CN105399808A - Sebastes schlegeli immunological enhancing protein (HNGB1) gene as well as coded protein and application - Google Patents

Sebastes schlegeli immunological enhancing protein (HNGB1) gene as well as coded protein and application Download PDF

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CN105399808A
CN105399808A CN201510821237.0A CN201510821237A CN105399808A CN 105399808 A CN105399808 A CN 105399808A CN 201510821237 A CN201510821237 A CN 201510821237A CN 105399808 A CN105399808 A CN 105399808A
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protein
albumen
hngb1
phmgb1
immunostimulant
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CN105399808B (en
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张敏
赵鑫鹏
王光花
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Qingdao Agricultural University
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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
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to the field of molecular biology, particularly to a Sebastes schlegeli immunological enhancing protein (HNGB1) coding gene as well as preparation and application of recombinant protein of the coding gene. The coding gene is shown as the amino acid sequence in SEQ ID No.1 in the sequence table, and the coding gene of the protein is shown as the base sequence in SEQ ID No.2 in the sequence table. A preparation method for the recombinant HNGB1 protein comprises the following steps: using Sebastes schlegeli head-kidney cDNA stimulated by vibrio anguillarum as a template, conducting PCR to amplify the HNGB1 coding gene, and constructing a plasmid pHMGB1; converting escherichia coli BL21Transetta (DE3) to the pHMGB1, conducting induced expression on the converter, extracting and purifying the protein so as to obtain the recombinant HNGB1 protein. The recombinant HNGB1 protein can be combined with double-strand DNA, can obviously improve the immunological competence of Sebastes schlegeli head-kidney macrophage, and can improve the sterilization function on vibrio anguillarum and the resistance of Sebastes schlegeli to vibrio anguillarum. The recombinant HNGB1 protein can be used as an immunological enhancing agent, and can be applied to control of vibrio anguillarum diseases of the fish.

Description

A kind of Xu Shi flat rockfish immunostimulant albumen HMGB1 gene and proteins encoded and application
Technical field
The present invention relates to biology field, specifically a kind of Xu Shi flat rockfish immunostimulant protein coding gene and albumen Synthesis and applications thereof.
Background technology
High mobility group protein (HMGB) family comprises HMGB1, HMGB2, HMGB3 and HMGB4, and wherein studying more in fish is HMGB1, is secondly HMGB2.HMGB1 is that one is present in the endonuclear nonhistone chromosomal associated proteins of eukaryotic cell, gains the name because its travelling speed in polyacrylamine gel electrophoresis is fast.Research finds, HMGB1 is present in most animals body.The principal character of this proteinoid is them all containing two HMG structural domains and an acidic carboxypolymer end regions.In Mammals, nuclear HMGB1 plays an important role in stable, the reparation of DNA mismatch of nucleosomal structure and the regulation and control of genetic expression etc.Current research confirms, after host is subject to pathogen infection, nuclear HMGB1 can move to tenuigenin, moving to cytoplasmic HMGB1 can stimulate body to produce a series of immune response, comprise respiratory burst and the phagolysis of enhancing scavenger cell and promote lymphocytic proliferation function, the generation etc. of enhance TNF-α inducible factor.Therefore, HMGB1 plays a significant role in the natural immunity of host versus Infected with Pathogenic Fungi.
Summary of the invention
The object of the invention is to provide a kind of Xu Shi flat rockfish immunostimulant albumen HMGB1 encoding gene and the albumen Synthesis and applications thereof with nucleic acid binding properties.
For achieving the above object, the technical solution used in the present invention is:
HMGB1 of the present invention is the aminoacid sequence in sequence table SEQ IDNo.1, and its encoding gene is the base sequence in sequence table SEQ IDNo.2.
