CN104694547A - Penaeus monodon heat shock protein 40 gene and protein coded by same - Google Patents

Penaeus monodon heat shock protein 40 gene and protein coded by same Download PDF

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CN104694547A
CN104694547A CN201510082048.6A CN201510082048A CN104694547A CN 104694547 A CN104694547 A CN 104694547A CN 201510082048 A CN201510082048 A CN 201510082048A CN 104694547 A CN104694547 A CN 104694547A
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gene
heat shock
shock protein
protein
hsp
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邱丽华
傅明骏
赵超
史进选
江世贵
周发林
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South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
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South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
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Abstract

The invention discloses a penaeus monodon heat shock protein 40 gene and a protein coded by the same, and belongs to the technical field of biological genes. The gene is used for laying a foundation for studying the effect of penaeus monodon heat shock protein 40 in penaeus monodon, and providing a theoretical base for various physicochemical irritation response effects happening in the process of culturing penaeus monodon in the molecular level. According to technical scheme, the gene sequence is as shown in SEQ ID NO:1; and the amino acid sequence of the coded protein is as shown in SEQ ID NO:2.

Description

The albumen of tigar prawn heat shock protein(HSP) 40 gene and coding thereof
Technical field
The invention discloses the albumen of tigar prawn heat shock protein(HSP) 40 gene and coding thereof, belong to biological gene technical field.
Background technology
Heat shock protein(HSP) (heat shock proteins, HSPs) also known as stress protein, that body cell is under the induction of some stressors (as high ambient temperature, anoxic, heavy metal poisoning, oxidative stress, infection, hunger, wound, metabolic poison etc.), HSP gene is activated, and one of high expression group of protein of high conservative on evolving, from the infringement of stressors, there is vital role to body.
Hsp40 albumen is the important molecular chaperones of a class, has important physiological function.Research show Hsp40 albumen organism normal and heat shock, salt, heavy metal, toxicant metabolism etc. coerce the effect such as degraded of folding, the transhipment of lower participation protein, secretion and target protein.Hsp40 can by regulating the ATPase of Hsp40/Hsp70 active, and the substrate polypeptide that Hsp70 is combined occurs folding, and the protein renaturation (again folding) of promotion partially denaturing is protected the cell of coercing infringement and made it recover normal function.Hsp40 participates in the renaturation of the stable of Protein requirement structure and metaprotein, is a kind of and the closely-related key protein of the multiple vital movement of cell.
The cultivation density of tigar prawn is low, the poor important factor being puzzlement spot always and saving aquaculture of resistance, for improving its speed of growth, cultivation density and resistance, and then improve the output of tigar prawn, to the growth of its inherence, molecule mechanism that resistance is relevant is studied is that tigar prawn is bred, cultivated requisite work.
Summary of the invention
For the problems referred to above, the object of the invention is the pcr amplification primer designed based on the est sequence of laboratory high-flux sequence acquisition, and be cloned into the full expressed sequence of tigar prawn heat shock protein(HSP) 40 gene by RACE technology; In addition by tigar prawn heat shock protein(HSP) 40 gene design prokaryotic expression primer, carry out recombinant expressed in vitro to heat shock protein(HSP) 40, for the effect of research tigar prawn heat shock protein(HSP) 40 in tigar prawn lays the foundation, and can be occur in cultivating penaeus monodon process various physicochemical irritation response effect is provided fundamental basis from molecule aspect.
Heat shock protein(HSP) 40 gene provided by the invention, this gene order is as shown in SEQ ID NO:1.
The preparation method of this gene comprises the steps: successively
1, the extraction of total serum IgE
