CN105566484A - Novel recombinant perforin protein and preparation method and application thereof - Google Patents
Novel recombinant perforin protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses novel recombinant perforin protein and a preparation method and application thereof. The amino acid sequence of the recombinant perforin protein is shown as SEQ ID NO.2, and the encoding nucleotide sequence of the recombinant perforin protein is shown as SEQ ID NO.1. A modern gene engineering technique is adopted, a perforin protein sequence is designed and synthesized and is connected to an expression vector, host bacteria are converted, the vector is provided with a GST label, the protein can be eluted and purified through an affinity chromatography method, and a large amount of purified recombinant perforin protein is prepared. According to the protein, perforating rings can be formed on gram negative bacteria and eukaryocyte, a membrane dissolving effect is generated, and good application prospects are achieved in the field of bioexperiments and the field of medicine preparation. The recombinant protein obtained through the method is good in stability, high in purity and activity, simple in process, short in consumed time, low in cost, not high in equipment requirement and convenient to produce on a large scale.
Description
Technical field
The invention belongs to biological technical field.More specifically, a kind of novel recombinant perforin albumen and its preparation method and application is related to.
Background technology
Lampetra japonica (Martens). (
lampetrajaponicum) belong to Cyclostomata, Chordata, be the existing ancient biology of one, in recent years, the research for the unique physiological mechanism of Lampetra japonica (Martens). causes extensive attention.
Pore-forming protein albumen is the distinctive immune effector molecule of Lampetra japonica (Martens)., there is lectin (Lectin) structural domain and cell pore-forming toxins (toxin) structural domain, can be anchored on cytolemma, a ring-type complex body is formed by self-polymerization, this complex body is formed on cytolemma hydrophilicly wears membrane channel, and then causes entocyte to outflow, and reaches the object killing heterologous cells, there is potential application foreground widely, need further development research.
Summary of the invention
The technical problem to be solved in the present invention overcomes the existing correlative study of pore-forming protein albumen and the deficiency of application, Lampetra japonica (Martens). pore-forming protein albumen that a kind of novel restructuring is provided and preparation method thereof, the pore-forming protein albumen prepared can form duct at bacterium surface, bacterium content is caused to outflow, can be used as a kind of novel antibacterial material of non-antibiotic, have huge application prospect; And can in the punching of eukaryotic cell surface, cell can be made to carry out film quality separation, is effective novel method of extraction and isolation organoid, in cell experiment research, also have wide using value.
The object of this invention is to provide the nucleotide sequence of a kind of novel recombinant perforin albumen and this albumen of coding expression.
Another object of the present invention is to provide the preparation method of above-mentioned recombinant perforin albumen.
Another object of the present invention is to provide the application of above-mentioned recombinant perforin albumen, comprises application and purposes that this restructuring perforin and nucleotides sequence thereof are listed 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, its aminoacid sequence is as shown in SEQIDNO.2.This albumen is the immune important effect molecule of Lampetra japonica (Martens)., and its structural domain is divided into Lectin domain and cytotoxin structural domain two parts.
Meanwhile, the nucleotide sequence of above-mentioned recombinant perforin albumen is encoded also within protection scope of the present invention.
Further, the nucleotide sequence of above-mentioned coding recombinant perforin albumen is as shown in SEQIDNO.1, or described nucleotides sequence is classified as sequence shown in SEQIDNO.1 and carries out one or several Nucleotide replacement and the nucleotide sequence of the codon same sense mutation obtained.
Comprise a nucleotide sequence for coding recombinant perforin albumen, or by the nucleotide sequence of coding recombinant perforin albumen and expression vector through the recombinant expression vector obtained of recombinating, also within protection scope of the present invention.
Preferably, above-mentioned recombinant expression vector comprises nucleotide sequence shown in SEQIDNO.1.
Preferably, above-mentioned expression vector is the empty carrier containing the element needed for the encoding sequence transcribing and translate insertion.
More preferably, above-mentioned expression vector is pGEX-6p1 etc.