1) structure of plasmid pHMGB1:
The Xu Shi flat rockfish head-kidney cDNA stimulated with Vibrio anguillarum, for template, carries out pcr amplification with primer HMGB1F1 and HMGB1R1.Be connected with carrier pEasy-T1 after PCR primer purifying, be built into plasmid pT1HMGB1.PT1HMGB1 and plasmid pET259 is cut with restriction enzyme EcoRV and SwaI enzyme respectively, reclaims 0.621kb and 5.4kb fragment respectively, these two fragment T4DNA ligase enzymes are connected, is built into plasmid pHMGB1.
2) the recombinant expressed and protein purification of HMGB1:
By step 1) plasmid pHMGB1 transformation of E. coli BL21Transetta (DE3), screening transformant BL21/pHMGB1 containing on the LB substratum of kantlex and paraxin.BL21/pHMGB1 is inoculated in the LB substratum containing kantlex and paraxin, is about 0.50 37 DEG C of shaking culture to OD600, adds 1mM isopropyl-β-D-thiogalactoside(IPTG), continue cultivation 4 hours, carry out protein induced expression.Use nickel-nitrilotriaceticacid affinity column purification of recombinant proteins subsequently.Be the restructuring HMGB1 albumen in sequence table SEQ IDNo.1 shown in aminoacid sequence.
Immunostimulant albumen can be used as immunostimulant for strengthening the resistivity to Vibrio anguillarum of fish body.
Tool of the present invention has the following advantages: immunostimulant albumen, can acute activation host innate immunity when host is by pathogen infection.
Accompanying drawing illustrates:
The electrophoretogram of the combination of HMGB1 albumen and DNA after the restructuring that Fig. 1 provides for the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, the invention will be further described.Embodiment is intended to carry out citing to the present invention and describes, but not limits the invention in any form.
Embodiment 1
HMGB1 of the present invention is the aminoacid sequence in sequence table SEQ IDNo.1, and its encoding gene is the base sequence in sequence table SEQ IDNo.2.
Sequence table SEQ IDNo.1 is:
MVKEPGKPRGKMSSYAYFVQTCREEHKKKHPEASVNFSEFSKKCSERWKMMSAKEKGKFEDLARSDKVRYEREMMSYIPPRGSKTKKFKDPNAPKRPPSAFFVFCAEYRPKVKGEAPGLTIGEVAKRLGEMWNGTASEDKQPFEKKAAKLKEKYEKDVAAYRAKGKPGAAPTAAAPSKAPAKVEKKDDDDDDDDDEDDDDFDDDDD
(a) sequence signature:
● length: 206
● type: aminoacid sequence
● chain: strand
● topological framework: linear
(b) molecule type: protein
C () is supposed: no
(d) antisense: no
E () is originated at first: the flat rockfish of Xu Shi
(f) constitutional features: the expection of this albumen is containing two HMG structural domains (being made up of amino acid 7-79 and 93-163 respectively).
Sequence table SEQ IDNo.2 is:
ATGGTGAAAGAACCAGGAAAGCCGAGGGGCAAGATGTCCTCCTATGCCTATTTTGTCCAGACTTGTCGGGAGGAGCACAAGAAGAAGCACCCCGAAGCTTCGGTCAACTTCTCTGAGTTCTCCAAGAAGTGCTCTGAGCGGTGGAAGATGATGTCTGCCAAGGAGAAGGGCAAATTTGAGGACCTGGCCAGGTCGGACAAGGTACGCTATGAGAGGGAGATGATGAGCTACATACCACCCAGGGGATCCAAGACGAAGAAGTTCAAGGACCCTAATGCCCCCAAGAGACCCCCATCTGCCTTCTTCGTCTTTTGCGCGGAGTATCGCCCCAAGGTGAAAGGCGAGGCCCCTGGTCTGACCATTGGAGAAGTTGCCAAGAGGCTGGGTGAGATGTGGAACGGCACCGCTTCAGAGGACAAGCAGCCCTTTGAGAAGAAGGCAGCTAAATTGAAGGAGAAGTATGAGAAGGACGTCGCAGCATACCGCGCTAAGGGCAAACCAGGCGCCGCACCAACAGCAGCAGCACCAAGCAAAGCCCCGGCCAAGGTAGAGAAGAAGGACGACGACGACGACGATGATGATGACGAGGACGACGACGACTTCGATGATGACGACGACTAG
