CN105399808B - A kind of flat Rockfish Immune-enhancing effect albumen HMGB1 gene of Xu Shi and coding albumen and application - Google Patents
A kind of flat Rockfish Immune-enhancing effect albumen HMGB1 gene of Xu Shi and coding albumen and application Download PDFInfo
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- CN105399808B CN105399808B CN201510821237.0A CN201510821237A CN105399808B CN 105399808 B CN105399808 B CN 105399808B CN 201510821237 A CN201510821237 A CN 201510821237A CN 105399808 B CN105399808 B CN 105399808B
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Abstract
The present invention relates to molecular biology field, flat Rockfish Immune-enhancing effect albumen (HMGB1) encoding gene of specifically a kind of Xu Shi and its recombinant protein preparation and application.HMGB 1 is shown in the amino acid sequence in sequence table SEQ ID No.1, and the encoding gene of the albumen is shown in the base sequence in sequence table SEQ ID No.2.Preparation method: using the flat Rockfish head-kidney cDNA of Xu Shi of Vibrio anguillarum stimulation as template, PCR amplification HMGB1 encoding gene constructs plasmid pHMGB1；PHMGB1 is converted into e. coli bl21 Transetta (DE3), after carrying out inducing expression to transformant, extracts and purifying protein is to get recombination HMGB1 albumen.The recombination HMGB1 albumen is remarkably improved the immunocompetence of the flat Rockfish head-kidney macrophage of Xu Shi in combination with double-stranded DNA, improves its resistivity to the flat Rockfish of sterilizing function and Xu Shi of Vibrio anguillarum to Vibrio anguillarum.Recombination HMGB1 albumen can be used as the prevention and treatment that immunopotentiator is applied to fish Vibrio anguillarum disease.
Description
Technical field
The present invention relates to molecular biology field, the flat Rockfish Immune-enhancing effect protein coding gene of specifically a kind of Xu Shi and
The preparation of its albumen and application.
Background technique
High mobility group protein (HMGB) family includes HMGB1, HMGB2, HMGB3 and HMGB4, wherein studying in fish
That more is HMGB1, followed by HMGB2.HMGB1 is that one kind is present in the endonuclear nonhistone chromosomal knot of eukaryocyte
Hop protein, because its in polyacrylamine gel electrophoresis migration velocity it is fast due to gain the name.The study found that HMGB1 be present in it is most of
In animal body.The albuminoid is characterized mainly in that they contain there are two HMG structural domain and an acidic carboxypolymer end regions.
In mammals, the HMGB1 of nucleus is in the stabilization of nucleosomal structure, the reparation of DNA mismatch and regulation of gene expression etc.
Aspect plays an important role.Research confirms that after host is by pathogen infection, the HMGB1 of nucleus can move to cell at present
Matter, moving to cytoplasmic HMGB1 can stimulate body to generate a series of immune response, and the breathing including enhancing macrophage is quick-fried
Hair and phagocytosis and the proliferation function for promoting lymphocyte enhance the generation etc. of TNF-α inducible factor.Therefore, HMGB1 exists
It plays a significant role in the innate immunity of host versus Infected with Pathogenic Fungi.
Summary of the invention
It is an object of that present invention to provide a kind of flat Rockfish Immune-enhancing effect albumen HMGB1 codings of Xu Shi with nucleic acid binding properties
Gene and its albumen preparation and application.
To achieve the above object, the technical solution adopted by the present invention are as follows:
HMGB1 of the invention is the amino acid sequence in sequence table SEQ ID No.1, and encoding gene is sequence table SEQ
Base sequence in ID No.2.
1) building of plasmid pHMGB1:
Using the flat Rockfish head-kidney cDNA of Xu Shi of Vibrio anguillarum stimulation as template, PCR expansion is carried out with primer HMGB1F1 and HMGB1R1
Increase.PCR product is connect with carrier pEasy-T1 after purification, is built into plasmid pT1HMGB1.By pT1HMGB1 and plasmid pET259
Restriction enzyme EcoRV and SwaI digestion is used respectively, 0.621kb and 5.4kb segment is separately recovered, this two segments are used
The connection of T4DNA ligase, is built into plasmid pHMGB1.
