CN104480126A - Trachinotus ovatus peroxiredoxin gene - Google Patents
Trachinotus ovatus peroxiredoxin gene Download PDFInfo
- Publication number
- CN104480126A CN104480126A CN201410735312.7A CN201410735312A CN104480126A CN 104480126 A CN104480126 A CN 104480126A CN 201410735312 A CN201410735312 A CN 201410735312A CN 104480126 A CN104480126 A CN 104480126A
- Authority
- CN
- China
- Prior art keywords
- peroxiredoxin
- trachinotus ovatus
- prx1
- gene
- trachinotus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a cDNA of a trachinotus ovatus peroxiredoxin gene Prx1. The nucleotide of the cDNA is as shown in the SEQ ID NO.1 in the description. The invention further discloses an expression vector containing the trachinotus ovatus peroxiredoxin gene, a recombinant microorganism transformed by utilizing the vector, and a method for preparing the trachinotus ovatus peroxiredoxin gene by utilizing the microorganism. By adopting a genetic engineering technology, a recombinant trachinotus ovatus peroxiredoxin protein can be prepared.
Description
Technical field
The invention belongs to gene engineering technology field, what be specifically related to is a kind of Trachinotus ovatus peroxiredoxin gene and containing the expression vector of this gene and recombinant bacterial strain, and by recombinant protein that recombinant bacterial strain obtains.
Background technology
Peroxiredoxin (Peroxiredoxins, Prxs) is the antioxidant reductase that in organism, a class is important, by catalysis H
2o
2with lipid hydroperoxide reduction, cell is avoided to be subject to further damage.All Prx contain a conservative Cys residue at N-terminal, are under the jurisdiction of peroxiredoxins, and compared with other peroxidase, maximum feature is most Prx is unique hydrogen donor with Trx in vivo.1988, in Kim yeast saccharomyces cerevisiae, find Prx.After this, people in succession in protobiont, plant, animal, parasite, and even have found this enzyme in archeobacteria and eubacterium body.Different according to contained Cys number of residues, peroxiredoxin can be divided into 2 classes: (1) 1-Cys Prx.This type has the Cys residue of a high conservative, as the Prx6 in human body at 52; (2) typical 2-Cys type.52 and 172 have respectively the Cys residue that conservative, simultaneously C-end has a conservative Cys, as Prx1-4; (3) atypia 2-Cys type.C-end is containing extra two less conservative Cys residues, and N-end band positive charge amino-acid residue is scattered in hydrophobic amino acid sequence, as Prx5.Therefore, peroxiredoxin development and application research has become domestic and has crossed forage science and pharmaceutical research focus, has wide prospect at animals and plants transgenic engineering and course of drug development.
Trachinotus ovatus (Trachinotus ovatus) is subordinate to Chordata (Chordata), Osteichthyes (Osteichthyes), Perciformes (Perciformes), Anabantoidei (Percoidei), Scad section (Carangidae), for famous and precious edible fishes, have in south China marine site and suitable propagate scale artificially.But Trachinotus ovatus disease takes place frequently in recent years, cause higher mortality ratio, become one of technical bottleneck of restriction Trachinotus ovatus aquaculture industry Sustainable development.At present, people is not also had to study Trachinotus ovatus peroxiredoxin gene.
Summary of the invention
First object of the present invention is the cDNA providing a kind of Trachinotus ovatus peroxiredoxin gene Prx1 (Peroxiredoxin 1 type) and the aminoacid sequence of inferring thus.
Second object of the present invention is the expression vector that providing package contains above-mentioned Trachinotus ovatus peroxiredoxin gene Prx1 and the recombinant microorganism utilizing this expression vector to transform.
3rd object of the present invention is to provide a kind of process from described microorganism preparation restructuring Trachinotus ovatus peroxiredoxin gene Prx1.
First object of the present invention is achieved by the following technical solution: the cDNA of a kind of Trachinotus ovatus peroxiredoxin gene Prx1, its nucleotide sequence is as shown in SEQ ID NO.1.
The aminoacid sequence of the Trachinotus ovatus peroxiredoxin gene Prx1 inferred by it is as shown in SEQ ID:2.
The full-length gene of Trachinotus ovatus peroxiredoxin gene Prx1 of the present invention obtains Trachinotus ovatus peroxiredoxin sequence with transcript profile order-checking, the cDNA obtained with the reverse transcription of Trachinotus ovatus liver total RNA is for template, then obtain end sequence through the amplification of Smart-RACE method, obtain Trachinotus ovatus peroxiredoxin cDNA full length gene sequence finally by sequence assembly.