Get healthy tigar prawn, dissect, get ovary tissue, and 1mL Trizol (Invitrogen will be organized in, Japan) middle homogenate, extract tigar prawn total serum IgE by Trizol test kit service manual and use DEPC aqueous suspension, electrophoresis observation extract the integrity of RNA, and be placed in-80 DEG C of preservations.
2, the synthesis of cDNA first chain
(oligo-dT/adaptor (5 '-GGCCACGCGACT AGTAC (T) 16-3 ') 1 μ L (10pmol/L) is blended in ThermoScript II (M-MLV to get above-mentioned tigar prawn total serum IgE 2 μ L and reverse transcription primer, Promega, USA) cDNA mono-chain is synthesized under effect, reaction conditions: 42 DEG C of 60min, 70 DEG C of 15min.
3, design of primers foundation, the method for primer synthesis
Est sequence design primer according to existing tigar prawn heat shock protein(HSP) 40:
Pm-HSP40cDNA total length increases and expresses primer used
Hua Da genome company is submitted to synthesize after design of primers.
The pcr amplification of 4 tigar prawn heat shock protein(HSP) 40 genes, clone
Using the first chain cDNA of above-mentioned synthesis as template, the 3 ˊ ends of cDNA end rapid amplifying technology to goal gene are utilized to carry out pcr amplification.
In 3 ˊ RACE, utilize touchdown PCR (touchdown PCR) method, carry out pcr amplification with adapter-primer: 5>GGCCACGCGTCGACTAGTAC<3 and HSP40-F1, HSP40-F2.
After the PCR primer obtained carries out separation detection on the agarose gel electrophoresis of 1%, after PCR primer purifying, be cloned in pMD-18T carrier, transformation of E. coli DH5 α competent cell, picking positive colony, submits to Shanghai Ying Jun Bioisystech Co., Ltd to check order.
5, to the mensuration of tigar prawn heat shock protein(HSP) 40 gene
After the PCR primer obtained carries out separation detection on the agarose gel electrophoresis of 1%, after PCR primer purifying, be cloned in pMD-18T carrier, transformation of E. coli (Escherichia coli) DH5 α competent cell, picking positive colony checks order.Sequencing result BLAST software carries out homology mensuration, and be defined as tigar prawn heat shock protein(HSP) 40 DNA homolog sequence, result is as shown in SEQ ID NO:1.
6, tigar prawn heat shock protein(HSP) 40 utilizes intestinal bacteria to carry out in-vitro recombination expression
According to amplification institute heat shock protein(HSP) 40 gene order total length, in open reading frame, design primer H40-F and H40-R of prokaryotic expression, add Nde I and BamH I restriction enzyme site in primer and protect base.Carry out pcr amplification, then cut heat shock protein(HSP) 40 goal gene with NdeI and BamH I enzyme, same enzyme cuts pET21a plasmid, and heat shock protein(HSP) 40 gene fragment and pET21a are reclaimed in gel electrophoresis, and carry out connecting of endonuclease bamhi with T4 ligase enzyme, 16 DEG C are spent the night.Transformation of E. coli DH5 α, screening positive clone on the LB solid medium containing penbritin, positive colony is again with PCR reaction, order-checking qualification.Correct positive colony is carried out the extraction of heat shock protein(HSP) 40-Pet21a recombinant plasmid, transformation of E. coli BL21, coated plate, choose mono-clonal, order-checking.Get recombinant bacterium liquid and be inoculated in LB liquid nutrient medium containing penbritin, 37 DEG C of about shaking culture 2h, make bacterium liquid OD value reach between 0.4-0.6, adding isopropylthio semi-lactosi sweet (IPTG) is 0.6mmol/L to ultimate density, and 28 DEG C are continued to cultivate abduction delivering.Every 2h gets 2.0ml sample, bacteria recovered by centrifugation, add the resuspended thalline of PBS of 200ul, mixing, adds the sample-loading buffer of 50ul, puts in boiling water bath and boil 10min, the centrifugal 1min of 10000 × g, get supernatant 15ul electrophoresis on the SDS-PAGE of 12%, coomassie brilliant blue staining, Taking Pictures recording result under white light.
The albumen of above-mentioned heat shock protein(HSP) 40 genes encoding, the aminoacid sequence of described albumen is as shown in SEQ ID NO:2
This aminoacid sequence is encoding to the terminator codon TGA at 1304bp place with the initiator codon ATG at SEQ ID NO:1116bp place, 396 aminoacid sequences of encoding.
Compared with prior art, technical scheme provided by the invention has following technological merit:
1, gene provided by the invention is that the effect of research tigar prawn heat shock protein(HSP) 40 in tigar prawn lays the foundation, and can be providing fundamental basis from molecule aspect to various physicochemical irritation response effect of occurring in cultivating penaeus monodon process.
2, albumen provided by the invention is bred at spot joint, be can be used as a kind of immune protein in breeding process, to the heavy metal in preventing from cultivating to the vital role of the injury of tigar prawn.Simultaneously also can using HSP40 as detecting the molecular indexes that in spot joint shrimp aquaculture environment, a huge sum of money pollutes, there is directive significance to the improvement of cultivating penaeus monodon environment and monitoring.
3, gene provided by the invention improves cultivation density, the enhancing resistance of tigar prawn.
Accompanying drawing explanation
Fig. 1 tigar prawn heat shock protein(HSP) 40 utilizes intestinal bacteria to carry out in-vitro recombination expression detection figure;
Wherein: 1,2,3 be respectively PET28a empty plasmid do not induce, induce 3h, induction 6h, 4,5,6 be respectively PET21a-pmHSP40 does not induce, induce 3h, induction 6h; Right figure is pmHSP40western blot result.
Embodiment
Below in conjunction with embodiment; claim of the present invention is described in further detail; but do not form any limitation of the invention, the amendment of anyone limited number of time made within the claims in the present invention scope, still within claims of the present invention.
In following examples unless otherwise indicated, the normal experiment method in this area and operation steps is.
Embodiment 1
Tigar prawn heat shock protein(HSP) 40 gene provided by the invention, this gene order is as shown in SEQ ID NO:1.
Its preparation method is as follows
1, the extraction of total serum IgE
Test male and female tigar prawn (fresh weight about 200g) used and be all taken from Sanya, Hainan, support three days temporarily in indoor aquarium, water temperature controls between 24 ~ 25 DEG C.The Trizol (invitrogen company of the U.S.) organizing about 50mg scissors to shred needed for getting to add 1ml carries out homogenate, and extracts total serum IgE according to the following steps.
Total RNAs extraction step:
(1) add 200 μ L precooling chloroforms, vortex oscillation 1min mixes, and leaves standstill 5min, 12000g, 4 DEG C, 15min.
(2) get 1/3rd supernatant solution, add the Virahol of isopyknic precooling, put upside down mixing, place 10min on ice, 12000g, 4 DEG C, 10min.
(3) outwell supernatant, add the resuspended precipitation of 1mL 75% ethanol, 7500g, 4 DEG C of centrifugal 5min.
(4) repeating step 3 is once, and with rifle head sucking-off supernatant, dry 5 ~ 10min
(5) 40 μ LddH2O dissolution precipitations are added
(6) electrophoresis detection of RNA: electrophoresis chamber, comb etc. are placed in 3%H2O2 and spend the night immersion, the sepharose (with 0.5 × TBE solution preparation) of configuration 1.5%, get 5 μ L total serum IgE and add 1 μ L sample-loading buffer, carry out 30min electrophoresis detection, the RNA quality extracted is measured.
2, the synthesis of cDNA first chain
The reverse transcription experiment of cDNA is carried out according to related kit (PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa)) working method.
Slight mixing, ice bath 2min after 70 DEG C of incubation 10min, put back on ice after the centrifugal several seconds
To be mixed evenly and of short duration centrifugal; After PCR instrument 42 DEG C reaction 1h, 70 DEG C, 15min takes out and places on ice, and-20 DEG C save backup.
3, design of primers foundation, the method for primer synthesis
With the est sequence of laboratory high-flux sequence gained for template carries out RACE design of primers.Obtain primer as follows:
4, the clone of tigar prawn heat shock protein(HSP) 40 gene cDNA complete sequence
Using the first chain cDNA of above-mentioned synthesis as template, pcr amplification is carried out with the RACE primer of design, in 3 ' RACE, Semi-nested (half-nest type) PCR method is utilized to carry out amplified reaction, first respectively PCR is carried out to expansion reaction by HSP40-F1 and AP (adapter-primer: Adaptor primer), products therefrom gets 1 μ L as template after carrying out 100 times of dilutions, utilizes HSP40-F2 and Adaptor primer to carry out PCR bis-amplifications.
