CN103319575A - Haemophilus parasuis (Hps) immunoprotecive antigen CdtB - Google Patents
Haemophilus parasuis (Hps) immunoprotecive antigen CdtB Download PDFInfo
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Abstract
The invention relates to identification of a Haemophilus parasuis (Hps) immunoprotecive antigen, separation and cloning of a protein gene and application of a protein coded by the Hps immunoprotecive antigen in a vaccine. According to the invention, a new protein CdtB having immunogenicity is separated from Hps (the culture collection number is CVCC3361), and the nucleotide sequence is shown as SEQ ID NO:2 in a sequence table and is formed by coding 277 amino acids. The CdtB is a new protein having immunogenicity and can provide effective immunoprotection for Hps infection in mice. The invention also comprises preparation of an Escherichia coli recombinant bacterium BL21/Hps-CdtB expressing the immunogenicity protein gene CdtB. The recombinant Hps CdtB protein expressed by the invention has favorable safety and protection efficacy, and the immunoprotection effect is up to 70%.
Description
Technical field
The present invention relates to zoonosis subunit vaccine preparing technical field.Be specifically related to a kind of application of albumen in vaccine of separation, clone and its coding of evaluation, protein gene of haemophilus parasuis immune protective antigen.
Background technology
Cell-lethal expansion toxin is a member of bacterioprotein toxin family latest find, be found in the earliest in the colibacillary nutrient solution supernatant, interaction in vitro is in Chinese hamster ovary cell (Chinese hamster ovary cells, CHO) can cause target cell to be expanded and death, so this toxin is named as " cell-lethal expansion toxin ".At present, CDT is the active toxin of the unique DNA of having enzyme (DNase I) in the bacteriotoxin, has hereditary toxic action, causes the dna double splitting of chain, causes irreversible cell-cycle arrest and apoptosis in mammalian cell.
Now confirm multiple gram negative pathogenic bacteria, all can produce this toxin.Crystalline structure studies show that the CDT holotoxin is the trimer compositions that is made of CdtA, CdtB and CdtC, and three subunits are respectively by cdtA, cdtB, cdtC genes encoding.
Research is found cell-lethal expansion detoxifying function in eukaryotic cell, and Giemsa staining is found cell is expanded behind the cultivation 72-96h; Find compared to control group behind the function cells 72h, the experimental group cell number changes not obvious, and test confirms that this toxin can make the cell cycle cycle arrest in the G2/M phase; By microinjection technique the CDTs holotoxin is expelled in the cell, finds the dna double splitting of chain, show that this toxin has the DNA enzymic activity.The retardance of cell expansion and loop cycle can not occur in inoblast, epithelial cell, bone-marrow-derived lymphocyte, T lymphocyte and dendritic cell under the effect of CDTs, but directly causes apoptosis and be cracked into cell debris.Research thinks that the CdtB subunit is the conservative subunit of CDT holotoxin, it also is the function subunit, have the structure and function similar with DNase I, this subunit extracorporeal treatment plasmid DNA, can cause the linearizing of DNA, the CdtB microinjection to cell, is found that this subunit not only can cause expansion and the retardance in cell cycle cycle of target cell, and can cause target cell dna double chain break, this is DNA with regard to the target that CdtB is described.The DNA enzymic activity of CdtB subunit is the key of CDT holotoxin performance cytotoxic effect, CdtA and CdtC do not have the activity of similar DNase, expansion and the apoptosis that can not cause target cell, the Main Function of the two is to be incorporated into surface of cell membrane, promote CdtB to enter nucleus and bring into play its cytotoxic effect, but they be how to play a role and how still unclear finish after the mission their whereabouts.
Haemophilus parasuis (Haemophilus parasuis, Hps) is a kind of Gram-negative bacteria, often is settled in the upper respiratory tract of pig, is a kind of conditioned pathogen, can cause the Haemophilus parasuis of pig.
This bacterium of reported first in 1910, therefore should disease be called again leather draw Se Shi sick (
Disease).Hps infected pigs, can affect 2 the week age to 4 monthly ages pig, the morbidity be common in 5～8 the week age pig.Sickness rate is 10%～15%, and mortality ratio can reach 50% when serious, brings massive losses to pig industry, and this sick principal character is fibrinous pleurisy, peritonitis, sacroiliitis, pericarditis and meningitis.