The present invention is for template with Trachinotus ovatus peroxiredoxin cDNA for expressing the peroxiredoxin mature peptide gene order of Trachinotus ovatus peroxiredoxin recombinant protein, obtain through PCR method amplification, it derives from the gene fragment corresponding to mature peptide region of Trachinotus ovatus peroxiredoxin full-length gene.
According to the mature peptide gene order of above-mentioned Trachinotus ovatus peroxiredoxin recombinant protein, it is just in time the fragment of Trachinotus ovatus peroxiredoxin full-length gene 128bp to 721bp (being equivalent to the 43rd to the 240th amino acids), its nucleotide sequence and coded aminoacid sequence as follows.
The cDNA of Trachinotus ovatus peroxiredoxin gene, its nucleotide sequence is as follows:
Wherein bottom underscore part is the DNA sequence dna of the mature peptide of Trachinotus ovatus peroxiredoxin.
The aminoacid sequence of Trachinotus ovatus peroxiredoxin gene is as follows:
The aminoacid sequence of the mature peptide of Trachinotus ovatus peroxiredoxin.
Amino acid sequence analysis of the present invention shows that Trachinotus ovatus peroxiredoxin is participated in by 197 amino acid and forms, and no signal peptide sequence, 197 aminoacid sequences are mature peptide sequence.
Second object of the present invention is achieved by the following technical solution: a kind of expression vector, and it is the expression vector of the cDNA containing above-mentioned Trachinotus ovatus peroxiredoxin gene Prx1; And utilize the recombinant microorganism of above-mentioned vector.
The construction process of expression vector of the present invention is according to a conventional method, by the Trachinotus ovatus peroxiredoxin gene synthesized by PCR method through enzyme cut with separation and purification after, between the corresponding restriction enzyme site (i.e. BamH I and Hind III) being connected to existing respective carrier, be namely built into the required expression vector containing Trachinotus ovatus peroxiredoxin gene.
The Trachinotus ovatus peroxiredoxin gene that the above-mentioned coli expression carrier containing Trachinotus ovatus peroxiredoxin gene of the present invention is preferentially synthesized by the present invention and the recombinant expression vector that coli expression carrier pRSET A is built into, called after pRSET-Prx1.
3rd object of the present invention is achieved by the following technical solution: the method preparing restructuring Trachinotus ovatus peroxiredoxin gene Prx1 provided by the invention, comprise and transform host cell with above-mentioned expression vector, cultivate transformant, obtain the Trachinotus ovatus peroxiredoxin gene of restructuring.
Wherein host cell is preferably from intestinal bacteria, first constructs a kind of recombinant strain, and it is the intestinal bacteria containing Trachinotus ovatus peroxiredoxin gene.The above-mentioned corresponding expression vectors containing Trachinotus ovatus peroxiredoxin gene is adopted to construct the E. coli recombinant stain of energy high expression Trachinotus ovatus peroxiredoxin.
The bacterial strain that the above-mentioned E. coli recombinant stain expressing Trachinotus ovatus peroxiredoxin of the present invention is preferentially obtained by the expression vector pRSET-Prx1 transformation of E. coli BL21 containing Trachinotus ovatus peroxiredoxin gene, called after pRSET-Prx1-BL21.
Wherein prepare the method for restructuring Trachinotus ovatus peroxiredoxin gene Prx1, specific as follows: to choose E. coli recombinant stain pRSET-Prx1-BL21 and be seeded in 10mL and contain in the LB liquid nutrient medium of penbritin, 37 DEG C, spend the night, as kind of a daughter bacteria, next day is inoculated in identical substratum by 1:100 inoculum size again, when recombinant bacterium grows into logarithmic phase, adding 100mM IPTG to final concentration is 0.4mM, 220rpm shaking culture, thalline is collected after recombinant bacterium grows into plateau, restructuring Trachinotus ovatus peroxiredoxin product is obtained through separation and purification.