(1) one expands PCR reaction system formula: 10 × Ex Taq Buffer 2.5 μ L; DNTP Mixture (2.5mM) 2 μ L; HSP40-F1 (10 μMs) 1 μ L; Adaptor primer (10 μMs) 1 μ L; Ex Taq (5U/ μ L) 0.25 μ L; MgCL21.5 μ L; CDNA 1 μ L; DdH2O 15.75 μ L; Total 25 μ L.
The response procedures of one expansion PCR experiment is as follows:
(2) two expand PCR reaction system formula: 10 × Ex Taq Buffer 2.5 μ L; DNTP Mixture (2.5mM) 2 μ L; HSP40-F2 (10 μMs) 1 μ L; Adaptor primer (10 μMs) 1 μ L; MgCL21.5 μ L; Ex Taq (5U/ μ L) 0.25 μ L; Dilute 100 times one expands PCR primer 1 μ L; DdH2O 15.75 μ L; Total 25 μ L.
The response procedures of two expansion PCR experiment is as follows:
After the PCR primer obtained carries out separation detection on the agarose gel electrophoresis of 1%, after PCR primer purifying, be cloned in pMD-18T carrier, transformation of E. coli (Escherichia coli) DH5 α competent cell, picking positive colony, submits to Shanghai Ying Jun Bioisystech Co., Ltd to check order.
5, to the mensuration of tigar prawn heat shock protein(HSP) 40 gene
After the PCR primer obtained carries out separation detection on the agarose gel electrophoresis of 1%, after PCR primer purifying, be cloned in pMD-18T carrier, transformation of E. coli (Escherichia coli) DH5 α competent cell, picking positive colony, checks order.Sequencing result BLAST software carries out homology mensuration, is defined as the homologous sequence of tigar prawn heat shock protein(HSP) 40 gene.
6, tigar prawn heat shock protein(HSP) 40 utilizes intestinal bacteria to carry out in-vitro recombination expression
According to amplification institute heat shock protein(HSP) 40 gene order total length, in open reading frame, design primer H40-F and H40-R of prokaryotic expression, add Nde I and BamH I restriction enzyme site in primer and protect base.Carry out pcr amplification, then cut heat shock protein(HSP) 40 goal gene with NdeI and BamH I enzyme, same enzyme cuts pET21a plasmid, and heat shock protein(HSP) 40 gene fragment and pET21a are reclaimed in gel electrophoresis, and carry out connecting of endonuclease bamhi with T4 ligase enzyme, 16 DEG C are spent the night.Transformation of E. coli DH5 α, screening positive clone on the LB solid medium containing penbritin, positive colony is again with PCR reaction, order-checking qualification.Correct positive colony is carried out the extraction of heat shock protein(HSP) 40-Pet21a recombinant plasmid, transformation of E. coli BL21, coated plate, choose mono-clonal, order-checking.Get recombinant bacterium liquid and be inoculated in LB liquid nutrient medium containing penbritin, 37 DEG C of about shaking culture 2h, make bacterium liquid OD value reach between 0.4-0.6, adding isopropylthio semi-lactosi sweet (IPTG) is 0.6mmol/L to ultimate density, and 28 DEG C are continued to cultivate abduction delivering.Every 2h gets 2.0ml sample, bacteria recovered by centrifugation, add the resuspended thalline of PBS of 200ul, mixing, adds the sample-loading buffer of 50ul, puts in boiling water bath and boil 10min, the centrifugal 1min of 10000 × g, get supernatant 15ul electrophoresis on the SDS-PAGE of 12%, coomassie brilliant blue staining, Taking Pictures recording result under white light.
Conventionally SDS-PAGE is carried out to the sample before IPTG induction and after induction, then transferring film, close, wash film, antibody incubation, develop the color, take pictures.Antibody used is 6 × His Tag HRP antibody kit of Huaan biotech firm, and what color reaction detected is HRP-DAB enhancement type colouring reagents box.Consult Fig. 1, wherein, PET21a-pmHSP10 SDS-PAGE, 1,2,3 are respectively PET28a empty plasmid does not induce, induces 3h, induction 6h result; 4,5,6 be respectively PET21a-pmHSP10 and do not induce, induce 3h, induction 6h result.
The albumen of above-mentioned heat shock protein(HSP) 40 gene sequences encode, the aminoacid sequence of described albumen is as shown in SEQ ID NO:2
This aminoacid sequence is encoding to the terminator codon TGA at 1304bp place with the initiator codon ATG at SEQ ID NO:1116bp place, and 396 aminoacid sequences of encoding, the nucleotides sequence of the amino acid whose gene fragment of specific coding is classified as SEQ ID NO:3.