More preferably, above-mentioned recombinant expression vector can be obtained through restructuring by expression vectors such as the nucleotide sequence insertion pGEX-6p-1 shown in SEQIDNO.1.
In addition, the preparation method of above-mentioned recombinant perforin albumen comprises the steps:
S1. the nucleotide sequence of clones coding recombinant perforin albumen;
S2. the nucleotide sequence that step S1 obtains is built into expression vector, obtains recombinant expression vector;
S3. the recombinant expression vector that step S2 obtains is cultivated through conversion, bacterium liquid, collects nutrient solution, purification recombinant perforin albumen.
Further preferably, preparation method's step of above-mentioned recombinant perforin albumen is as follows:
S1., based on Lampetra japonica (Martens). cDNA, clone obtains target gene sequence (shown in SEQIDNO.1 sequence);
S2. the sequence clone obtained by step S1 by primer P1 and P2, on prokaryotic fusion expression vector pGEX-6p1, obtains recombinant expression vector pGEX-6p1-LPFP;
S3. by recombinant expression vector pGEX-6p1-LPFP transformation of E. coli Rosetta(DE3);
S4. the intestinal bacteria Rosetta(DE3 after transforming) after cultivating, carry out ultrasonic degradation, collect supernatant, utilize the method for GST label affinity chromatography to carry out protein purification;
Or step S4 is operating as: the intestinal bacteria Rosetta(DE3 after conversion) after cultivating, the centrifugal 10min of 7000rpm, resuspended under thalline being placed in damping fluid, use mechanical high pressure crusher at 4 DEG C of temperature by bacterial cell disruption, utilize the method for GST label affinity chromatography to carry out protein purification;
Described damping fluid is 20mMTris-HCl, 50mMNaCl, pH7.5.
The application of above-mentioned recombinant perforin albumen in cell perforation field, also within protection scope of the present invention, is specifically preparing the application in cytolysis preparation.
Therefore, when directed toward bacteria or fungi, this recombinant perforin albumen can be used as novel antibacterial material or the preparation antibacterials of non-antibiotic.Described antimicrobial substance or antibacterials can form duct at bacterium surface, cause bacterium content to outflow, thus play antibacterial effect.
And when for cancer cells, this recombinant perforin albumen described can make cancer cells occur significantly boring a hole dissolution phenomena, and then kill and wound cancer cells, play the object of Therapeutic cancer, therefore, can be applicable to prepare cancer treatment drugs.
Further preferably, above-mentioned bacterium is intestinal bacteria.Preferably, above-mentioned cancer cells is HeLa cell.
In addition, described recombinant perforin albumen can in the punching of eukaryotic cell surface, and cell can be made to carry out film quality separation, is effective novel method of extraction and isolation organoid, in cell experiment research, also have wide using value.
In the research of pore-forming protein albumen, we find, pore-forming protein albumen can form duct on the cell walls of Gram-negative bacteria, causes the content of cell to flow out, and lysis is dead, reaches antibacterial bacteriostatic effect.Due to antibiotic abuse, the resistance of pathogenic bacteria generally strengthens, and the antibacterial mechanisms of pore-forming protein albumen is different from microbiotic, germ there is no tolerance to it, and meta-bolites is amino acid, to environment and human-body safety harmless, have broad application prospects as a kind of novel antimicrobial drug.Another function of perforin is, at red corpuscle, the eukaryotic cells such as HeLa cell can forming duct, providing a kind of new tool of safety non-toxic for extracting entocyte in Biochemistry Experiment.
The present invention adopts modern genetic engineering technology, design and synthesize out pore-forming protein protein sequence, and be connected on expression vector and transform Host Strains again, carrier is with GST label, the method of affinity chromatography can be adopted to carry out eluting to albumen, prepare recombinant perforin albumen purified in a large number.This albumen can form perforation ring on gram negative bacterium and eukaryotic cell, produces molten film effect.And the restructuring perforin that the inventive method obtains also overcomes the various shortcomings of the albumen of natural extract, be convenient to the batch production of pore-forming protein albumen, and effectively can control difference between batch and be not conducive to pore-forming protein albumen be core material the preparation of all ingredients and the industrial applications of pore-forming protein albumen.
