CN105755030B - A kind of preparation method of pinctada fucata martensii meat anti-oxidizing peptide - Google Patents

A kind of preparation method of pinctada fucata martensii meat anti-oxidizing peptide Download PDF

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CN105755030B
CN105755030B CN201610130757.1A CN201610130757A CN105755030B CN 105755030 B CN105755030 B CN 105755030B CN 201610130757 A CN201610130757 A CN 201610130757A CN 105755030 B CN105755030 B CN 105755030B
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pinctada fucata
pfmap
oxidizing
martensii meat
peptide
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CN105755030A (en
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吴燕燕
马永凯
李来好
杨贤庆
王锦旭
林婉玲
赵永强
戚勃
邓建朝
马海霞
荣辉
胡晓
杨少玲
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South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
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Abstract

The invention discloses a kind of preparation methods of pinctada fucata martensii meat anti-oxidizing peptide, the following steps are included: the amino acid sequence of (1) according to pinctada fucata martensii meat anti-oxidizing peptide, construct its target gene, after double digestion, it is connected with the prokaryotic expression plasmid PGEX-6P-1 equally through double digestion, pronuclear recombination expression vector is made, recombinant strain is made to competent escherichia coli cell through heat-shock transformed in pronuclear recombination expression vector；(2) by recombinant strain culture, and after adding IPTG Fiber differentiation, thalline were collected by centrifugation, and precipitating will be collected after thallus ultrasonication, obtains the precipitating containing destination protein；(3) it is isolated and purified using GST agarose affinity chromatography to get pinctada fucata martensii meat anti-oxidizing peptide is arrived.This method is efficient, specificity is strong, at low cost, the pinctada fucata martensii meat anti-oxidizing peptide purity is high of acquisition, and antioxidant activity is strong.

Description

A kind of preparation method of pinctada fucata martensii meat anti-oxidizing peptide
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of preparation side of pinctada fucata martensii meat anti-oxidizing peptide Method.
Background technique
With many natural protein-based anti-oxidation active substances in marine organisms body, China's oceanic area is wide, horizontal Across the torrid zone, subtropical zone, three, temperate zone climate zone, marine species are abundant, provide a natural marine activity for researcher Larger molecular organics library source, it can be found that more efficient, single-minded macromoleculars or small molecule anti-oxidation active substance, and then it is more preferable Ground is applied to actual production come the healthy living level for improving people's dietary nutrition structure, improving the people.It deeply develops and utilizes sea Foreign living resources are not only able to satisfy and ensure the higher value application of domestic food supply and some low value fish and shellfish, but also can bring More job opportunities promote related industry quickly transition and upgrade, and to maintenance national marine equity, construction ocean power Also there is certain positive effect.
Pinctada fucata is also known as pteria martensii, is female shellfish of China's sea-farming production " Nan Zhu ", belongs to Mollusca Lamellibranchiata pearl shell mesh Pteriidae Pinctada is mainly distributed on Guangxi, Guangdong, Hainan and Sound Velocity in Southern Taiwan Strait is coastal, day This region following the line of the sea；Pinctada fucata meat is a kind of sea in the high high-quality protein source of high protein, low fat, low sugar, nutritive value Product.Its crude protein dry weight content is between 66.71%~81.2%, value of exploiting and utilizing with higher.
Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst has been devoted to the exploitation of pinctada fucata meat in the past 10 years It utilizes, the exploration of the anti-oxidation peptide sterling functional character of thick enzymolysis liquid drinks from the beginning by now has accumulated a system The achievement of column.But pinctada fucata martensii meat anti-oxidizing peptide food, medicine and cosmetic field application then due to research and develop the time Short, upstream pearl aquaculture transition and upgrade is slow, industrial chain the degree of automation is low, chemical synthesis safety problem, shellfish meat extraction purification The reasons such as rate is low, process costs are high cause its application still in primary laboratory theoretical research stage.So also needing further to tie It closes the raising of modern biology technology to prepare anti-oxidation peptide speed and purity, its clear structure-activity relationship, reduce process costs, and utilizes Genetic engineering carries out protokaryon or eukaryotic expression to clearly rear pinctada fucata source anti-oxidation peptide sequential structure, to realize big rule Mould preparation production pinctada fucata source anti-oxidation peptide, the fields such as final Transformation Application to food, medicine, cosmetics.
