CN105566484B - A kind of novel recombinant perforin albumen and its preparation method and application - Google Patents
A kind of novel recombinant perforin albumen and its preparation method and application Download PDFInfo
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- CN105566484B CN105566484B CN201610075535.4A CN201610075535A CN105566484B CN 105566484 B CN105566484 B CN 105566484B CN 201610075535 A CN201610075535 A CN 201610075535A CN 105566484 B CN105566484 B CN 105566484B
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- albumen
- perforin
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- recombinant perforin
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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
Abstract
The invention discloses a kind of novel recombinant perforin albumen and its preparation method and application.The amino acid sequence of the recombinant perforin albumen is as shown in SEQ ID NO.2, and coding nucleotide sequence is as shown in SEQ ID NO.1.The present invention uses modern genetic engineering technology, avidin sequence of perforating is designed and synthesized out, and is connected on expression vector and converts host strain again, carrier has GST label, albumen can be purified by flash using the method for affinity chromatography, prepare a large amount of purified recombinant perforin albumen.The albumen can form perforation ring on gramnegative bacterium and eukaryocyte, generate molten film effect, have a good application prospect in Bioexperiment field and field of medicine preparation.And good, with high purity, the active height of recombinant protein stability that the method for the present invention harvests, simple process, time-consuming is short, at low cost, equipment requirement is not high, convenient for large-scale production.
Description
Technical field
The invention belongs to field of biotechnology.More particularly, to a kind of novel recombinant perforin albumen and its preparation side
Method and application.
Background technique
Lamprey (Lampetra japonicum) belong to Cyclostomata, Chordata, it is a kind of existing ancient biology,
In recent years, the research of lamprey uniqueness physiological mechanism is caused to pay attention to extensively.
Fibroin of perforating is the distinctive immune effector molecule of lamprey, with agglutinin (Lectin) structural domain and carefully
Born of the same parents' pore-forming toxins (toxin) structural domain, can be anchored on cell membrane, form a cyclic annular complex by self-polymerization, this is multiple
Zoarium formed on cell membrane it is hydrophilic wear membrane channels, and then cellular content is caused to outflow, reaches and kill heterologous cells
Purpose has extensive potential application foreground, needs further developmental research.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies of correlative study and the application of existing perforation fibroin, provide
Lamprey perforation fibroin of a kind of novel recombination and preparation method thereof, the perforation fibroin being prepared can be in bacterium surface
Duct is formed, causes bacterium content to outflow, can be used as a kind of novel antibacterial substance of non-antibiotic, before having huge application
Scape；And can be punched on eukaryocyte surface, cell can be made to carry out film quality separation, be the effective new side for extracting separation organelle
Method also has wide application value in cell experiment research.
The object of the present invention is to provide the nucleotides sequences that a kind of novel recombinant perforin albumen and coding express the albumen
Column.
It is a further object of the present invention to provide the preparation methods of above-mentioned recombinant perforin albumen.
Another object of the present invention is to provide the application of above-mentioned recombinant perforin albumen, including the recombination perforin and its
Nucleotides sequence is listed in the application and use in cell perforation field.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of novel recombinant perforin albumen, amino acid sequence is as shown in SEQ ID NO.2.The albumen is exempted from for lamprey
The important effect molecule of epidemic disease system, structural domain are divided into two parts of Lectin domain and cytotoxin structural domain.
Meanwhile encoding the nucleotide sequence of above-mentioned recombinant perforin albumen also within protection scope of the present invention.
Further, the nucleotide sequence of above-mentioned coding recombinant perforin albumen is as shown in SEQ ID NO.1 or the core
Nucleotide sequence is that sequence shown in SEQ ID NO.1 carries out codon same sense mutation obtained from one or several nucleotide substitutions
Nucleotide sequence.
A kind of nucleotide sequence comprising encoding recombinant perforin albumen, or the nucleotide by coding recombinant perforin albumen
Sequence and expression vector are through recombinating obtained recombinant expression carrier, also within protection scope of the present invention.
Preferably, above-mentioned recombinant expression carrier includes nucleotide sequence shown in SEQ ID NO.1.
Preferably, above-mentioned expression vector is the empty carrier of element needed for the coded sequence being inserted into containing transcription and translation.
It is highly preferred that above-mentioned expression vector is pGEX-6p1 etc..