2) recombinant expression and protein purification of HMGB1:
The plasmid pHMGB1 of step 1) is converted into e. coli bl21 Transetta (DE3), mould containing kanamycins and chlorine
Transformant BL21/pHMGB1 is screened on the LB culture medium of element.BL21/pHMGB1 is inoculated in containing kanamycin and chloramphenicol
It is about 0.50 in 37 DEG C of shaken cultivations to OD600 in LB culture medium, 1mM isopropyl-β-D-thiogalactoside is added, continues
Culture 4 hours, carries out protein induced expression.Then use nickel-nitrilotriacetic acid affinity chromatography column purification weight
Histone.HMGB1 albumen is recombinated in as sequence table SEQ ID No.1 shown in amino acid sequence.
Immune-enhancing effect albumen can be used as immunopotentiator for enhancing the resistivity to Vibrio anguillarum of fish body.
The present invention has the advantage that Immune-enhancing effect albumen is when host is by pathogen infection, it can acute activation host day
Right immune response.
Detailed description of the invention:
Fig. 1 be recombination provided in an embodiment of the present invention after HMGB1 albumen and DNA combination electrophorogram.
Specific embodiment
Below with reference to embodiment, the invention will be further described.Embodiment is intended to carry out citing description to the present invention, and
It is non-to limit the invention in any form.
Embodiment 1
HMGB1 of the invention is the amino acid sequence in sequence table SEQ ID No.1, and encoding gene is sequence table SEQ
Base sequence in ID No.2.
Sequence table SEQ ID No.1 are as follows:
MVKEPGKPRGKMSSYAYFVQTCREEHKKKHPEASVNFSEFSKKCSERWKMMSAKEKGKFEDLARSDKVR
YEREMMSYIPPRGSKTKKFKDPNAPKRPPSAFFVFCAEYRPKVKGEAPGLTIGEVAKRLGEMWNGTASEDKQPFEKK
AAKLKEKYEKDVAAYRAKGKPGAAPTAAAPSKAPAKVEKKDDDDDDDDDEDDDDFDDDDD
(a) sequence signature:
● length: 206
● type: amino acid sequence
● chain: single-stranded
● topological structure: linear
(b) molecule type: protein
(c) assume: no
(d) antisense: no
(e) initial source: the flat Rockfish of Xu Shi
(f) structure feature: the albumen is expected containing there are two HMG structural domains (respectively by amino acid 7-79 and 93-163 group
At).
Sequence table SEQ ID No.2 are as follows:
ATGGTGAAAGAACCAGGAAAGCCGAGGGGCAAGATGTCCTCCTATGCCTATTTTGTCCAGACTTGTCGG
GAGGAGCACAAGAAGAAGCACCCCGAAGCTTCGGTCAACTTCTCTGAGTTCTCCAAGAAGTGCTCTGAGCGGTGGAA
GATGATGTCTGCCAAGGAGAAGGGCAAATTTGAGGACCTGGCCAGGTCGGACAAGGTACGCTATGAGAGGGAGATGA
TGAGCTACATACCACCCAGGGGATCCAAGACGAAGAAGTTCAAGGACCCTAATGCCCCCAAGAGACCCCCATCTGCC
TTCTTCGTCTTTTGCGCGGAGTATCGCCCCAAGGTGAAAGGCGAGGCCCCTGGTCTGACCATTGGAGAAGTTGCCAA
GAGGCTGGGTGAGATGTGGAACGGCACCGCTTCAGAGGACAAGCAGCCCTTTGAGAAGAAGGCAGCTAAATTGAAGG
AGAAGTATGAGAAGGACGTCGCAGCATACCGCGCTAAGGGCAAACCAGGCGCCGCACCAACAGCAGCAGCACCAAGC
AAAGCCCCGGCCAAGGTAGAGAAGAAGGACGACGACGACGACGATGATGATGACGAGGACGACGACGACTTCGATGA
TGACGACGACTAG
(a) sequence signature:
● length: 621
● type: base sequence
(b) molecule type: gene