This research is passed through the clone, is expressed the gene of coding CdtB, and with the protein immunization mouse behind the purifying, finds that it can provide effective immune protective efficiency to the mouse infection haemophilus parasuis, can be used as the candidate antigens of haemophilus parasuis subunit vaccine exploitation.
Summary of the invention
The albumen and the encoding gene thereof that the purpose of this invention is to provide the candidate antigens that can be used in preparation haemophilus parasuis subunit vaccine.
The present invention identifies the CdtB albumen with immune protective effect, and can provide effective immune protective efficiency to the mouse infection haemophilus parasuis, can be used as the candidate antigens of Haemophilus parasuis subunit vaccine exploitation.
In order to realize the object of the invention; the present invention has at first cloned the CdtB gene of haemophilus parasuis; then this gene is connected in recombinant expressed CdtB albumen in the expression vector; the albumen of the rehabilitation serum that uses pig after to purifying carries out western-blot hybridization and finds that it has immunogenicity; to carry out the immunoprotection test of mouse behind the protein purification of expressing, the result shows that it has certain immanoprotection action.
The present invention protects a kind of method of the CdtB of preparation albumen simultaneously, and described method comprises cultivates the recombination bacillus coli that contains the CdtB albumen coded sequence, obtains CdtB albumen.The method of the described CdtB of preparation albumen specifically can be: 37 ℃ of described recombinant bacteriums are cultivated 3h, OD
600=0.7 o'clock, add IPTG to final concentration 0.8 μ M, go to 37 ℃ and continue to cultivate 4h.
CdtB albumen of the present invention has the immunoprotection activity.
CdtB albumen of the present invention can be applied to the preparation of Haemophilus parasuis subunit vaccine.
More specifically, the invention provides the following:
1. haemophilus parasuis immune protective antigen, its aminoacid sequence is shown in SEQ ID NO:1.
2. coding is according to the nucleic acid of 1 described haemophilus parasuis immune protective antigen, and its sequence is shown in SEQ ID NO:2.
3. recombinant vectors, it comprises according to 2 described nucleic acid.
4. comprise according to 2 described nucleic acid or according to the cell of 3 described recombinant vectorss.
5. according to 1 described haemophilus parasuis immune protective antigen, it is as the haemophilus parasuis subunit vaccine.
According to 1 described haemophilus parasuis immune protective antigen, according to 2 described nucleic acid, according to 3 described recombinant vectorss with according to the purposes of 4 described cells in preparation haemophilus parasuis subunit vaccine.
According to 1 described haemophilus parasuis immune protective antigen, according to 2 described nucleic acid, according to 3 described recombinant vectorss and according to 4 described cells for the preparation of the purposes in the medicine of the disease that caused by haemophilus parasuis of prevention.
8. according to 7 described purposes, the wherein said disease that is caused by haemophilus parasuis is Haemophilus parasuis.
9. vaccine composition, it comprises according to 1 described haemophilus parasuis immune protective antigen.
10. method for preparing the haemophilus parasuis immune protective antigen said method comprising the steps of:
Structure comprises the recombinant expression vector of the nucleotide sequence shown in SEQ ID NO:2;
Described recombinant expression vector is transformed in the host cell;
Cultivate described host cell through conversion and induce it to express described haemophilus parasuis immune protective antigen; With
The host cell of inducing from described process separates and the described haemophilus parasuis immune protective antigen of purifying.
11. according to 10 described methods, wherein said host cell is intestinal bacteria.
Description of drawings
Fig. 1: hybridization figure after the CdtB transferring film.
Fig. 2: attack malicious mouse survival rate figure after the CdtB immunity.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.