The present invention has following beneficial effect:
The present invention by recombinating to the clone of Lampetra japonica (Martens). pore-forming protein protein gene, successfully gives expression to the activated pore-forming protein albumen of tool in prokaryotic cell prokaryocyte, and by grope and optimization obtains a set of simple and effective protein expression and purification method.The pore-forming protein albumen that the present invention prepares can form duct at bacterium surface, causes bacterium content to outflow, and as a kind of novel antibacterial material of non-antibiotic, has huge application prospect; And can in the punching of eukaryotic cell surface, cell can be made to carry out film quality separation, is effective novel method of extraction and isolation organoid, in cell experiment research, also have wide using value.
In addition, protokaryon protein purification expression method results of the present invention obtain recombinant protein good stability, and purity is high, active high.And the method technique is simple, consuming time short, cost is low, and equipment requirements is not high, is convenient to scale operation.
Accompanying drawing explanation
Fig. 1 is that recombinant perforin albumen is to the perforation of HeLa cell and lethal effect.
Fig. 2 is the relation of the cell perforation rate of recombinant perforin protein concentration and HeLa cell.
Fig. 3 is pore-forming protein albumen to colibacillary perforation effect and intestinal bacteria are molten breaks once journey.
Embodiment
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are commercial.
the structure of embodiment 1 recombinant perforin protein expressing plasmid
1, the extraction of Lampetra japonica (Martens). total serum IgE
Get Lampetra japonica (Martens). liver organization, with the cracking of Trizol reagent, chloroform, isopropanol precipitating obtains RNA.
2, the amplification of the fragment of pore-forming protein albumen
(1) according to the SMARTerRACE5 '/3 ' test kit of Clontech company, above-mentioned RNA reverse transcription is synthesized the first chain, then carries out pcr amplification.
The primer that described pcr amplification uses is: P1 and P2, is the two ends sequent synthesis according to perforin genes, and wherein, upstream primer P1 is containing BamHI cleavage site, and downstream primer P2 is containing XhoI cleavage site.
Upstream primer P1:5'-CGCGGATCCATGGTGTACCCGACCACAC
Downstream primer P2:3-CCGCTCGAGGCTCTCGGTAGCAACGGCGGTCAT
(2) fragment that PCR obtains to be connected in carrier T and to be transformed in bacillus coli DH 5 alpha, selects positive colony and checks order.
(3) sequence obtained that increased by 3 ' RACE and 5 ' RACE is spliced, obtain recombinant perforin protein coding gene total length, called after LPFP, altogether 1189bp, the nucleotide sequence of coding head of district 942bp(coding region is as shown in SEQIDNO.1), 313 amino acid of encoding.
3, the structure of recombinant perforin protein expressing plasmid
With the pGEX plasmid containing pore-forming protein protein gene for template, P1, P2 are primer, carry out pcr amplification, and obtain the single band of specific amplified, product size is at about 1656bp.
Obtained pcr amplification product is cloned on prokaryotic fusion expression vector pGEX-6p-1, obtains recombinant expression vector pGEX-6p1-LPFP.
Foreign gene in recombinant expression vector pGEX-6p1-LPFP is correct through order-checking qualification.
the expression and purification of embodiment 2 recombinant perforin albumen
1, the expression of recombinant perforin albumen
By recombinant expression vector pGEX-6p1-LPFP transformation of E. coli Rosetta(DE3) in, through cultivating and after induction, adopting the method for ultrasonication to obtain the broken supernatant liquor of genetic engineering bacterium.
Show through the analysis of SDS-Page protein electrophoresis, containing significantly solvable specifically expressing product band in its supernatant liquor.The recombinant perforin molecular weight of albumen that protein electrophoresis obtains is about 61.9kDa.