(c) assume: no
(d) antisense: no
(e) initial source: the flat Rockfish of Xu Shi
Embodiment 2
The preparation method of High mobility group box-1:
1) building of plasmid pHMGB1:
The corresponding amino acid sequence of HMGB1 encoding gene is analyzed, to remove encoded signal peptide and termination codon
Gene order is template, designs forward primer HMGB1F1 and reverse primer HMGB1R1.The flat Rockfish head of Xu Shi stimulated with Vibrio anguillarum
Kidney cDNA is template, carries out PCR amplification with primer HMGB1F1 and HMGB1R1.Amplification condition are as follows: 94 DEG C of 60s initial denaturation templates
DNA, then 94 DEG C of 40s, 55 DEG C of 60s, 72 DEG C of 30s are changed to 94 DEG C of 40s, 61 DEG C of 60s, 72 DEG C of 30s, 25 circulations after 5 circulations
Afterwards again in 72 DEG C of extension 7min.PCR product (is purchased from that " Quan Shijin (Beijing) biotechnology is limited with carrier pEasy-T1 after purification
Company ") it is connected 10 minutes in room temperature, containing ampicillin (100 μ g/ml), Xgal after connection mixed liquor conversion Escherichia coli
It is cultivated 18-24 hours on the LB culture medium of (40 μ g/ml), isopropyl-β-D-thiogalactoside (24 μ g/ml), screening conversion
Son extracts plasmid, as plasmid pT1HMGB1.
By above-mentioned plasmid pT1HMG1 and plasmid pET259, (plasmid pET259 building process is referring to Zheng WJ, HuYH, Sun
L.Cloning and analysis of a ferritin subunit from turbot(Scophthalmus
maximus).Fish Shellfish Immunol.2010a；28 (5-6), 829-836.) restriction enzyme is used respectively
0.62kb and 5.4kb segment is recycled after EcoRV and SwaI digestion, this two segments T4DNA ligase is connected, liquid will be connected
Escherichia coli are transformed into, are cultivated 18-24 hours on the LB culture medium containing kanamycins (50 μ g/ml), screening transformant extracts
Plasmid, as pHMGB1.
The HMGB1F1 is 5 '-gatatcATGGTGAAAGAACCAGGAAAG-3 '；HMGB1R1 is 5 '-
gatatcGTCGTCGTCATCATCGAAG-3’。
2) expression and preparation of HMGB1 albumen are recombinated:
Plasmid pHMGB1 conversion e. coli bl21 Transetta (DE3) of step 1) (is purchased from " Quan Shijin (Beijing)
Bioisystech Co., Ltd "), it cultivates, sieves on the LB culture medium of the chloramphenicol of kanamycins and 50 μ g/ml containing 50 μ g/ml
Selecting transformant is BL21/pHMGB1.BL21/pHMGB1 is inoculated in containing kanamycin and chloramphenicol LB culture medium,
In 37 DEG C of cultures to OD600About 0.5, the isopropyl-β-D-thiogalactoside of 1mM, 37 DEG C of continuation shaken cultivation 4-5 are added
Hour, it is then pure with the nickel-nitrilotriacetic acid affinity column of GE Healthcare (U.S.) company
Change recombinant protein.The recombinant protein of purifying is subjected to n terminal amino acid sequencing, it was demonstrated that it is respectively in sequence table SEQ ID No.1
HMGB1 albumen shown in amino acid sequence.