Compared with prior art, the present invention has the following advantages:
(1) the present invention obtains the cDNA of Trachinotus ovatus peroxiredoxin gene Prx1 from Trachinotus ovatus, comprises expression vector and the recombinant bacterial strain of this cDNA, and restructuring Trachinotus ovatus peroxiredoxin gene Prx1;
(2) this restructuring Trachinotus ovatus peroxiredoxin gene Prx1 has good reducing activity and reduction effect, is expected to development and application in the Disease epizootic aspect of Trachinotus ovatus.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that in embodiment 1, Trachinotus ovatus liver organization extracts total serum IgE;
Fig. 2 is the electrophorogram of the amplified production of the cDNA gene of reverse transcription Trachinotus ovatus peroxiredoxin Prx1 in embodiment 1;
Fig. 3 is the electrophorogram of the synthesis pcr amplification product of Trachinotus ovatus peroxiredoxin Prx1 mature peptide gene in embodiment 2;
Fig. 4 is the plasmid enzyme restriction figure of the pRSET-Prx1 containing Trachinotus ovatus peroxiredoxin Prx1 gene and coli expression carrier in embodiment 3;
Fig. 5 is the SDS-PAGE gel electrophoresis figure of the pRSET-Prx1-BL21 shown in embodiment 5 through induction;
Fig. 6 is the immunoblot results figure of mouse-anti 6 × His antibody in embodiment 5;
Fig. 7 is the SDS-PAGE gel electrophoresis figure of the restructuring shape silvery pomfret Scad peroxiredoxin albumen of purifying in embodiment 6;
Fig. 8 is the Trachinotus ovatus peroxiredoxin protein-active detected result figure that recombinates in embodiment 6.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, but do not limit the present invention in any form.
The acquisition of the cDNA gene of embodiment 1 Trachinotus ovatus peroxiredoxin Prx1
Trachinotus ovatus peroxiredoxin Prx1 full-length gene of the present invention obtains Trachinotus ovatus peroxiredoxin cDNA sequence, specifically as shown in SEQ ID NO.1 with transcript profile order-checking.Be template (see Fig. 2) with the cDNA that Trachinotus ovatus liver total RNA (Fig. 1 shown in) obtains according to the reverse transcription of M-MLV ThermoScript II specification sheets.Reverse transcription reaction condition: hatch 5min for 70 DEG C, ice bath 5min, adds M-MLV ThermoScript II premixed liquid, hatches 90min for 42 DEG C, 70 DEG C of deactivation 15min.
The aminoacid sequence of Trachinotus ovatus peroxiredoxin Prx1 is predicted by software DNAStar7.1 and is obtained.Specifically as shown in SEQ ID NO.2.
The synthesis of embodiment 2 Trachinotus ovatus peroxiredoxin Prx1 gene
According to the cDNA complete sequence of embodiment 1 Trachinotus ovatus peroxiredoxin Prx1 gene, design and synthesis pair of primers, upstream primer before the 128th bit base plays 21 bases, adds the restriction enzyme site of BamH I and 3 protection bases (5 '-GGTGGATCCATGTCTGCTGGAAATGCTAAG-3 '), 30bp altogether; Downstream primer is restriction enzyme site 2 protection bases (5 '-CGAAGCTTTTACTGCTTGGAGAAGAACTC-3 ') that front 21 bases risen at the 701st bit base add Hind III, 29bp altogether.With the cDNA library of Trachinotus ovatus peroxiredoxin Prx1 for template, through PCR method amplification Trachinotus ovatus peroxiredoxin the 1st to the corresponding DNA sequence dna of the 197th amino acids sequence, be the mature peptide corresponding sequence of Trachinotus ovatus peroxiredoxin Prx1, pcr amplification condition is: 94 DEG C of denaturation 2min; Then 94 DEG C of sex change 30s, 55 DEG C of annealing 45s, 72 DEG C extend 50S, totally 35 circulations; Last 72 DEG C of 10min.
The electrophorogram of pcr amplification product is as Fig. 3.Pcr amplification has gone out the fragment being about 591 as seen from Figure 3.
Embodiment 3 is containing the structure of the coli expression carrier of Trachinotus ovatus peroxiredoxin gene
Above-described embodiment 2 increases the PCR primer that obtains after BamH I and Hind III enzyme is cut, and digestion products AxyGen company PCR primer purification kit reclaims, and separation and purification is about the Trachinotus ovatus peroxiredoxin gene fragment of 590bp, after expression vector pRSET A cuts with BamH I and Hind III enzyme too, digestion products carries out agarose gel electrophoresis, the large fragment of separation and purification 2.9kb, mix by 1:3 with the Trachinotus ovatus peroxiredoxin gene fragment of 590bp, with T4 ligase enzyme 16 DEG C connect spend the night after proceed in e. coli bl21 with standard chlorination calcium conversion method, screening has the transformant of amicillin resistance, plasmid is extracted with standard method, screening size is about the restructuring quality of 3.5kb, by restriction enzyme BamH I and Hind III double digestion restructuring quality, obtain two fragments of 590bp and 2.9bp, size is identical with expression vector pRSET A size with Trachinotus ovatus peroxiredoxin gene respectively, prove that Trachinotus ovatus peroxiredoxin gene has been cloned in coli expression carrier pRSET A, restructuring quality called after pRSET-Prx1.Plasmid enzyme restriction figure is as Fig. 4.