(a) sequence signature:
● length: 621
● type: base sequence
(b) molecule type: gene
C () is supposed: no
(d) antisense: no
E () is originated at first: the flat rockfish of Xu Shi
Embodiment 2
The preparation method of High mobility group box-1:
1) structure of plasmid pHMGB1:
The aminoacid sequence corresponding to HMGB1 encoding gene is analyzed, to remove the gene order of coded signal peptide and termination codon for template, and design forward primer HMGB1F1 and reverse primer HMGB1R1.The Xu Shi flat rockfish head-kidney cDNA stimulated with Vibrio anguillarum, for template, carries out pcr amplification with primer HMGB1F1 and HMGB1R1.Amplification condition is: 94 DEG C of 60s denaturation template DNAs, then 94 DEG C of 40s, 55 DEG C of 60s, 72 DEG C of 30s, changes 94 DEG C of 40s into, 61 DEG C of 60s, 72 DEG C of 30s after 5 circulations, extends 7min again after 25 circulations at 72 DEG C.10 minutes are connected with carrier pEasy-T1 (being purchased from " Quan Shijin (Beijing) Bioisystech Co., Ltd ") in room temperature after PCR primer purifying, on the LB substratum containing penbritin (100 μ g/ml), Xgal (40 μ g/ml), isopropyl-β-D-thiogalactoside(IPTG) (24 μ g/ml), 18-24 hour is cultivated after connecting mixed solution transformation of E. coli, screening transformant extracts plasmid, is plasmid pT1HMGB1.
By above-mentioned plasmid pT1HMG1 and plasmid pET259, (plasmid pET259 building process is see ZhengWJ, HuYH, SunL.Cloningandanalysisofaferritinsubunitfromturbot (Scophthalmusmaximus) .FishShellfishImmunol.2010a; 28 (5 – 6), 829 – 836.) cut rear recovery 0.62kb and 5.4kb fragment with restriction enzyme EcoRV and SwaI enzyme respectively, these two fragment T4DNA ligase enzymes are connected, connecting fluid is transformed into intestinal bacteria, 18-24 hour is being cultivated containing on the LB substratum of kantlex (50 μ g/ml), screening transformant extracts plasmid, is pHMGB1.
Described HMGB1F1 is 5 '-gatatcATGGTGAAAGAACCAGGAAAG-3 '; HMGB1R1 is 5 '-gatatcGTCGTCGTCATCATCGAAG-3 '.
2) to recombinate the expression of HMGB1 albumen and preparation:
By step 1) plasmid pHMGB1 transformation of E. coli BL21Transetta (DE3) (being purchased from " Quan Shijin (Beijing) Bioisystech Co., Ltd "), LB substratum containing the kantlex of 50 μ g/ml and the paraxin of 50 μ g/ml is cultivated, and screening transformant is BL21/pHMGB1.BL21/pHMGB1 is inoculated in the LB substratum containing kantlex and paraxin, is cultured to OD in 37 DEG C 600be about 0.5, add the isopropyl-β-D-thiogalactoside(IPTG) of 1mM, 37 DEG C are continued shaking culture 4-5 hour, then use the nickel-nitrilotriaceticacid affinity column purification of recombinant proteins of GEHealthcare (U.S.) company.The recombinant protein of purifying is carried out n terminal amino acid order-checking, confirms that it is respectively the HMGB1 albumen in sequence table SEQ IDNo.1 shown in aminoacid sequence.