Claims (7)

1. tigar prawn heat shock protein(HSP) 40 gene, is characterized in that, described gene order is as shown in SEQ ID NO:1.
2. the albumen of tigar prawn heat shock protein(HSP) 40 genes encoding according to claim 1, is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ ID NO:2.
3. containing the recombinant plasmid of gene described in claim 1 or 2.
4. recombinant plasmid according to claim 3, is characterized in that, the recombinant plasmid that described recombinant vectors obtains for the multiple clone site that gene described in claim 1 or 2 is inserted pET21a plasmid.
5. the recombinant bacterium containing gene described in claim 1 or 2.
6. recombinant bacterium according to claim 5, is characterized in that, recombinant plasmid transformed e. coli bl21 described in claim 4 obtains by described recombinant bacterium.
7. tigar prawn heat shock protein(HSP) 40 in-vitro recombination expression in intestinal bacteria described in right 1 or 2.
CN201510082048.6A 2015-02-13 2015-02-13 Penaeus monodon heat shock protein 40 gene and protein coded by same Pending CN104694547A (en)

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Cited By (5)

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CN105296654A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp60 gene expression by utilizing fluorescent RT-PCR technique
CN105296651A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp90 gene expression by adopting fluorescence RT-PCR technology
CN105296652A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp40 gene expression by utilizing fluorescent RT-PCR technique
CN105296653A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp83 gene expression by utilizing fluorescent RT-PCR technique
CN109468327A (en) * 2018-11-09 2019-03-15 中国科学院南海海洋研究所 40 gene lvHSP40 of litopenaeus vannamei heat shock protein and its application in the inhibition prawn development of ovary

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105296654A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp60 gene expression by utilizing fluorescent RT-PCR technique
CN105296651A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp90 gene expression by adopting fluorescence RT-PCR technology
CN105296652A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp40 gene expression by utilizing fluorescent RT-PCR technique
CN105296653A (en) * 2015-11-26 2016-02-03 天津师范大学 Method for detecting Chouioia cunea Yang Hsp83 gene expression by utilizing fluorescent RT-PCR technique
CN105296652B (en) * 2015-11-26 2018-07-06 天津师范大学 The method that chalcid fly Hsp40 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi
CN105296654B (en) * 2015-11-26 2018-07-06 天津师范大学 The method that chalcid fly Hsp60 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi
CN105296653B (en) * 2015-11-26 2018-07-06 天津师范大学 The method that chalcid fly Hsp83 gene expressions are nibbled using fluorescent RT-PCR technology detection Zhou Shi
CN109468327A (en) * 2018-11-09 2019-03-15 中国科学院南海海洋研究所 40 gene lvHSP40 of litopenaeus vannamei heat shock protein and its application in the inhibition prawn development of ovary

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