The structure of the E. coli recombinant stain pRSET-Prx1-BL21 of embodiment 4 high expression Trachinotus ovatus peroxiredoxin
By standard chlorination calcium method by pRSET A-Prx1 transformation of E. coli BL21, transformant is being screened containing on the LB flat board of penbritin, obtain the sub-pRSET A-Prx1-BL21 of recombinant conversion containing pRSET A-Prx1 through plasmids detection and restriction analysis, last sequence verification, its sequencing result is correct.
Embodiment 5 utilizes recombination bacillus coli pRSET-Prx1-BL21 genetic engineering bacterium Restruction Trachinotus ovatus peroxiredoxin
Picking E. coli recombinant stain pRSET-Prx1-BL21, first strain inoculation is contained in the LB liquid nutrient medium of penbritin at 10mL, 37 DEG C, 220rpm shaking culture is spent the night, as kind of a daughter bacteria, next day is inoculated in identical substratum by 1:100 inoculum size again, (the A when recombinant bacterium grows into logarithmic phase
600=0.5-0.6), adding 100mM IPTG to final concentration is 0.4mM, 37 DEG C, receives bacterium after 220rpm vibration inducing culture 3.5h.The Trachinotus ovatus peroxiredoxin of synthesis obtains high expression in Recombinant organism pRSET-Prx1-BL21.
The cell that takes a morsel adds 2 × electrophoresis upstream damping fluid, SDS-PAGE gel electrophoresis is run by standard method after boiling 10min, result is as Fig. 5, and a new fusion rotein band appears in the pRSET-Prx1-BL21 shown through inducing on the position of about 28kDa, and this place's band without induction is very light.Then ordinary method is utilized to carry out immunoblotting (Western blot) analysis.Primary antibodie is mouse-anti 6 × His antibody, and two resist for sheep anti-mouse igg-HRP.Result, as Fig. 6, shows the fusion peroxiredoxin albumen of our use escherichia coli expression of mouse-anti 6 × His antibody capable identification.These results prove that the albumen obtained is restructuring Trachinotus ovatus peroxiredoxin Prx1.
Embodiment 6 is recombinated Trachinotus ovatus peroxiredoxin protein purification and Activity determination
By the method for above-described embodiment 4, E. coli recombinant stain pRSET-Prx1-BL21 genetic engineering bacterium is carried out enlarged culturing, 10, the centrifugal 10min of 000g collects thalline, after ultrasonic disruption, utilize His-BindPurification Kit Protocol purification of Recombinant Trachinotus ovatus peroxiredoxin albumen, through SDS-PAGE detected through gel electrophoresis, result is as Fig. 7, and display obtains the higher Trachinotus ovatus peroxiredoxin recombinant protein of purity.
Substrate Regular Insulin (1mg/mL) is added in 96 orifice plates, divide and add dithiothreitol (DTT) (200mM) and pRSET-Prx1-BL21 group, add pRSET-Prx1-BL21 and do not add dithiothreitol (DTT) group, with reductive glutathione (200mM) for positive control, albumen damping fluid is blank.Utilize microplate reader to measure reducing agent dithiothreitol and deposit OD in case
650the light absorption value at place.Often kind of sample repeats 3 times, and the final absorption value of each sample gets the average result of 5 times.Result as shown in Figure 8.As can be seen from Figure 8, under dithiothreitol (DTT) exists situation, its light absorption value is higher, shows that substrate Regular Insulin consumes in a large number, reflects that pRSET-Prx1-BL21 has good reducing activity and reduction effect.
Claims (7)
1. a cDNA of Trachinotus ovatus peroxiredoxin gene Prx1, its nucleotide sequence is as shown in SEQID NO.1.
2. a Trachinotus ovatus peroxiredoxin gene Prx1, its aminoacid sequence is as shown in SEQ ID NO.2.
3. comprise the expression vector of cDNA according to claim 1.
4. comprise the expression vector pRSET-Prx1 of cDNA according to claim 1.