The clone of embodiment 1, CdtB protein coding gene
One, the extraction of the total DNA of Hps
(available from national veterinary microorganism DSMZ, bacterium numbering: centrifugal 1 minute of incubated overnight bacterium liquid 12000rpm CVCC3361) abandons supernatant with 1mL haemophilus parasuis (Haemophilus parasuis, Hps).In bacterial sediment, add 40 μ L DB liquid (TIANamp Bacteria DNA Kit, the biological company limited of day root), 160 μ L N,O-Diacetylmuramidases and 8 μ L RNaseA.Concuss mixes.37 ℃ of temperature were bathed 30～60 minutes, constantly put upside down centrifuge tube for several times.Add 200 μ L DLT liquid (TIANamp Bacteria DNA Kit, the biological company limited of day root) and 25 μ L Proteinase Ks (TIANamp Bacteria DNAKit, the biological company limited of day root), leniently put upside down immediately mixing.Put in 65 ℃ of water-baths at least 30 minutes, and constantly put upside down centrifuge tube for several times.Centrifugal 3～5 minutes of 12000rpm draws in whole supernatants to clean centrifuge tube with pipettor.Add 200 μ L dehydrated alcohols, all suck adsorption column, 12000rpm, centrifugal 30 seconds behind the mixing.Outwell the liquid in the collection tube, put back to adsorption column.Add 500 μ L W1 liquid (TIANamp Bacteria DNA Kit, the biological company limited of day root), left standstill 12000rpm, centrifugal 30 seconds 1 minute.Outwell the liquid in the collection tube, put back to adsorption column.Add 500 μ L W1 liquid, 12000rpm, centrifugal 30 seconds.Outwell the liquid in the collection tube, put back to adsorption column.12000rpm, centrifugal 1 minute.Adsorption column is put into a 1.5mL centrifuge tube.Add 100 μ L T1 liquid (TIANamp Bacteria DNA Kit, the biological company limited of day root) in adsorption film central authorities, 65 ℃ of water-baths 5 minutes, centrifugal 1 minute of 12000rpm.
Two, the preparation of CdtB gene
According to the nucleotide sequence (shown in the SEQ ID NO:2 of sequence table) of CdtB gene, the design primer pair is as follows:
Forward primer: 5 '-ATA
CATATGACGTTATTTAATCGAGCTAT-3 '
Reverse primer: 5 '-AAT
GGATCCTTAACGTTTTTTTAC-3 '
The underscore of forward primer partly is the restriction enzyme site of NdeI, and the underscore of reverse primer partly is the BamHI restriction enzyme site.
Take total DNA of Hps as template, carry out pcr amplification with the primer pair of design.
Amplification system:
The PCR reaction conditions: 94 ℃ of denaturations 5 minutes, then 30 circulations in 45 seconds are extended in-72 ℃ of 94 ℃ of sex change-52 ℃ of annealing in 30 seconds 30 seconds, and last 72 ℃ were extended 7 minutes.
The PCR product detects output and specificity with 1% agarose gel electrophoresis, and with DNA purification kit (ultrathin centrifugal column type, day root company production) purifying.The PCR product of purifying is checked order, and the result shows that having obtained sequence is the dna fragmentation of sequence table 2, is the gene of coding haemophilus parasuis CdtB albumen.
Three, the structure of recombinant expression vector
The PCR product that 1, will check order correct reclaims enzyme and cuts product with NdeI and BamHI double digestion, agarose electrophoresis.
2, with plasmid pET28a (article No. 69864-3, Novagen) BamHI and NdeI double digestion, agarose electrophoresis reclaims enzyme and cuts product.
3, the enzyme of step 1 is cut enzyme that product is connected with step and cut product and connect, obtain recombinant plasmid.
Recombinant plasmid is checked order.The result shows, having inserted sequence between the BamHI of pET28a and NdeI restriction enzyme site is the gene of the coding haemophilus parasuis CdtB albumen shown in the sequence table 2, with this recombinant plasmid called after pET28a-CdtB.
Four, the preparation of engineering bacteria
Transform with pET28a-CdtB electric shock that to coat the LB that contains 50 μ g/ml kantlex behind the e. coli bl21 (DE3) (article No. CB105, TIANGEN) dull and stereotyped, 37 ℃ of incubated overnight obtain containing the recombinant bacterium of pET28a-CdtB, are engineering bacteria.
Replace pET28a-CdtB with pET28a, transform e. coli bl21 (DE3), step is the same, obtains containing the recombinant bacterium of pET28a, in contrast bacterium.
Embodiment 2, CdtB protein expression purifying
One, the preparation of CdtB albumen and purifying
Embodiment 1 (four) the middle recombinant for preparing in the LB substratum that contains 50 μ g/ml kantlex, is cultivated 3h for 37 ℃; During OD600=0.7, add IPTG to final concentration 0.8 μ M, go to 37 ℃ and continue to cultivate 4h.