Recent studies have found that by being oriented enzymatic hydrolysis, available small molecule antioxidation active peptides, warp to pearl oyster shellfish meat Mass spectroscopy structural parses to obtain corresponding amino acid sequence Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg；With Corresponding amino acid sequence be abbreviated as GAGLPGKRER (referring particularly in the patent of invention application No. is 201410040473.4 It is described)；The peptide antioxidant activity is strong and more stable, in order to by this anti-oxidation peptide mass production application, if only using enzyme The method for solving pearl oyster meat, containing various other peptides and small-molecule substance, purifies cost since enzymolysis process side reaction is more Higher, yield is again low.Therefore, it is badly in need of finding Hepu pearl oyster of strong, the at low cost method of a species specificity to produce this sequence Shellfish meat anti-oxidation peptide (PFMAP, Pinctada fucata meat antioxidant peptides).
Therefore, the quasi- one section small molecule isolated with South China Sea Inst .of Aquatic Products, Chinese Academy of aquatic Products early period of the present invention is anti- Based on aoxidizing peptide (Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg), pass through Escherichia coli (E.coli) original Nuclear expression prepares pinctada fucata martensii meat anti-oxidizing peptide, this is compared with prepared by traditional enzyme solution to prepare anti-oxidation peptide sterling pure There to be unrivaled advantage in terms of degree, preparation speed, cost, the degree of automation！
Summary of the invention
The purpose of the present invention is to provide a kind of sides of recombination pinctada fucata meat anti-oxidation peptide fusion gene pronucleus expression Method, this method is efficient, specificity is strong, at low cost, and the recombination pinctada fucata meat anti-oxidation peptide fusion purity is high of acquisition resists Oxidation activity is strong.
Above-mentioned purpose of the invention is achieved through the following technical solutions: a kind of pinctada fucata martensii meat anti-oxidizing peptide Preparation method, comprising the following steps:
(1) according to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP, its target gene PFMAP is constructed, it will PFMAP is through restriction enzyme Bam HI and Xho I double digestion, with the prokaryotic expression equally through Bam HI and Xho I double digestion Plasmid PGEX-6P-1 is connected, and pronuclear recombination expression vector PGEX-6P-1-PFMAP is made, pronuclear recombination expression vector is passed through It is heat-shock transformed to Escherichia coli Rosetta (DE3) competent cell, recombinant strain DE3-PGEX-6P-1-PFMAP is made；
(2) recombinant strain DE3-PGEX-6P-1-PFMAP is contained into ampicillin and chloramphenicol sterilized LB Escherichia coli culture medium in cultivate, add IPTG Fiber differentiation after, thalline were collected by centrifugation, will collect after thallus ultrasonication Precipitating, and detected with SDS-PAGE, the precipitating containing destination protein is spare；
(3) precipitating containing destination protein is isolated and purified using GST agarose affinity chromatography, as pinctada fucata meat is anti- Aoxidize peptide.
The present invention utilizes the method for molecular biology and genetic engineering building recombination pinctada fucata meat anti-oxidation peptide for the first time The engineered strain of fusion；For its later period efficiently, stablize expression anti-oxidation peptide, industrial-scale production and its cosmetics, The application of the fields such as health food lays the foundation.
In the preparation method of above-mentioned pinctada fucata martensii meat anti-oxidizing peptide:
According to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP in step (1), its target gene is constructed The detailed process of PFMAP are as follows: according to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP, according to Escherichia coli preference Codon designs its corresponding mRNA sequence, and further constructs its nucleotide full length sequence, according to its full length sequence, synthesis Corresponding two single-stranded, by two single-stranded progress PCR reactions of degeneration (RD), constructs pinctada fucata martensii meat anti-oxidizing peptide target gene PFMAP。
The process for further constructing its nucleotide full length sequence is: mRNA sequence both ends be added restriction endonuclease Bam HI and The base sequence of Xho I, and 3~5 protection bases are added respectively at the base sequence both ends of restriction endonuclease Bam HI and Xho I, To construct the nucleotide full length sequence of pinctada fucata martensii meat anti-oxidizing peptide.