5. prepare the method for restructuring Trachinotus ovatus peroxiredoxin gene Prx1, it is characterized in that: comprise and transform host cell with expression vector according to claim 3, cultivate transformant, obtain the Trachinotus ovatus peroxiredoxin gene of restructuring.
6. method according to claim 5, is characterized in that: wherein host cell is intestinal bacteria.
7. a restructuring Trachinotus ovatus peroxiredoxin gene Prx1, comprise and transform host cell with expression vector according to claim 3, cultivate transformant, obtain the method preparation of the step of the Trachinotus ovatus peroxiredoxin gene Prx1 of restructuring from culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410735312.7A CN104480126A (en) | 2014-12-05 | 2014-12-05 | Trachinotus ovatus peroxiredoxin gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410735312.7A CN104480126A (en) | 2014-12-05 | 2014-12-05 | Trachinotus ovatus peroxiredoxin gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104480126A true CN104480126A (en) | 2015-04-01 |
Family
ID=52754732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410735312.7A Pending CN104480126A (en) | 2014-12-05 | 2014-12-05 | Trachinotus ovatus peroxiredoxin gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104480126A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107686841A (en) * | 2017-09-20 | 2018-02-13 | 江苏师范大学 | A kind of IbANR genes of coding sweet potato anthocyanin reductase and its application |
| CN110551732A (en) * | 2019-08-30 | 2019-12-10 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide LEAP-2 gene and application thereof |
| CN110643612A (en) * | 2019-08-30 | 2020-01-03 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide NK-lysin gene and application thereof |
-
2014
- 2014-12-05 CN CN201410735312.7A patent/CN104480126A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| LONGWANG ET AL.: "Molecular characterization and functional analysis of a peroxiredoxin 1 cDNA from golden pompano (Trachinotus ovatus)", 《DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY》 * |
| NCBI: "GenBank: ACQ58196.1", 《NCBI GENBANK》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107686841A (en) * | 2017-09-20 | 2018-02-13 | 江苏师范大学 | A kind of IbANR genes of coding sweet potato anthocyanin reductase and its application |
| CN110551732A (en) * | 2019-08-30 | 2019-12-10 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide LEAP-2 gene and application thereof |
| CN110643612A (en) * | 2019-08-30 | 2020-01-03 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide NK-lysin gene and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Song et al. | Cultivation at 6–10 C is an effective strategy to overcome the insolubility of recombinant proteins in Escherichia coli | |
| CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
| CN110408635A (en) | A kind of application of the nucleic acid constructs containing Streptavidin element in protein expression, purifying | |
| CN104480125B (en) | Trachinotus ovatus thioredoxin gene | |
| CN104480126A (en) | Trachinotus ovatus peroxiredoxin gene | |
| CN112851792A (en) | Preparation method and application of grass carp TNF-alpha recombinant protein | |
| CN108998458B (en) | Process for preparing recombinant human insulin | |
| CN102206282B (en) | Efficient method for producing Trx-hCTRP2 | |
| Gorokhovets et al. | Rational design of recombinant papain-like cysteine protease: Optimal domain structure and expression conditions for wheat-derived enzyme triticain-α | |
| CN107937408B (en) | Epinephelus coioidesinsulinGene, encoded protein and application thereof | |
| CN102321643B (en) | Optimized DNA (Deoxyribonucleic Acid) molecule for coding ADI (Aiginine Deiminase) and engineering bacteria for expressing ADI | |
| CN103397038B (en) | Production method of human interleukin-38 | |
| CN104878036B (en) | A kind of models fitting and genetic modification improve method and the application of protein expression efficiency | |
| CN103555739A (en) | Recombined microorganism glutamine transaminase gene and preparation method thereof | |
| CN103709242B (en) | A kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene, preparation method and application | |
| CN103724415B (en) | Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof | |
| CN103131718A (en) | Cloning of hypertonicity-resistant functional gene CgHog1 from Candida glycerinogenes and application thereof | |
| CN105838724A (en) | Malate dehydrogenase gene RGMDH1 and recombinant expression vector containing same | |
| Wang et al. | Cloning and characterization of a phosphomevalonate kinase gene from Sanghuangporus baumii | |
| CN110819645B (en) | Fancy carp Gtpch2 gene, coded protein and application thereof | |
| CN109371047A (en) | Method for constructing and expressing heat-resistant antibacterial peptide fusion protein by using protein IHF- α | |
| CN102199202B (en) | Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof | |
| Liu et al. | Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917 | |
| CN101962654B (en) | Overexpression of thymidylate synthase in colon bacillus | |
| CN105368802A (en) | Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150401 |