It is highly preferred that above-mentioned recombinant expression carrier can the insertion of the nucleotide sequence as shown in SEQ ID NO.1 pGEX-6p-1
Equal expression vectors are obtained through recombination.
In addition, the preparation method of above-mentioned recombinant perforin albumen includes the following steps:
S1. the nucleotide sequence of clone's coding recombinant perforin albumen；
S2. the step S1 nucleotide sequence obtained is built into expression vector, obtains recombinant expression carrier；
S3. step S2 obtain recombinant expression carrier is inverted, bacterium solution culture, collect culture solution, purification recombination is worn
Hole fibroin.
It is further preferred that steps are as follows for the preparation method of above-mentioned recombinant perforin albumen:
S1. based on lamprey cDNA, clone obtains target gene sequence (sequence shown in SEQ ID NO.1)；
S2. the step S1 sequence obtained is cloned on prokaryotic fusion expression vector pGEX-6p1 by primer P1 and P2,
Obtain recombinant expression carrier pGEX-6p1-LPFP；
S3. recombinant expression carrier pGEX-6p1-LPFP is converted into Escherichia coli Rosetta(DE3)；
S4. the Escherichia coli Rosetta(DE3 after converting) after culture, ultrasonic cracking is carried out, supernatant is collected, is utilized
The method of GST label affinity chromatography carries out protein purification；
Or the operation of step S4 are as follows: the Escherichia coli Rosetta(DE3 after conversion) after culture, 7000rpm centrifugation
Thallus is placed under buffer and is resuspended by 10min, and bacterial cell disruption is utilized GST at a temperature of 4 DEG C using mechanical high pressure crusher
The method of label affinity chromatography carries out protein purification；
The buffer is 20mMTris-HCl, 50mMNaCl, pH7.5.
Application of the above-mentioned recombinant perforin albumen in cell perforation field also within protection scope of the present invention, specifically
It is the application in preparation cell dissolution preparation.
Therefore, in directed toward bacteria or fungi, which can be used as the novel antibacterial substance of non-antibiotic
Or preparation antibacterials.The antibacterial material or antibacterials can form duct in bacterium surface, cause outside bacterium content
Stream, to play the role of antibacterial.
And when being directed to cancer cell, it is existing that the described recombinant perforin albumen can make cancer cell apparent perforation dissolution occur
As, and then cancer cell is killed, the purpose for the treatment of cancer is played, therefore, can be applied to preparation cancer treatment drugs.
It is further preferred that above-mentioned bacterium is Escherichia coli.Preferably, above-mentioned cancer cell is HeLa cell.
In addition, the recombinant perforin albumen can punch on eukaryocyte surface, cell can be made to carry out film quality separation, be to mention
The effective new method for taking separation organelle also has wide application value in cell experiment research.
Perforation fibroin research in, it has been found that perforation fibroin can on the cell wall of Gram-negative bacteria shape
At duct, the content of cell is caused to flow out, cell cracking is dead, reaches antibacterial bacteriostatic effect.Due to the abuse of antibiotic, disease
The drug resistance of opportunistic pathogen generally enhances, and the antibacterial mechanisms for fibroin of perforating are different from antibiotic, and germ has no tolerance, and generation to it
Thanking to product is amino acid, safe and harmless to environment and human body, is had broad application prospects as a kind of novel antimicrobial.It wears
Another function of porin is that it is possible to form duct on red blood cell, the eukaryocytes such as HeLa cell, is Biochemistry Experiment
The middle cellular content that extracts provides a kind of safe and non-toxic new tool.
The present invention uses modern genetic engineering technology, designs and synthesizes out avidin sequence of perforating, and be connected to table
Host strain is converted on up to carrier again, carrier has GST label, albumen can be purified by flash using the method for affinity chromatography,
Prepare a large amount of purified recombinant perforin albumen.The albumen can be formed on gramnegative bacterium and eukaryocyte and be worn
Orifice ring generates molten film effect.And the recombination perforin that the method for the present invention obtains also overcomes the various of the albumen naturally extracted
Disadvantage convenient for the batch production for fibroin of perforating, and can be effectively controlled difference between batch and not be conducive to using fibroin of perforating as core original
The industrial applications of the preparation of the various reagents of material and fibroin of perforating.