Tri- protection bases of CGC are added on the left side of restriction endonuclease Bam HI；CGG tri- are added on the right of restriction endonuclease XhoI Protect base.
Temperature when PCR reaction of degeneration (RD) is preferably 92~96 DEG C, and denaturation time is preferably 2~5min.
In step (2) when addition IPTG Fiber differentiation, temperature is preferably 30~40 DEG C, preferably in 200~250rpm of revolving speed 3~5h of Fiber differentiation；Preferably, temperature is 37 DEG C, in revolving speed 220rpm Fiber differentiation 4h.
In step (3) preferably by GST agarose affinity chromatography twice isolate and purify the precipitating containing destination protein to get To recombination pinctada fucata meat anti-oxidation peptide fusion.
Sample after the purifying of first time GST agarose affinity chromatography and digestion is crossed into second of GST agarose affinity chromatography, Efflux is collected, the component being collected into is subjected to Tricine-SDS-PAGE detection, the sample containing target gene PFMAP is Pinctada fucata martensii meat anti-oxidizing peptide.
Compared with prior art, the present invention has the advantage that
(1) present invention preparation product purity advantage: the present invention with regard to pinctada fucata antioxidant peptide active small molecule peptide fragment and Speech, relative to the purity is high that traditional enzyme solution obtains, this is because the fusion egg of escherichia expression system inducing expression GST agarose affinity chromatography isolates and purifies purity of protein height to Bai Jing twice, and specificity is strong, and more easy purification；And conventional method The enzymolysis liquid of acquisition Degree of Enzymatic Hydrolysis control different batches will receive raw material, operator, environment influence and cause to digest Degree it is different so that pinctada fucata meat enzymatic hydrolysis small active peptides it is not of uniform size, at the purifying in later period Reason is made troubles；
(2) present invention preparation speed advantage: the present invention is lured since e. coli host bacteria repoductive time used is 20min It is short to lead incubation time, greatlys save the time, however traditional enzyme solution is due to by conditions such as cost of material, working process modes Limitation many limits such as have in technique yield in unit time for E. coli system in the actual production process System；
(3) preparation cost advantage of the present invention: since Escherichia coli Growth culture medium raw material is cheap, it is easy to extensive Production obtains a large amount of antioxidant activity peptide material, has economy relative to traditional enzyme solution in cost；
(4) compared with traditional zymolysis technique, chemical solid phase synthetic technology preparation antioxidant activity polypeptide, the method for the present invention is more Quickening is fast, efficient, and the method for the present invention the degree of automation arrives, and whole-course automation unattended may be implemented, people is greatly saved Power cost；
(5) operation of the present invention is simple, can recombinate pinctada fucata meat anti-oxidation peptide fusion gene pronucleus table with rapid build The engineered strain reached, the DPPH oxidation resistance of example proof PFMAP improve 5 times before being more transformed, this is later period small molecule The application in cosmetics, field of health care food of active peptide lays the foundation.
Detailed description of the invention
Fig. 1 is first time GST agarose affinity chromatography purifying SDS-PAGE analysis chart, wherein M:Protein marker； 1: loading；2: outflow；3:Binding buffer rinses component；4-7:20mM GSH elution fraction；
Fig. 2 is second of GST agarose affinity chromatography Tricine-SDS-PAGE analysis chart, wherein M:Protein marker；1: before cutting；2: after cutting；3: outflow；4:20mM GSH elution.
Specific embodiment
Embodiment 1
The method of recombination pinctada fucata meat anti-oxidation peptide fusion gene pronucleus expression provided in this embodiment, including it is following Step:
(1) pinctada fucata martensii meat anti-oxidizing peptide target gene PFMAP is constructed
According to the amino acid sequence (Gly-Ala-Gly-Leu-Pro-Gly- of pinctada fucata martensii meat anti-oxidizing peptide PFMAP Lys-Arg-Glu-Arg, refering to application No. is 201410040473.4 patents), according to the codon of coli strain Preference is designed and PFMAP amino acid sequence (Gly-Ala-Gly- with computer software Primer Premier 5.0 Leu-Pro-Gly-Lys-Arg-Glu-Arg, refering to the patent of application number 201410040473.4) corresponding mRNA sequence.