Draw after incubation time, inductive condition, temperature etc. are optimized, the culture condition of genetic engineering bacterium is: selected substratum is optimized on the basis of LB substratum, add the glucose of 1% and the ammonia benzyl microbiotic of 100mg/L, genetic engineering bacterium bacterium liquid is added in this substratum with the ratio of 1:100, at 37 DEG C, the CMC model of 220rpm, OD value to substratum reaches in the scope of 0.4 ~ 0.6, IPTG inductor is added according to the concentration of 0.1mmol/L, at 18 DEG C, overnight induction under the condition of 120rpm, centrifugal, collect thalline.
2, the purifying of recombinant perforin albumen
Draw after purification condition is optimized, by total thalline bindingbuffer solution, (described bindingbuffer solution is: 20mMTris.Cl, 50mMNaCl, pH7.5) wash, carry out resuspended with appropriate bindingbuffer solution again, adopt the method cracking engineering bacteria (preferred condition, when needing a large amount of preparation restructuring perforins, adopts the method cracking engineering bacteria of high-pressure machinery fragmentation) of ultrasonication.
After cracking, by centrifugation supernatant liquor, the centrifugal condition of optimization is 8000rpm, 3min, to supernatant liquor affinity chromatography purifying.Expression and the chromatography of recombinant protein is analyzed by SDS-PAGE.
Reach a conclusion: carrier with GST label can effectively affinely be adsorbed on chromatography column, and by with 10mM reductibility paddy sweet Guang peptide wash-out, target protein to be eluted.After collection obtains target protein elution peak, adopt the method removing reductive glutathione of dialysis, and concentrated by albumen super filter tube (Millipore), obtain the recombinant perforin albumen of high purity and high density.
embodiment 3 recombinant perforin albumen is to the cytolysis of HeLa cell
1, get HeLa cell, 800rpm is centrifugal, and 10min removes enchylema, re-uses PBS(pH7.4,130mMNaCl, 2.5mMKCl, 10mMNa
2hPO
4, 2mMKH
2pO
4) fully wash 3 times, remove supernatant through the centrifugal 10min of 800rpm, basic medium is resuspended, is made into HeLa cell suspending liquid.
2, in above-mentioned HeLa cell suspending liquid, add the recombinant perforin albumen of different amount, result shows, after adding albumen, dissolution phenomena (as shown in Figure 1) appears significantly boring a hole in HeLa cell.And, the amount that the kill rate of HeLa cell is white with perforation has obvious dose-dependence, the pore-forming protein albumen of 1 μ g/ml can make the HeLa cell perforation of 80%, causes the tenuigenin of HeLa cell to outflow thus obviously the killing and wounding (as shown in Figure 2) of mediated cell.
embodiment 4 recombinant perforin albumen is to colibacillary cytolysis
1, get intestinal bacteria incubated overnight, the centrifugal 10min of 7000rpm precipitates intestinal bacteria, by about 2 × 10
7intestinal bacteria PBS(pH7.5) fully wash 3 times, remove supernatant through the centrifugal 10min of 7000rpm, re-use Cell Basal Medium resuspended, be made into bacterium re-suspension liquid.
2, in above-mentioned bacterium re-suspension liquid, add recombinant perforin albumen, hatch 1h with intestinal bacteria, intestinal bacteria recentrifuge is precipitated.
After treatment, carry out morphologic observation under being placed in Electronic Speculum, as shown in Figure 3, colibacillary cell walls reorganized pore-forming protein albumen is molten broken, and tenuigenin outflows, necrocytosis.
