeze protein

1. the structure of external prokaryotic expression system

Sequences Design specificity amplification primer according to SEQ ID No.1, AFP IV-F 5 '-ACGAATTCATGAAGTTCTCCCTCGTTG-3 ', AFP IV-R 5 '-ACCTCGAGGAGTGAAACAGGAGCCCGAG-3 ', with lefteye flounder cDNA for template amplification AFP IV gene, reaction system is as follows:

PCR response procedures: 1.94 DEG C of 5min

2.94℃ 30s

3.55℃ 30s

4.72℃ 1min

5.2-435 circulation

6.72℃ 10min

Reaction terminate after, get 2 μ L PCR primer through 1% agarose gel electrophoresis detect, determine band single and with expection clip size consistent.PCR primer is reclaimed, together with prokaryotic expression carrier pET-30a (Fig. 1), all carries out double digestion with EcoR I and Xho I.Be connected after gene fragment is reclaimed with linearized vector fragment glue, transform Trans-T1 competent escherichia coli cell and carry out indigo plant screening in vain, adopting plasmid enzyme restriction and bacterium liquid PCR two kinds of method screening positive clones.The positive colony filtered out send corresponding bacterium liquid to check order to Beijing Liuhe Huada Genomics Technology Co., Ltd, and checking goal gene direction of insertion also determines that AFP IV gene reading frame remains unchanged.

The preparation of 2.BL21 (DE3) competent cell and recombinant expression plasmid transform

Use TSS solution to prepare fresh BL21 (DE3) competent cell, method is as follows:

(1) frozen BL21 (DE3) cell is taken out, streak culture dull and stereotyped in not adding any antibiotic LB, 37 DEG C of incubated overnight;

(2) choose single bacterium colony and be connected to 10mL LB liquid nutrient medium, in 37 DEG C, in 200rpm shaking table, be cultured to OD ₆₀₀0.4-0.5 (attention may not exceed 0.5);

(3) nutrient solution is poured into 50mL centrifuge tube, 2500 × g, 4 DEG C of centrifugal 15min collect thalline, TSS solution [1% peptone (weightmeasurement ratio) of 1ml precooling on ice, 0.5% yeast powder (weightmeasurement ratio), 0.5%NaCl (weightmeasurement ratio), 10% polyethylene glycol (Mr 3350) (weightmeasurement ratio), 5%DMSO (volume percent), 0.05M MgCl2, pH6.5] the resuspended thalline of pressure-vaccum gently.

Get 100 μ L competent cells immediately and do Plastid transformation, or competent cell is preserved in 2-3h on ice and used.

3. the recombinate abduction delivering of antifreeze protein and SDS-PAGE detects

(1) recombinant bacterium obtained above is chosen single colony inoculation contains kantlex LB liquid nutrient medium (50ug/mL kantlex) in 2mL, 37 DEG C of incubator overnight are cultivated;

(2) get 0.5mL bacterium liquid next day to transfer in 50mL containing in the LB liquid nutrient medium (50ug/mL kantlex) of kantlex, 37 DEG C of shaking tables are cultured to OD ₆₀₀0.5-1.0 (being best when 0.8), take out 1mL bacterium liquid as non-induced control sample, the centrifugal 5min of 12000rpm, abandon after supernatant by the resuspended precipitation of 50 μ L PBS+50 μ L 2 × SDS-PAGE sample buffer, after boiling 5min, the centrifugal 5min of 12000rpm, is stored in-20 DEG C by supernatant;

(3) in remaining bacterium liquid, add IPTG to final concentration 0.6mM inducing culture 4h, after getting 1mL induction, bacterium liquid is by upper one step process process, as induced samples;

(4) get non-induced control sample and each 10 μ L of induced samples carry out SDS-PAGE, PAGE glue adopts 15% separation gel, arrange current stabilization 7mA until sample instruction forward position reach on separation gel along time, regulate electric current to 10mA until electrophoresis terminates;

(5) albumin glue dyes 30min in staining fluid, then decolours in destainer, and carries out scanning record to albumin glue.