5000rpm, 10 minutes centrifugal collection thalline are suspended among the PBS in the solution ultrasonication in ice bath (300w, 10 minutes; Ultrasonic 3s stops 5s), centrifugal 10 minutes collecting precipitations of 15000rpm will precipitate with 30ml solution A (NaH afterwards
2PO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L transfer pH to 8.0) dissolving, can of short duration ultrasonication urge molten.Add the resin (Ni-NTA His-Bind Resin, Novagen) that 2mL handles well in advance, 4 ℃ of low-speed oscillations made resin and the abundant combination of target protein in 30 minutes, in conjunction with complete, added in the adsorption column.Treat that liquid flows to end, add 10mL solution B (NaH
2PO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L transfer pH to 6.3) wash 6 times.Add 2mL solution C (NaH
2PO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L transfer pH to 5.9) wash 1 time.Add 10mL solution D (NaH
2PO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L transfer pH to 4.5), collect elutriant by every pipe 1mL.
The molecular weight of the CdtB albumen of SDS-PAGE electrophoresis showed purifying is about 32kDa (comprising histidine-tagged), and basic symbols rationally opinion is inferred.Collection contains the fraction of CdtB albumen.
Adopt identical step to cultivate and purifying the contrast bacterium of preparation among the embodiment 1 (four), the solution that obtains is enzyme liquid in contrast.
Two, the Western blotting of CdtB albumen is identified
Recombinant C dtB albumen to purifying in above carries out the Western blotting analysis, at first prepares 12% polyacrylamide gel, and CdtB albumen loading is carried out SDS-PAGE; After electrophoresis finishes, gel is immersed in balance 15min in the electrophoresis liquid (39mM glycine, 48mM Tris alkali, 0.037%SDS, 20% methyl alcohol); Cut out the nitrocellulose filter slightly larger than gel, be positioned over electrophoresis liquid and be dipped to fully moistening; Cut out two thick filter paper, upper strata filter paper is slightly smaller than gel, and lower floor's filter paper will be less times greater than nitrocellulose filter, and is immersed in the electrophoresis liquid to fully moistening; Order according to filter paper-film-glue-filter paper is added on the Graphite Electrodes, drives bubble away with glass stick; Plugged, 15v constant voltage, transfer printing 30min.Transfer printing is taken off nitrocellulose filter after finishing; Film is packed in the little valve bag, add 4 ℃ of sealings of 5% skimming milk and spend the night; Take out film, wash 5 times with PBST, each 10min; Then add the primary antibodie (pig rehabilitation serum) of 5% skimming milk dilution in 1: 200, hatch 1h for 37 ℃; Take out film, wash 5 times with PBST, each 10min; Then add two anti-(anti-pig two resists the rabbit of HRP mark) of 5% skimming milk dilution in 1: 5000, hatch 1h for 37 ℃; Take out film, wash again 5 times with PBST, each 10min; Then with the colour developing of ECL chemical luminescence reagent kit, the result shows the protein band in the same size of CdtB albumen and expection, as shown in Figure 1.
The evaluation of embodiment 3, CdtB protein immunization protectiveness
1, Hps is to kunming mice LD
50Mensuration
With Hps (available from national veterinary microorganism DSMZ, bacterium numbering: CVCC3361) be inoculated in the TSB liquid nutrient medium and (contain 10% horse serum, 0.01%NAD) 37 ℃ of 200rpm/min cultivated 14～16 hours, then being coated with the TSA solid medium next day (contains 10% horse serum, 0.01%NAD) cultivates 24～36h for 37 ℃.Wash lawn with PBS, be diluted to 5 * 10
8CFU/mL (OD
600Be about 1.0), then 2 times concentrate step by step, are concentrated into the backward injected in mice of required dosage (as shown in table 1).8～10 week female kunming mices in age (being purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center) are divided into 5 groups, 10 every group.Every mouse peritoneal is injected the poisonous substance of attacking of 100 μ L, and attacks the rear Continuous Observation of poison 5 days, and death condition is respectively organized in record.Calculate LD according to the Kou Shi formula
50Formula is as follows:
LD
50＝lg
1{Xm-i〔∑P-(3-Pm-Pn)/4〕}
Xm: the logarithmic value of maximal dose group dosage
I: two adjacent groups dosage (d) logarithmic value poor
∑ P: the summation of each treated animal mortality ratio
P: each treated animal mortality ratio, decimally expression
Pm: maximum dose level treated animal mortality ratio, decimally expression
Pn: minimum dose treated animal mortality ratio, decimally expression
N: every treated animal number
Attack poison after 5 days, the 5th group dead 10, the 1st group dead 2.It is concrete that respectively to organize mortality ratio as shown in the table.