SEQUENCELISTING
<110> Zhongshan University
Novel recombinant perforin albumen of <120> mono-kind and its preparation method and application
<130>
<160>2
<170>PatentInversion3.3
<210>1
<211>942
<212>DNA
<213> Lampetra japonica (Martens). (Lampetrajaponicum) recombinant perforin protein coding gene sequence
<400>1
atggtgtacccgaccacacttcacatcattggcggccaaggtggaaacgcgttctcgttc60
aacgggcaggagaatgcggcgacgctgcagaagctctctgtgagcgttgggggatggcag120
gtgaggggcgtgcaggtgtggctgacggacgggcgcagggagacattcggcgccatggac180
tcctccgctaaggagttcgaattcgagtcgggcgagttcatcaagagcctctcgctgtgg240
ggcaacggagccggcactcgcctgggcgctatcaagttcataacgagccgcagccgcgag300
ttctttgccaagatgacggactgggggctcaagaccgagtacaagatcgacgtgggctct360
ggcatctgcctgggtgttcagggccgaggggggtccgacattgactccatgggcttcatc420
ttcatcaatgccataaaatcgtcggtgatccaggacatgaagtacccgaccatgcaccaa480
attctgcccaacgtgcagatggaggagaacaaagaaatggagtacaagaacgacaccagc540
atcgtgcaatcgtacacattcgagagctccaagaagatcattaaaaagtcatcgtggtcc600
accaccaacaagatcgagtccaccttcagcctgtcggtgaaggccggcatccccgaggtc660
atggaggtggagaccggattcagcttcaccgtgggcagtgagagcacgcacgcggtggag720
gagtccgaggagaagacggaaacgctcacgttccccgtcactgtcccgacgcacaagacc780
gtcaccgtggtcgccaacatcgggcgcgccgacatcgaccttccgtacacggccctgctg840
cgcatcacctgcgtgaacggcgcatcccttgacgctcccctgagcggcatctacaagggg900
ctcacctacaccaagatgaccgccgttgctaccgagagctag942
<210>2
<211>313
<212>PRT
<213> Lampetra japonica (Martens). (Lampetrajaponicum) recombinant perforin protein amino acid sequence
<400>2
MetValTyrProThrThrLeuHisIleIleGlyGlyGlnGlyGlyAsn
151015
AlaPheSerPheAsnGlyGlnGluAsnAlaAlaThrLeuGlnLysLeu
202530
SerValSerValGlyGlyTrpGlnValArgGlyValGlnValTrpLeu
354045
ThrAspGlyArgArgGluThrPheGlyAlaMetAspSerSerAlaLys
505560
GluPheGluPheGluSerGlyGluPheIleLysSerLeuSerLeuTrp
65707580
GlyAsnGlyAlaGlyThrArgLeuGlyAlaIleLysPheIleThrSer
859095
ArgSerArgGluPhePheAlaLysMetThrAspTrpGlyLeuLysThr
100105110
GluTyrLysIleAspValGlySerGlyIleCysLeuGlyValGlnGly
115120125
ArgGlyGlySerAspIleAspSerMetGlyPheIlePheIleAsnAla
130135140
IleLysSerSerValIleGlnAspMetLysTyrProThrMetHisGln
145150155160
IleLeuProAsnValGlnMetGluGluAsnLysGluMetGluTyrLys
165170175
AsnAspThrSerIleValGlnSerTyrThrPheGluSerSerLysLys
180185190
IleIleLysLysSerSerTrpSerThrThrAsnLysIleGluSerThr
195200205
PheSerLeuSerValLysAlaGlyIleProGluValMetGluValGlu
210215220
ThrGlyPheSerPheThrValGlySerGluSerThrHisAlaValGlu
225230235240
GluSerGluGluLysThrGluThrLeuThrPheProValThrValPro
245250255
ThrHisLysThrValThrValValAlaAsnIleGlyArgAlaAspIle
260265270
AspLeuProTyrThrAlaLeuLeuArgIleThrCysValAsnGlyAla
275280285
SerLeuAspAlaProLeuSerGlyIleTyrLysGlyLeuThrTyrThr
290295300
LysMetThrAlaValAlaThrGluSer
305310
Claims (10)
1. a novel recombinant perforin albumen, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.2.
2. recombinant perforin albumen according to claim 1, it is characterized in that, this albumen is the immune important effect molecule of Lampetra japonica (Martens)., and its structural domain is divided into Lectin domain and cytotoxin structural domain two parts.
3. the nucleotide sequence of recombinant perforin albumen described in coding claim 1 or 2.