4. restructuring antifreeze protein purifying

(1), according to step inducing culture 400mL bacterium liquid noted earlier, after induction 4h, bacterium liquid is collected thalline in the centrifugal 5min of 11000rpm;

(2) according to the amount adding 20-30mL B-PER (Thermo, USA) in often liter of bacterium liquid, add 12mL B-PER solution and blow and beat resuspended thalline, incubated at room 30min;

(3) 11000rpm (whizzer maximum speed of revolution) centrifugal 30min, moves supernatant to new pipe;

(4) in residue precipitation, add 6mL B-PER solution and blow and beat resuspended thalline, incubated at room 30min;

(5) the centrifugal 30min of 11000rpm, moves supernatant to new pipe;

(6) repeat (4), (5) two steps, obtain clean supernatant protein suspension (lysates);

(7) in purification column, add the affine resin (Clontech, USA) of 2mL 50%TALON metal, open end cap, allow ethanol in filler flow out;

(8) add 4mL B-PER diluent (B-PER diluent is that B-PER and lavation buffer solution mix in the ratio of 1:1 (v/v)), open end cap, allow liquid in filler flow out; Lavation buffer solution is 50mMNaH ₂pO4; 300mM NaCl; 20mM imidazoles.

(9) in lysates, add 4mL lavation buffer solution, mix gently.Get 4mL upper prop, after His-tag albumen and filler fully combine, open end cap, allow liquid in filler flow out;

(10) pillar is washed with 20mL PBS;

(11) successively with the lavation buffer solution washing pillar that 10mL imidazole concentration is 20mM, 40mM, 100mM, last outflow component is collected;

(12) with elution buffer (50mM NaH ₂pO4; 300mM NaCl; 250mM imidazoles) wash-out is carried out to albumen, use 0.5mL, co-elute 8 times at every turn, collect each outflow component respectively;

(13) SDS-PAGE detection is carried out.

5. restructuring antifreeze protein desalination and concentration

Use ultra-15 centrifugal filter, is added beyond filter device by purified protein samples, the filtration unit building lid is put into centrifugal rotor, with the centrifugal 30min of 4000 × g.Add PBS degerming after filtration subsequently and wash 2 times, all use 4000 × g centrifugal 30min desalination and unnecessary damping fluid at every turn.Then, rifle head is stretched into bottom filtration unit, absorption sample of vacillating now to the left, now to the right, to reclaim concentrated rear albumen completely.After degerming after filtration, measuring AFP IV purifying protein concentration is 0.56mg/mL.

Embodiment 3: the analysis of recombinant large-tooth flounder AFP IV proteobacteria low-temperature protection effect

E. coli Trans T1 is cultured to OD ₆₀₀be about 1.0, with LB substratum gradient dilution 10 ⁶doubly, draw 4ml in centrifuge tube, adding final concentration is recombinant large-tooth flounder AFP IV albumen (degerming after filtration) after the purifying of 50 μ g/ml, not add the equivalent bacterium liquid of AFP IV albumen for contrast.Process 0,24,48,72,96,120 and 144h respectively at 4 DEG C, get 100 μ l respectively in the corresponding time and coat on LB flat board, carry out enumeration after 12-16h is cultivated in 37 DEG C of inversions, often organize 5 repetitions.With colony number and deepfreeze time for parameter is mapped, process 48h, the colony number of experimental group and control group is all on a declining curve with the prolongation of deepfreeze time, the two there was no significant difference; 72h-96h, experimental group colony number has obvious rising, is significantly higher than control group, but declines again consistent as control group at 96h; After 96h, experimental group colony number is in rising trend, and the control group not adding recombinant large-tooth flounder AFP IV albumen continues on a declining curve, and experimental group colony number is significantly higher than control group.Result shows recombinant large-tooth flounder AFP IV albumen to add in the bacterial cultures of deepfreeze, can significantly improve bacterium survival ability at low temperatures, and recombinant expressed lefteye flounder AFP IV albumen has obvious low-temperature protection effect.

Above-mentioned intestinal bacteria also can be replaced by other bacterium, and then adopt protective material of the present invention all can significantly improve bacterium survival ability at low temperatures to other bacterium.

Claims (3)

1. an application for lefteye flounder antifreeze protein, is characterized in that: lefteye flounder IV type antifreeze protein is as the application preparing cryoprotective agent.

2., by the application of lefteye flounder antifreeze protein according to claim 1, it is characterized in that: described lefteye flounder AFP IV albumen is for the preparation of the application of the cryoprotective agent outside bacterial body.

3., by the application of the lefteye flounder antifreeze protein described in claim 1 or 2, it is characterized in that:

Described lefteye flounder AFP IV is by shown in sequence table SEQ ID No.1 nucleotide sequence.