The invention has the following advantages:
The present invention is recombinated by the clone to lamprey perforin protein gene, is successfully expressed and is provided in prokaryotic cell
Active perforation fibroin, and by groping and optimization obtains a set of simple and effective protein expression and purification method.The present invention
The perforation fibroin being prepared can form duct in bacterium surface, cause bacterium content to outflow, as a kind of non-antibiosis
The novel antibacterial substance of element, there is huge application prospect；And can be punched on eukaryocyte surface, cell can be made to carry out film quality
Separation is the effective new method for extracting separation organelle, also has wide application value in cell experiment research.
In addition, protokaryon protein purification expression of the invention harvests to obtain recombinant protein stability good, purity is high, activity
It is high.And this method simple process, time-consuming short, at low cost, equipment requirement is not high, convenient for large-scale production.
Detailed description of the invention
Fig. 1 is perforation and lethal effect of the recombinant perforin albumen to HeLa cell.
Fig. 2 is the relationship of the cell perforation rate of recombinant perforin protein concentration and HeLa cell.
Fig. 3, which is that perforation fibroin acts on the perforation of Escherichia coli and Escherichia coli are molten, breaks once journey.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The building of 1 recombinant perforin protein expressing plasmid of embodiment
1, the extraction of lamprey total serum IgE
Lamprey liver organization is taken, is cracked with Trizol reagent, chloroform, isopropanol precipitating obtains RNA.
2, the amplification of the segment of perforation fibroin
(1) according to 5 '/3 ' kit of SMARTer RACE of Clontech company, above-mentioned RNA reverse transcription is synthesized first
Chain, then carry out PCR amplification.
Primer used in the PCR amplification are as follows: P1 and P2 is the both ends sequent synthesis according to perforin genes,
Wherein, upstream primer P1 cleavage site containing BamHI, downstream primer P2 cleavage site containing XhoI.
Upstream primer P1:5'-CGCGGATCCATGGTGTACCCGACCACAC
Downstream primer P2:3-CCGCTCGAGGCTCTCGGTAGCAACGGCGGTCAT
(2) segment that PCR is obtained is connected in carrier T and is transformed into bacillus coli DH 5 alpha, selects positive colony progress
Sequencing.
(3) 3 ' RACE and 5 ' the RACE sequence expanded are spliced, obtains recombinant perforin encoding histone base
Because of overall length, it is named as LPFP, total 1189bp, encodes the nucleotide sequence such as SEQ ID NO.1 institute of the code area head of district 942bp(
Show), encode 313 amino acid.
3, the building of recombinant perforin protein expressing plasmid
Using the pGEX plasmid containing perforin protein gene as template, P1, P2 are primer, carry out PCR amplification, are obtained special
The single band of amplification, primer size is in 1656bp or so.
Pcr amplification product obtained is cloned on prokaryotic fusion expression vector pGEX-6p-1, recombinant expression is obtained and carries
Body pGEX-6p1-LPFP.
Foreign gene in recombinant expression carrier pGEX-6p1-LPFP is correct through sequencing identification.
The expression and purification of 2 recombinant perforin albumen of embodiment
1, the expression of recombinant perforin albumen
Recombinant expression carrier pGEX-6p1-LPFP is converted into Escherichia coli Rosetta(DE3) in, after culture and induction,
The broken supernatant of genetic engineering bacterium is obtained using the method for ultrasonication.
Analysis shows, apparent soluble specifically expressing product item is contained in supernatant through SDS-Page protein electrophoresis
Band.The recombinant perforin molecular weight of albumen that protein electrophoresis obtains is about 61.9kDa.
It is obtained after being optimized to incubation time, inductive condition, temperature etc., the condition of culture of genetic engineering bacterium are as follows: selected
It selects culture medium to optimize on the basis of LB culture medium, 1% glucose and the ammonia benzyl antibiotic of 100mg/L is added, by gene
Engineering bacteria bacterium solution is added in the culture medium with the ratio of 1:100, in 37 DEG C, the CMC model of 220rpm, until the OD value of culture medium
In the range of reaching 0.4~0.6, IPTG inducer is added according to the concentration of 0.1mmol/L, under conditions of 18 DEG C, 120rpm
Thallus is collected in overnight induction, centrifugation.
2, the purifying of recombinant perforin albumen
It is obtained after being optimized to purification condition, by total thallus binding buffer solution (binding
Buffer solution are as follows: 20mM Tris.Cl, 50mM NaCl, pH7.5) it is washed, then it is molten with suitable binding buffer
Liquid is resuspended, and cracking engineering bacteria using the method for ultrasonication, (preferred condition needs a large amount of preparation and reorganization perforation egg
In the case where white, engineering bacteria is cracked using the method that high-pressure machinery is crushed).