4. nucleotide sequence according to claim 3, it is characterized in that, described nucleotide sequence is as shown in SEQIDNO.1, or described nucleotides sequence is classified as sequence shown in SEQIDNO.1 and carries out one or several Nucleotide replacement and the nucleotide sequence of the codon same sense mutation obtained.
5. a recombinant expression vector, is characterized in that, described recombinant expression vector comprises the nucleotide sequence described in claim 3 or 4, or described recombinant expression vector is obtained through recombinating by the nucleotide sequence described in claim 3 or 4 and expression vector.
6. recombinant expression vector according to claim 5, it is characterized in that, described expression vector is pGEX-6p1.
7. the preparation method of recombinant perforin albumen described in claim 1 or 2, is characterized in that, comprise the steps:
S1. the nucleotide sequence of clone described in claim 3 or 4;
S2. the nucleotide sequence that step S1 obtains is built into expression vector, obtains recombinant expression vector;
S3. the recombinant expression vector that step S2 obtains is cultivated through conversion, bacterium liquid, collects nutrient solution, purification recombinant perforin albumen.
8. preparation method according to claim 7, is characterized in that, step is as follows:
S1., based on Lampetra japonica (Martens). cDNA, clone obtains target gene sequence (shown in SEQIDNO.1 sequence);
S2. the sequence clone obtained by step S1 by primer P1 and P2, on prokaryotic fusion expression vector pGEX-6p1, obtains recombinant expression vector pGEX-6p1-LPFP;
S3. by recombinant expression vector pGEX-6p1-LPFP transformation of E. coli Rosetta(DE3);
S4. the intestinal bacteria Rosetta(DE3 after transforming) after cultivating, carry out ultrasonic degradation, collect supernatant, utilize the method for GST label affinity chromatography to carry out protein purification;
Or step S4 is operating as: the intestinal bacteria Rosetta(DE3 after conversion) after cultivating, the centrifugal 10min of 7000rpm, resuspended under thalline being placed in damping fluid, use mechanical high pressure crusher at 4 DEG C of temperature by bacterial cell disruption, utilize the method for GST label affinity chromatography to carry out protein purification;
Described damping fluid is 20mMTris-HCl, 50mMNaCl, pH7.5.
9. the application of recombinant perforin albumen described in claim 1 or 2 in preparation cytolysis preparation.
10. recombinant perforin albumen described in claim 1 or 2 is to the punching of eukaryotic cell surface, the application that makes cell carry out in film quality separation, extraction and isolation organoid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109496914A (en) * | 2018-09-30 | 2019-03-22 | 中山大学 | A kind of artificial breeding method of wild Japanese lamprey |
| CN109576275A (en) * | 2018-12-16 | 2019-04-05 | 中国水产科学研究院黄海水产研究所 | A kind of Cynoglossus semilaevis antibacterium disease related gene and its application method |
| CN113121667A (en) * | 2021-03-31 | 2021-07-16 | 中山大学 | Cell membrane pore-forming protein LjGSDM and expression and application thereof |
| CN114711433A (en) * | 2022-03-09 | 2022-07-08 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of food and medicine for treating obesity and improving cold resistance |
-
2016
- 2016-02-03 CN CN201610075535.4A patent/CN105566484B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109496914A (en) * | 2018-09-30 | 2019-03-22 | 中山大学 | A kind of artificial breeding method of wild Japanese lamprey |
| CN109576275A (en) * | 2018-12-16 | 2019-04-05 | 中国水产科学研究院黄海水产研究所 | A kind of Cynoglossus semilaevis antibacterium disease related gene and its application method |
| CN113121667A (en) * | 2021-03-31 | 2021-07-16 | 中山大学 | Cell membrane pore-forming protein LjGSDM and expression and application thereof |
| CN113121667B (en) * | 2021-03-31 | 2022-04-01 | 中山大学 | Cell membrane pore-forming protein LjGSDM and expression and application thereof |
| CN114711433A (en) * | 2022-03-09 | 2022-07-08 | 辽宁师范大学 | Application of lamprey LIP protein in preparation of food and medicine for treating obesity and improving cold resistance |
| 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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