3) combination of restructuring HMGB1 albumen and DNA
The restructuring HMGB1 albumen of different concns, the restructuring HMGB1 albumen of thermally denature or PBS and HMGB1 gene product are mixed in PBS, under room temperature condition, hatches 1h, reaction product is carried out DNA agarose gel analysis.Found that (Fig. 1), restructuring HMGB1 albumen can make the mobility speed of DNA fragmentation slow down, and the concentration of restructuring HMGB1 albumen is higher, and the mobility speed of DNA is slower.And the DNA mobility speed processed with the restructuring HMGB1 albumen (swimming lane 2) of thermally denature and PBS group (swimming lane 1) completely the same.These results illustrate that restructuring HMGB1 albumen can combine with DNA, and this characteristic contributes to HMGB1 and plays a role in stable nucleus minibody structure and regulate gene expression.
4) recombinating HMGB1 albumen can the generation of TNF-α in stimulating expression of macrophage
Extract Xu Shi flat rockfish head-kidney scavenger cell, 96 porocyte culture plates are seeded to by the density of 105 cells in every hole, add in Tissue Culture Plate by the restructuring HMGB1 albumen of restructuring HMGB1 albumen, thermally denature or PBS, final concentration is 80mg/ml, hatches 12h in 28 DEG C.Collecting cell culture supernatant, detects the generation of TNF-α by fish TNF-α detection kit.Result shows, and the TNF-alpha content in the cells and supernatant of restructuring HMGB1 albumen treatment group is 1.51 times of PBS group; And thermally denature restructuring HMGB1 albumen treatment group and PBS group are without significant difference (P<0.05).
5) HMGB1 albumen of recombinating can strengthen the fungicidal activity of Xu Shi flat rockfish head-kidney scavenger cell
Prepared by Vibrio anguillarum.In LB substratum, culturing eel vibrio is to OD 600be 0.5, then centrifugal 10 minutes of room temperature, collects thalline, is suspended in PBS damping fluid (PBS damping fluid (pH7.2 ~ 7.4): NaCl137mM, KCl2.7mM, Na 2hPO410mM, KH 2pO42mmol/L) in final concentration be 2 × 10 6cFU/ml.
HMGB1 is on the impact of scavenger cell fungicidal activity in restructuring.To recombinate HMGB1 albumen and negative control (final concentration is 80ng/ml) adds in Tissue Culture Plate and scavenger cell hatches 2 hours in PBS, and suck supernatant, every hole adds the above-mentioned Vibrio anguillarum suspension prepared of 50 μ l, hatches 4 hours for 25 DEG C.Remove supernatant, with PBS washing twice, every hole adds 50 μ l0.5%Tween20, and lysate is coated with LB flat board by lysing cell, is placed in 28 DEG C of incubator incubated overnight.Enumeration is carried out after cultivating 36h, enumeration result shows, the bactericidal index of restructuring HMGB1 albumen treatment group scavenger cell is 0.83, is 1.22 times (bactericidal index calculation formula is: 1-(total colony number of the quantity/indigenous bacteria suspension of enumeration)) of control group.Illustrate that restructuring HMGB1 albumen can strengthen the sterilizing ability of scavenger cell.
6) application of restructuring HMGB1 albumen in disease control
20 flat rockfish of Xu Shi (every bar heavily about 5g) are divided into 2 groups at random, often organize 10.By these 2 groups difference called after A and B group.Every bar fish abdominal injection 50 μ l restructuring HMGB1 albumen (10 μ g) of A group (experimental group), every bar fish abdominal injection 50 μ lPBS of B group (control group).After 2h, every bar experiment fish injects 3 respectively) the middle 50 μ l Vibrio anguillarum suspensions prepared, respectively at 12h and 24h after infection, A and B group fish (each time point respectively gets 5 fishes) is anaesthetized, get renal tissue, weigh, wash rear homogenate with sterilizing PBS, homogenate is coated LB solid plate, 28 DEG C of overnight incubation.Enumeration result shows, and compared with PBS group, the Vibrio anguillarum quantity in the flat rockfish liver of Xu Shi of injection restructuring HMGB1 albumen is 1.64 × 10 respectively 4cFU/g and 2.03 × 10 4cFU/g is 0.78 times and 0.76 times of control group, significantly lower than control group.Illustrate that restructuring HMGB1 albumen can make the resistivity of the flat rockfish of Xu Shi to Vibrio anguillarum significantly strengthen.