3) combination of HMGB1 albumen and DNA is recombinated
The recombination HMGB1 albumen of various concentration, the recombination HMGB1 albumen of thermal denaturation or PBS and HMGB1 gene product are existed
It is mixed in PBS, in being incubated for 1h under room temperature, reaction product is subjected to DNA agarose gel analysis.As a result, it has been found that (Fig. 1), weight
Group HMGB1 albumen can be such that the mobility speed of DNA fragmentation slows down, moreover, the concentration of recombination HMGB1 albumen is higher, the swimming of DNA
Speed is slower.And it is complete with the DNA mobility speed and PBS group (swimming lane 1) of the recombination HMGB1 albumen (swimming lane 2) of thermal denaturation processing
Unanimously.These results illustrate that recombinating HMGB1 albumen can combine with DNA, which facilitates HMGB1 and stablizing nucleosomal structure
And it plays a role in controlling gene expression.
4) recombination HMGB1 albumen can in stimulating expression of macrophage TNF-α generation
The flat Rockfish head-kidney macrophage of Xu Shi is extracted, is seeded to 96 porocyte culture plates by the density of every 105 cells in hole, it will
Recombination HMGB1 albumen, the recombination HMGB1 albumen of thermal denaturation or PBS are added in tissue culture plate, final concentration of 80mg/ml, in
28 DEG C of incubation 12h.Cells and supernatant is collected, with the generation of fish TNF-α detection kit detection TNF-α.The results show that recombination
TNF-α content in the cells and supernatant of HMGB1 albumen processing group is 1.51 times of PBS group；And thermal denaturation recombinates HMGB1 egg
White processing group and PBS group are without significant difference (P < 0.05).
5) recombination HMGB1 albumen can enhance the bactericidal activity of the flat Rockfish head-kidney macrophage of Xu Shi
Vibrio anguillarum preparation.Culturing eel vibrio is to OD in LB culture medium600It is 0.5, then room temperature is centrifuged 10 minutes, is collected
Thallus, be suspended in PBS buffer solution (PBS buffer solution (pH7.2~7.4): NaCl 137mM, KCl2.7mM,
Na2HPO410mM, KH2PO42mmol/L to final concentration of 2 × 10 in)6CFU/ml。
Recombinate influence of the HMGB1 to macrophage bactericidal activity.By recombination HMGB1 albumen and negative control, (final concentration is equal
It is added in tissue culture plate for 80ng/ml) and is incubated in PBS 2 hours with macrophage, suck supernatant, every hole is added on 50 μ l
The Vibrio anguillarum suspension prepared is stated, 25 DEG C are incubated for 4 hours.Supernatant is removed, is washed twice with PBS, 50 μ l 0.5% are added in every hole
Lysate is coated with LB plate, is placed in 28 DEG C of incubators and is incubated overnight by Tween 20, lytic cell.Bacterium colony is carried out after culture 36h
It counts, bacterium colony count results are shown, it is control group that the bactericidal index of recombination HMGB1 albumen processing group macrophage, which is 0.83,
1.22 times (bactericidal index calculation formula are as follows: 1- (the total clump count of quantity/indigenous bacteria suspension that bacterium colony counts)).Illustrate to recombinate
HMGB1 albumen can enhance the sterilizing ability of macrophage.
6) application of the recombination HMGB1 albumen in disease control
20 flat Rockfish of Xu Shi (every weighs about 5g) are randomly divided into 2 groups, every group 10.A and B are respectively designated as by this 2 groups
Group.50 μ l recombination HMGB1 albumen (10 μ g), every fish belly chamber note of B group (control group) is injected intraperitoneally in every fish of A group (experimental group)
Penetrate 50 μ l PBS.After 2h every experiment fish inject respectively 3) in prepare 50 μ l Vibrio anguillarum suspensions, after infection 12h and
A and B group fish (each time point respectively takes 5 fishes) is anaesthetized for 24 hours, takes renal tissue, weighing is homogenized after being washed with sterilizing PBS, will
Homogenate is coated on LB solid plate, 28 DEG C of overnight incubations.Bacterium colony count results are shown, compared with PBS group, injection recombination
Vibrio anguillarum quantity in the flat Rockfish liver of the Xu Shi of HMGB1 albumen is 1.64 × 10 respectively4CFU/g and 2.03 × 104CFU/g is pair
According to 0.78 times of group and 0.76 times, substantially less than control group.Illustrate that recombinating HMGB1 albumen can make the flat Rockfish of Xu Shi support Vibrio anguillarum
Anti- ability significantly increases.