MRNA sequence are as follows:
5’-AATGGTGGTGCAGGTCTGCCTGGTAAACGTGAGCGTAATGGTGGTGCTGGCCTGCCAGGTAAACG TGAACGCAACGGTGGCGCAGGTCTGCCGGGCAAGCGCGAACGTAACGGTGGTGCTGGTCTGCCGGGTAAACGCGAG CGCAACGGTGGTGCGGGTCTGCCTGGCAAACGTGAACGTAATGGTTAA-3’。
In the mRNA sequence, AT content is 0.4, G/C content 0.6, in the restriction enzyme site difference that active peptide both ends are added For restriction enzyme Bam HI and Xho I；In order to which later period restriction enzyme can smoothly cut restriction enzyme site, selection exists Both ends add 3~5 protection bases (on the left side Bam HI plus tri- protection bases of CGC respectively；On the right of XhoI restriction enzyme site In addition tri- protection bases of CGG).
Target gene full length sequence after the completion of design is as follows:
5’-CGCGGATCCATGAATGGTGGTGCAGGTCTGCCTGGTAAACGTGAGCGTAATGGTGGTGCTGGCCT GCCAGGTAAACGTGAACGCAACGGTGGCGCAGGTCTGCCGGGCAAGCGCGAACGTAACGGTGGTGCTGGTCTGCCG GGTAAACGCGAGCGCAACGGTGGTGCGGGTCTGCCTGGCAAACGTGAACGTAATGGTTAACTCGAGCGG-3’。
The objective gene sequence that above-mentioned analysis is completed, synthesize it is corresponding two it is single-stranded as follows:
PFMAP-P1:
5’-CGCGGATCCATGAATGGTGGTGCAGGTCTGCCTGGTAAACGTGAGCGTAATGGTGGTGCTGGCCT GCCAGGTAAACGTGAACGCAACGGTGGCGCAGGTCTGCCGGGCAAGCGCGAACGTAACGGTGGTGCTGGTCTGCCG GGTAAACGCGAGCGCAACGGTGGTGCGGGTCTGCCTGGCAAACGTGAACGTAATGGTTAACTCGAGCGG-3’。
PFMAP-P2:
5’-CCGCTCGAGTTAACCATTACGTTCACGTTTGCCAGGCAGACCCGCACCACCGTTGCGCTCGCGTT TACCCGGCAGACCAGCACCACCGTTACGTTCGCGCTTGCCCGGCAGACCTGCGCCACCGTTGCGTTCACGTTTACC TGGCAGGCCAGCACCACCATTACGCTCACGTTTACCAGGCAGACCTGCACCACCATTCATGGATCCGCG-3’。
Obtain target gene: by two single-stranded PFMAP-P1 and PFMAP-P2 of above-mentioned synthesis, at room temperature 10,000rpm It is centrifuged 1min；Then, be added DEPC processing water by concentration dilution to 50 μM, according to the PCR system of 25 μ L 95 DEG C in PCR instrument, It is denaturalized 5min, cooled to room temperature (1~2 DEG C/min is cooling preferably in instrument)；PCR product need to be solidifying through 2.5% agarose Gel electrophoresis verifying, obtains pinctada fucata martensii meat anti-oxidizing peptide PFMAP target gene, it is spare to be stored in 4 DEG C of refrigerators.
Double digestion: restriction enzyme Bam HI and Xho I double digestion target gene PFMAP is utilized: by above-mentioned acquisition Pinctada fucata martensii meat anti-oxidizing peptide PFMAP target gene is with restriction enzyme Bam HI and Xho I according to the digestion body of 20 μ L 37 DEG C of water-bath digestion 5h are lain in, the 10 × Loading Buffer for taking 5 μ L endonuclease reaction liquid that 0.5 μ L is added after the completion of digestion exists Verify whether that digestion is complete under 2.5% agarose gel concentration, if digestion is complete, according to invitrogene plastic recovery kit PFMAP genetic fragment after recycling double digestion.