Table 1
Calculate LD by the Kou Shi formula
50=1.46 * 10
9CFU.
2, the immunity of mouse with attack poison
With the kunming mice (being purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center) according to ages in CdtB proteantigen immunity 4～6 week of embodiment 2 purifying, 10 every group.Control group does not carry out immunity.For the first time immunity, 100 μ g/ only mix back multi-point injection immune mouse at 1: 1 with the Fu Shi Freund's complete adjuvant.The interval is after two weeks, carry out the immunity second time, 100 μ g/ only, mix at 1: 1 with freund 's incomplete adjuvant, back multi-point injection immune mouse is after 10 days, ELISA detects antibody horizontal, the mountain sheep anti mouse (Sigma) of HR mark is two anti-(1: 4000), and two exempt from 10 days by abdominal injection Hps (culture presevation numbering: CVCC3361), attack the LD that the toxic agent amount is above mensuration
505 times, observe the death condition of respectively organizing mouse in 5 days.As shown in Figure 2, the CdtB immune group has 70% survival rate.
Claims (11)
1. haemophilus parasuis immune protective antigen, its aminoacid sequence is shown in SEQ ID NO:1.
2. the encode nucleic acid of haemophilus parasuis immune protective antigen according to claim 1, its sequence is shown in SEQ ID NO:2.
3. recombinant vectors, it comprises nucleic acid according to claim 2.
4. the cell that comprises nucleic acid according to claim 2 or recombinant vectors according to claim 3.
5. haemophilus parasuis immune protective antigen according to claim 1, it is as the haemophilus parasuis subunit vaccine.
6. haemophilus parasuis immune protective antigen according to claim 1, nucleic acid according to claim 2, recombinant vectors according to claim 3 and cell according to claim 4 purposes in preparation haemophilus parasuis subunit vaccine.
7. haemophilus parasuis immune protective antigen according to claim 1, nucleic acid according to claim 2, recombinant vectors according to claim 3 and cell according to claim 4 are for the preparation of the purposes in the medicine of the disease that caused by haemophilus parasuis of prevention.
8. purposes according to claim 7, the wherein said disease that is caused by haemophilus parasuis is Haemophilus parasuis.
9. vaccine composition, it comprises haemophilus parasuis immune protective antigen according to claim 1.
10. method for preparing the haemophilus parasuis immune protective antigen said method comprising the steps of:
Structure comprises the recombinant expression vector of the nucleotide sequence shown in SEQ ID NO:2;
Described recombinant expression vector is transformed in the host cell;
Cultivate described host cell through conversion and induce it to express described haemophilus parasuis immune protective antigen; With
The host cell of inducing from described process separates and the described haemophilus parasuis immune protective antigen of purifying.
11. method according to claim 10, wherein said host cell is intestinal bacteria.
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Cited By (4)
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| CN104357458A (en) * | 2014-11-12 | 2015-02-18 | 四川农业大学 | Recombinant haemophilus parasuis immune protective antigen PotD and preparation method thereof |
| CN105617373A (en) * | 2014-11-06 | 2016-06-01 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and applications thereof |
| CN110229234A (en) * | 2019-06-05 | 2019-09-13 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | A kind of haemophilus parasuis fusion protein CdtB-OppA with immune protective |
| CN111793130A (en) * | 2019-03-20 | 2020-10-20 | 华中农业大学 | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105617373A (en) * | 2014-11-06 | 2016-06-01 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and applications thereof |
| CN105617373B (en) * | 2014-11-06 | 2019-07-09 | 普莱柯生物工程股份有限公司 | A kind of vaccine composition and its preparation method and application |
| CN104357458A (en) * | 2014-11-12 | 2015-02-18 | 四川农业大学 | Recombinant haemophilus parasuis immune protective antigen PotD and preparation method thereof |
| CN111793130A (en) * | 2019-03-20 | 2020-10-20 | 华中农业大学 | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody |
| CN110229234A (en) * | 2019-06-05 | 2019-09-13 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | A kind of haemophilus parasuis fusion protein CdtB-OppA with immune protective |
| CN110229234B (en) * | 2019-06-05 | 2022-09-27 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Haemophilus parasuis fusion protein CdtB-OppA with immune protection |
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Application publication date: 20130925 |