Table one, the influence for recombinating HMGB1 albumen Rockfish head-kidney macrophage bactericidal index flat to Xu Shi
| PBS group | HMGB1 group |
| 0.68±0.03 | 0.83±0.02 |
The influence (× 10 of table two, recombination HMGB1 albumen in the flat Rockfish body of Xu Shi to Vibrio anguillarum infection ability4CFU/g)
| PBS group | HMGB1 group |
| 2.11±0.24(12h) | 1.64±0.16(12h) |
| 2.67±0.27(24h) | 2.03±0.15(24h) |
Claims (4)
1. a kind of flat Rockfish Immune-enhancing effect albumen of Xu Shi, which is characterized in that its albumen is the amino acid in sequence table SEQ ID No.1
Shown in sequence.
2. the encoding gene of Immune-enhancing effect albumen described in claim 1, which is characterized in that the DNA sequence dna of the protein gene is such as
Shown in SEQ ID No.2.
3. the preparation method of Immune-enhancing effect albumen described in a kind of claim 1, it is characterised in that:
1) building of plasmid pHMGB1: using the flat Rockfish head-kidney cDNA of Xu Shi of Vibrio anguillarum stimulation as template, with primer HMGB1F1 and
HMGB1R1 carries out PCR amplification；PCR product is connect with carrier pEasy-T1 after purification, is built into plasmid pT1HMGB1；It will
PT1HMGB1 and plasmid pET259 uses restriction enzyme EcoRV and SwaI digestion respectively, and 0.621kb and 5.4kb is separately recovered
This two segments T4DNA ligase is connected, is built into plasmid pHMGB1 by segment；
2) the plasmid pHMGB1 of step 1) recombinant expression and protein purification of HMGB1: is converted into e. coli bl21 Transetta
(DE3), transformant BL21/pHMGB1 is screened on the LB culture medium containing kanamycins and chloramphenicol；BL21/pHMGB1 is inoculated with
It is about 0.50 in 37 DEG C of shaken cultivations to OD600 in LB culture medium containing kanamycin and chloramphenicol, 1mM isopropyl is added
Base-β-D- thiogalactoside continues culture 4 hours, carries out protein induced expression；Then use nickel-
Nitrilotriacetic acid affinity column purification of recombinant proteins；Amino acid sequence in as sequence table SEQ ID No.1
Shown in recombination HMGB1 albumen.
4. the preparation method of Immune-enhancing effect albumen according to claim 3, which is characterized in that in the step 1)
HMGB1F1 is 5 '-gatatcATGGTGAAAGAACCAGGAAAG-3 '；HMGB1R1 is 5 '-
gatatcGTCGTCGTCATCATCGA AG-3’。
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| CN111671895B (en) * | 2020-06-02 | 2022-03-08 | 江汉大学 | Application of anti-PfHMGB 1 antibody in anti-algal toxin reagent and anti-algal toxin reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102649816A (en) * | 2012-05-15 | 2012-08-29 | 浙江大学 | Application of high mobility group box (HMGB) protein and antibody to preparation of ostrea rivularis anti-infection immune preparation |
| CN102811734A (en) * | 2010-01-21 | 2012-12-05 | 阿肯色大学评议会 | Vaccine vectors and methods of enhancing immune responses |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102811734A (en) * | 2010-01-21 | 2012-12-05 | 阿肯色大学评议会 | Vaccine vectors and methods of enhancing immune responses |
| CN102649816A (en) * | 2012-05-15 | 2012-08-29 | 浙江大学 | Application of high mobility group box (HMGB) protein and antibody to preparation of ostrea rivularis anti-infection immune preparation |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
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