Utilize restriction enzyme Bam HI and Xho I double digestion plasmid PGEX-6P-1 (hereinafter referred to as PGEX): will be by GE The PGEX plasmid of company's purchase is equally used with restriction enzyme Bam HI and Xho I according to the digestion system of 20 μ L in 37 DEG C of water It bathes digestion for 24 hours, takes 5 μ L endonuclease reaction liquid that 10 × Loading Buffer of 0.5 μ L is added in 2.5% agarose after the completion of digestion Verify whether that digestion is complete under gum concentration, if digestion is complete, after water-bath 20min inactivates enzyme in 65 DEG C of remaining endonuclease reaction liquid It is recycled according to the TaKaRaMiniBEST DNAFragment Purification Kit Ver.4.0 kit of TAKARA company PGEX genetic fragment after double digestion.
Connection: by after double digestion obtained above PGEX and PFMAP according to the linked system of 20 μ L 16 in metal bath DEG C connection 16h, obtain connection liquid.
Conversion: connection liquid is transferred in DE3 competent cell.
It is gently shaken after taking 100 μ L Escherichia coli DE3 competent cells, ice bath to thaw, so that cell is suspended uniform.By 10 μ L It connects liquid to be added in competent cell, mix gently, ice bath 30min.42 DEG C heat shock 90 seconds, be then transferred quickly in ice bath, Stand 2 minutes.Be added 900 μ L LB liquid mediums, 37 DEG C, 150rpm concussion be incubated for 1h, 3000rpm is centrifuged 1min, in abandoning Clearly.A small amount of culture is taken to be spread evenly across LA-C (50 μ g/mL ampicillins and 34 μ g/mL chloramphenicol) solid medium tablets On.Plate is inverted into 37 DEG C of constant incubators, cultivates 10-16h, observes single colonie growing state.
Identification recombinant vector DE3-PGEX-GST-PFMAP: the single bacterium grown after culture in picking (4) is dropped down onto equipped with LA-C In the EP pipe of the 2mL of fluid nutrient medium, 37 DEG C, 180rpm culture 10h do bacterium colony PCR verifying, double digestion verifying or sequencing and test Card, wherein best with sequence verification, verification result shows that the present invention successfully constructs recombinant vector DE3-PGEX-GST-PFMAP.
Wherein:
The preparation of LB liquid medium: tryptone 10g is weighed, yeast extract 5g, sodium chloride 10g add deionized water It is cooled to room temperature spare to 1000mL, 121 DEG C of high pressure sterilization 20min.
The preparation of LA-C fluid nutrient medium: tryptone 10g is weighed, yeast extract 5g, sodium chloride 10g add deionization Ampicillin and chloramphenicol is added to final concentration of 50 μ g/mL ammonia to 1000mL, 121 DEG C of high pressure sterilization 20min in water after cooling Parasiticin and 50 μ g/mL, 4 DEG C save backup.
The preparation of LA-C solid medium: weighing tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, Add deionized water to 1000mL, 121 DEG C of high pressure sterilization 20min, ampicillin and chloramphenicol are added after cooling to final concentration of 50 μ g/mL ampicillins and 50 μ g/mL, 4 DEG C save backup.
(2) recombinant strain DE3-PGEX-GST-PFMAP inducing expression
It chooses single bacterium and falls within 100/500mL and contain in the taper triangular flask of LA-C fluid nutrient medium, in 37 DEG C, 220rpm condition It is lower to cultivate to OD600When about 0.6, addition IPTG induction makes its final concentration of 0.5mmol/L, continues culture induction 4h, this process Setting is not added with IPTG inducer as negative control.
4000rpm is centrifuged 10min and collects thallus, abandons supernatant；Thallus is suspended with 500 μ L PBS (PH7.4) buffers, ultrasound Broken 6min, super 0.5s stop 1.5s, are collected by centrifugation supernatant precipitating respectively, precipitating with 500 μ L solubilization of inclusion bodies liquid (8M Urea, 50mM Tris-HCl, 150mM NaCl, pH8.0) dissolution, 40 μ L samples and 10 μ 5 × protein of L loading are taken respectively Buffer is mixed, boiling water bath 10min.