After cracking, by being centrifugated supernatant, the centrifugal condition of optimization is 8000rpm, 3min, to the affine color of supernatant
Compose chromatographic purifying.Expression and the chromatography of recombinant protein are analyzed by SDS-PAGE.
Conclude that the GST label that carrier has effectively affine can be adsorbed on chromatographic column, and by being restored with 10mM
Property paddy sweet Guang peptide elution, target protein is eluted.After collection obtains target protein eluting peak, removed using the method for dialysis
Reductive glutathione, and be concentrated by albumen super filter tube (Millipore), obtain the recombinant perforin of high-purity and high concentration
Albumen.
Cytolysis of the 3 recombinant perforin albumen of embodiment to HeLa cell
1, HeLa cell is taken, 800rpm centrifugation 10min removes cell liquid, reuses PBS(pH7.4,130mMNaCl, 2.5mM
KCl, 10mM Na2HPO4, 2mM KH2PO4) sufficiently washing 3 times, supernatant, basal medium weight are removed through 800rpm centrifugation 10min
It is outstanding, it is made into HeLa cell suspending liquid.
2, different amounts of recombinant perforin albumen is added in above-mentioned HeLa cell suspending liquid, the results showed that, albumen is added
There is apparent perforation dissolution phenomena (as shown in Figure 1) in HeLa cell afterwards.Moreover, the killing rate of HeLa cell and perforation are white
Amount has apparent dose-dependence, and the perforation fibroin of 1 μ g/ml can make 80% HeLa cell perforation, cause HeLa cell
Cytoplasm outflow to mediated cell obvious killing (as shown in Figure 2).
Cytolysis of the 4 recombinant perforin albumen of embodiment to Escherichia coli
1, Escherichia coli are taken to be incubated overnight, 7000rpm is centrifuged 10min and precipitates Escherichia coli, will about 2 × 107Escherichia coli
With PBS(pH7.5) sufficiently washing 3 times, supernatant is removed through 7000rpm centrifugation 10min, Cell Basal Medium resuspension is reused, matches
At bacterium re-suspension liquid.
2, recombinant perforin albumen is added in above-mentioned bacterium re-suspension liquid, is incubated for 1h with Escherichia coli, again by Escherichia coli
Secondary centrifugation.
After processing, it is placed under Electronic Speculum and carries out morphologic observation, as shown in figure 3, the cell wall of Escherichia coli is perforated by recombination
Fibroin is molten broken, cytoplasm outflow, meronecrosis.
SEQUENCE LISTING
<110>Zhongshan University
<120>a kind of novel recombinant perforin albumen and its preparation method and application
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 942
<212> DNA
<213>lamprey (Lampetra japonicum) recombinant perforin protein coding gene sequence
<400> 1
atggtgtacc cgaccacact tcacatcatt ggcggccaag gtggaaacgc gttctcgttc 60
aacgggcagg agaatgcggc gacgctgcag aagctctctg tgagcgttgg gggatggcag 120
gtgaggggcg tgcaggtgtg gctgacggac gggcgcaggg agacattcgg cgccatggac 180
tcctccgcta aggagttcga attcgagtcg ggcgagttca tcaagagcct ctcgctgtgg 240
ggcaacggag ccggcactcg cctgggcgct atcaagttca taacgagccg cagccgcgag 300
ttctttgcca agatgacgga ctgggggctc aagaccgagt acaagatcga cgtgggctct 360
ggcatctgcc tgggtgttca gggccgaggg gggtccgaca ttgactccat gggcttcatc 420
ttcatcaatg ccataaaatc gtcggtgatc caggacatga agtacccgac catgcaccaa 480
attctgccca acgtgcagat ggaggagaac aaagaaatgg agtacaagaa cgacaccagc 540