Table one, restructuring HMGB1 albumen are on the impact of Xu Shi flat rockfish head-kidney scavenger cell bactericidal index
PBS group HMGB1 group
0.68±0.03 0.83±0.02
Table two, the impact (× 10 of restructuring HMGB1 albumen on Vibrio anguillarum infection ability in the flat rockfish body of Xu Shi 4cFU/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 (5)

1. a Xu Shi flat rockfish immunostimulant albumen, is characterized in that, its albumen is for shown in the aminoacid sequence in sequence table SEQ IDNo.1.
2. the encoding gene of immunostimulant albumen described in claim 1, is characterized in that, the DNA sequence dna of described protein gene is as shown in SEQIDNo.2.
3. a preparation for immunostimulant albumen described in claim 1, is characterized in that:
1) structure of plasmid pHMGB1:
The Xu Shi flat rockfish head-kidney cDNA stimulated with Vibrio anguillarum, for template, carries out pcr amplification with primer HMGB1F1 and HMGB1R1.Be connected with carrier pEasy-T1 after PCR primer purifying, be built into plasmid pT1HMGB1.PT1HMGB1 and plasmid pET259 is cut with restriction enzyme EcoRV and SwaI enzyme respectively, reclaims 0.621kb and 5.4kb fragment respectively, these two fragment T4DNA ligase enzymes are connected, is built into plasmid pHMGB1.
2) the recombinant expressed and protein purification of HMGB1:
By step 1) plasmid pHMGB1 transformation of E. coli BL21Transetta (DE3), screening transformant BL21/pHMGB1 containing on the LB substratum of kantlex and paraxin.BL21/pHMGB1 is inoculated in the LB substratum containing kantlex and paraxin, is about 0.50 37 DEG C of shaking culture to OD600, adds 1mM isopropyl-β-D-thiogalactoside(IPTG), continue cultivation 4 hours, carry out protein induced expression.Use nickel-nitrilotriaceticacid affinity column purification of recombinant proteins subsequently.Be the restructuring HMGB1 albumen in sequence table SEQ IDNo.1 shown in aminoacid sequence.
4., by the preparation of immunostimulant albumen according to claim 3, it is characterized in that, described step 1) in HMGB1F1 be 5 '-gatatcATGGTGAAAGAACCAGGAAAG-3 ': be 5 '-gatatcGTCGTCGTCATCATCGAAG-3 ' with HMGB1R1.
5. an application for immunostimulant albumen according to claim 1, is characterized in that, describedly can be used as immunostimulant for strengthening the resistivity to Vibrio anguillarum of fish body with the immunostimulant albumen shown in list SEQIDNo.1.
CN201510821237.0A 2015-11-23 2015-11-23 A kind of flat Rockfish Immune-enhancing effect albumen HMGB1 gene of Xu Shi and coding albumen and application Active CN105399808B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111575388A (en) * 2020-06-02 2020-08-25 江汉大学 Application of PfHMGB1 as molecular marker for algal toxin invasion and detection kit
CN111671895A (en) * 2020-06-02 2020-09-18 江汉大学 Application of anti-PfHMGB 1 antibody in anti-algal toxin reagent and anti-algal toxin reagent

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
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

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111575388A (en) * 2020-06-02 2020-08-25 江汉大学 Application of PfHMGB1 as molecular marker for algal toxin invasion and detection kit
CN111671895A (en) * 2020-06-02 2020-09-18 江汉大学 Application of anti-PfHMGB 1 antibody in anti-algal toxin reagent and anti-algal toxin reagent
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
CN111575388B (en) * 2020-06-02 2022-10-21 江汉大学 Application of PfHMGB1 as molecular marker of algal toxin invasion and detection kit

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