SDS-PAGE detection: prepare 12% SDS-PAGE, Tris-Gly electrophoretic buffer (Tris 30g, glycine 144.0g, SDS 10g, are settled to 1L), glue 80V 20min, separation gel 120V 60min, gel electrophoresis is concentrated in 10 μ L of applied sample amount Dye 20min is examined in end, and decoloration photographs to record.
The 4 DEG C of preservations of precipitating for the PFMAP containing destination protein that verifying is correctly gone out through prokaryotic expression are standby With.
The step can verify the host strain containing recombinant plasmid DE3-PGEX-6P-1-PFMAP after inducing expression Whether successful secretion goes out the small molecular protein containing destination protein PFMAP, according to the small molecular protein of SDS-PAGE electrophoresis result Molecular size range judgement.
(3) purifying of PFMAP
The above-mentioned precipitating containing destination protein is isolated and purified to obtain sterling PFMAP through GST agarose affinity chromatography twice, Wherein, crossing column purification for the first time is sterling GST-PFMAP in order to obtain, and second of purifying is sterling PFMAP in order to obtain.
First time GST agarose affinity chromatography purification step are as follows:
(a) 5mLGST column material is taken, cleans balance pillar, flow velocity 5mL/ with the Binding Buffer of 10 times of bed volumes min；
(b) upper prop, flow velocity 2mL/min, collection penetrate liquid；
(c) the Binding Buffer of 10 times of bed volumes cleans pillar, flow velocity 5mL/min；
(d) Elution Buffer is eluted, flow velocity 2mL/min, collects eluent.
Wherein: Binding Buffer includes: 1 × PBS, 20% glycerol, 2mM DTT, pH7.4；Elution Buffer It include: the glycerol of 20mM GSH, 1 × PBS, 20%, 2mM DTT, pH7.4.
The component being collected into is subjected to SDS-PAGE detection, correct destination protein GST-PFMAP will be verified, 3C egg is added White enzyme, digestion are overnight；SDS-PAGE result is as shown in Figure 1 after first time GST agarose affinity chromatography.
From figure 1 it appears that the protein band of 32KDa or so is exactly the purpose egg containing GST label protein in Fig. 1 It is white.
Second of GST agarose affinity chromatography purification step are as follows:
Sample after the purifying of first time GST agarose affinity chromatography and digestion is crossed into second of GST agarose affinity chromatography, Efflux is collected, the component being collected into is subjected to Tricine-SDS-PAGE detection；Correct PFMAP sample is detected to protect in 4 DEG C It deposits or sample is lyophilized and save.
Wherein the first step is crossed after column digestion in sample containing GST label protein and two kinds of destination protein PFMAP, through GST fine jade Lipolysaccharide affinity chromatography, GST label protein are adsorbed, and destination protein PFMAP outflow, purity is generally 90% or more.
SDS-PAGE result after second of GST agarose affinity chromatography is as shown in Fig. 2, from figure 2 it can be seen that 6.5kDa Place is destination protein PFMAP.
Embodiment 2
The activity verifying of pinctada fucata martensii meat anti-oxidizing peptide PFMAP
Using improved Rajapakse N et al. to the measuring method of DPPH clearance rate, while measuring preparation of the present invention Target gene PFMAP (being prepared in embodiment 1) and transformation before pinctada fucata martensii meat anti-oxidizing peptide DPPH remove energy The IC of power50Value, the results showed that the IC of the DPPH oxidation resistance of the anti-oxidation peptide before transformation50It is 0.132mg/mL, the present invention The IC of the DPPH oxidation resistance of the PFMAP of preparation50It is 0.0289mg/mL, the DPPH oxidation resistance of the PFMAP in the present invention Improve 4.6 times.
Specific embodiment listed above remarks additionally to the present invention, it should be pointed out that above embodiments are served only for The invention will be further described, does not represent protection scope of the present invention, other people are non-for prompting to make according to the present invention The modification and adjustment of matter, still fall within protection scope of the present invention.