atcgtgcaat cgtacacatt cgagagctcc aagaagatca ttaaaaagtc atcgtggtcc 600
accaccaaca agatcgagtc caccttcagc ctgtcggtga aggccggcat ccccgaggtc 660
atggaggtgg agaccggatt cagcttcacc gtgggcagtg agagcacgca cgcggtggag 720
gagtccgagg agaagacgga aacgctcacg ttccccgtca ctgtcccgac gcacaagacc 780
gtcaccgtgg tcgccaacat cgggcgcgcc gacatcgacc ttccgtacac ggccctgctg 840
cgcatcacct gcgtgaacgg cgcatccctt gacgctcccc tgagcggcat ctacaagggg 900
ctcacctaca ccaagatgac cgccgttgct accgagagct ag 942
<210> 2
<211> 313
<212> PRT
<213>lamprey (Lampetra japonicum) recombinant perforin protein amino acid sequence
<400> 2
Met Val Tyr Pro Thr Thr Leu His Ile Ile Gly Gly Gln Gly Gly Asn
1 5 10 15
Ala Phe Ser Phe Asn Gly Gln Glu Asn Ala Ala Thr Leu Gln Lys Leu
20 25 30
Ser Val Ser Val Gly Gly Trp Gln Val Arg Gly Val Gln Val Trp Leu
35 40 45
Thr Asp Gly Arg Arg Glu Thr Phe Gly Ala Met Asp Ser Ser Ala Lys
50 55 60
Glu Phe Glu Phe Glu Ser Gly Glu Phe Ile Lys Ser Leu Ser Leu Trp
65 70 75 80
Gly Asn Gly Ala Gly Thr Arg Leu Gly Ala Ile Lys Phe Ile Thr Ser
85 90 95
Arg Ser Arg Glu Phe Phe Ala Lys Met Thr Asp Trp Gly Leu Lys Thr
100 105 110
Glu Tyr Lys Ile Asp Val Gly Ser Gly Ile Cys Leu Gly Val Gln Gly
115 120 125
Arg Gly Gly Ser Asp Ile Asp Ser Met Gly Phe Ile Phe Ile Asn Ala
130 135 140
Ile Lys Ser Ser Val Ile Gln Asp Met Lys Tyr Pro Thr Met His Gln
145 150 155 160
Ile Leu Pro Asn Val Gln Met Glu Glu Asn Lys Glu Met Glu Tyr Lys
165 170 175
Asn Asp Thr Ser Ile Val Gln Ser Tyr Thr Phe Glu Ser Ser Lys Lys
180 185 190
Ile Ile Lys Lys Ser Ser Trp Ser Thr Thr Asn Lys Ile Glu Ser Thr
195 200 205
Phe Ser Leu Ser Val Lys Ala Gly Ile Pro Glu Val Met Glu Val Glu
210 215 220
Thr Gly Phe Ser Phe Thr Val Gly Ser Glu Ser Thr His Ala Val Glu
225 230 235 240
Glu Ser Glu Glu Lys Thr Glu Thr Leu Thr Phe Pro Val Thr Val Pro
245 250 255
Thr His Lys Thr Val Thr Val Val Ala Asn Ile Gly Arg Ala Asp Ile
260 265 270
Asp Leu Pro Tyr Thr Ala Leu Leu Arg Ile Thr Cys Val Asn Gly Ala
275 280 285
Ser Leu Asp Ala Pro Leu Ser Gly Ile Tyr Lys Gly Leu Thr Tyr Thr
290 295 300
Lys Met Thr Ala Val Ala Thr Glu Ser
305 310
Claims (2)
1. application of the amino acid sequence recombinant perforin albumen as shown in SEQ ID NO.2 in preparation cell dissolution preparation.
2. amino acid sequence recombinant perforin albumen as shown in SEQ ID NO.2 is preparing to the punching of eukaryocyte surface, is making
Application in terms of cell carries out film quality separation, extraction separates the drug of organelle.
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| CN109496914B (en) * | 2018-09-30 | 2022-02-08 | 中山大学 | Artificial breeding method of wild Japanese lamprey |
| CN109576275B (en) * | 2018-12-16 | 2020-05-22 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis antibacterial disease related gene and application method thereof |
| CN113121667B (en) * | 2021-03-31 | 2022-04-01 | 中山大学 | Cell membrane pore-forming protein LjGSDM and expression and application thereof |
| CN114711433B (en) * | 2022-03-09 | 2024-02-02 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of medicines for treating obesity and improving cold resistance |
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Non-Patent Citations (1)
| Title |
|---|
| Characterization, phylogenetic analysis and cDNA cloning of natterin-like gene from the blood of lamprey, Lampetra japonica;Zhuang Xue等，;《Immunology Letters》;20121231;第148卷(第1期);第1-10页 * |
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