Claims (8)

1. a kind of preparation method of pinctada fucata martensii meat anti-oxidizing peptide, it is characterized in that the following steps are included:
(1) according to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP, its target gene PFMAP is constructed, it will PFMAP is through restriction enzyme Bam HI and Xho I double digestion, with the prokaryotic expression equally through Bam HI and Xho I double digestion Plasmid PGEX-6P-1 is connected, and pronuclear recombination expression vector PGEX-6P-1-PFMAP is made, pronuclear recombination expression vector is passed through It is heat-shock transformed to Escherichia coli Rosetta(DE3) competent cell, recombinant strain DE3-PGEX-6P-1-PFMAP is made；
(2) by recombinant strain DE3-PGEX-6P-1-PFMAP in the sterilized LB containing ampicillin and chloramphenicol It is cultivated in Escherichia coli culture medium, after adding IPTG Fiber differentiation, thalline were collected by centrifugation, heavy by collecting after thallus ultrasonication It forms sediment, and is detected with SDS-PAGE, the precipitating containing destination protein is spare；
(3) precipitating containing destination protein, as pinctada fucata martensii meat anti-oxidizing are isolated and purified using GST agarose affinity chromatography Peptide；
Target gene full length sequence is as follows in step (1):
5’-
CGCGGATCCATGAATGGTGGTGCAGGTCTGCCTGGTAAACGTGAGCGTAATGGTGGTGCTGGCCTGCCAGGT AAACGTGAACGCAACGGTGGCGCAGGTCTGCCGGGCAAGCGCGAACGTAACGGTGGTGCTGGTCTGCCGGGTAAAC GCGAGCGCAACGGTGGTGCGGGTCTGCCTGGCAAACGTGAACGTAATGGTTAACTCGAGCGG-3’。
2. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 1, it is characterized in that: root in step (1) According to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP, the detailed process of its target gene PFMAP is constructed are as follows: root According to the amino acid sequence of pinctada fucata martensii meat anti-oxidizing peptide PFMAP, it is corresponding that its is designed according to Escherichia coli preferred codons MRNA sequence, and further construct its nucleotide full length sequence, according to its full length sequence, synthesize corresponding two it is single-stranded, by two The single-stranded progress PCR reaction of degeneration (RD) of item, constructs pinctada fucata martensii meat anti-oxidizing peptide target gene PFMAP.
3. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 2, it is characterized in that: further constructing it The process of nucleotide full length sequence is: in the base sequence of the both ends of mRNA sequence addition restriction endonuclease Bam HI and Xho I, and The base sequence both ends of restriction endonuclease Bam HI and Xho I add 3 ~ 5 protection bases respectively, to construct pinctada fucata meat The nucleotide full length sequence of anti-oxidation peptide.
4. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 3, it is characterized in that: in restriction endonuclease Bam The left side of HI adds tri- protection bases of CGC；Tri- protection bases of CGG are added on the right of restriction endonuclease XhoI.
5. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 2, it is characterized in that: PCR reaction of degeneration (RD) When temperature be 92 ~ 96 DEG C, denaturation time be 2 ~ 5 min.
6. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 1, it is characterized in that: adding in step (2) When adding IPTG Fiber differentiation, temperature is 30 ~ 40 DEG C, in 3 ~ 5h of revolving speed 200 ~ 250rpm Fiber differentiation.
7. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 1, it is characterized in that: sharp in step (3) The precipitating containing destination protein, as pinctada fucata martensii meat anti-oxidizing peptide are isolated and purified with GST agarose affinity chromatography twice.
8. the preparation method of pinctada fucata martensii meat anti-oxidizing peptide according to claim 7, it is characterized in that: by first time GST Sample crosses second of GST agarose affinity chromatography after agarose affinity chromatography purifying and digestion, collects efflux, will be collected into Component carry out Tricine-SDS-PAGE detection, the precipitating containing destination protein is pinctada fucata martensii meat anti-oxidizing peptide.
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CN103739693A (en) * 2014-01-27 2014-04-23 中国水产科学研究院南海水产研究所 Pinctada martensii meat antioxidant peptide sequence with antioxidant activity and application of Pinctada martensii meat antioxidant peptide sequence
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