CN102864157A

CN102864157A - Immune protective antigen of haemophilus parasuis

Info

Publication number: CN102864157A
Application number: CN2011101975039A
Authority: CN
Inventors: 金梅林; 周明光; 赵建平; 张强; 张安定; 康超; 徐高原
Original assignee: WUHAN KEQIAN ANIMAL BIOLOGICAL PRODUCTS CO Ltd; Huazhong Agricultural University
Current assignee: WUHAN KEQIAN BIOLOGICAL CO., LTD.; Huazhong Agricultural University
Priority date: 2011-07-05
Filing date: 2011-07-05
Publication date: 2013-01-09
Anticipated expiration: 2031-07-05
Also published as: CN102864157B

Abstract

The invention belongs to the technical field of animal-borne disease subunit vaccine preparation and relates to preparation and application of the immune protective antigen of the haemophilus parasuis. Outer membrane protein Hbp B genes of the haemophilus parasuis are cloned, a nucleotide sequence is indicated as SEQID NO:1, and the sequence of gene code is indicated as SEQ ID NO:2. Recombination Escherichia coli BL21/Pet-28a-Hbp B (preservation number is CCTCC NO:M2011228) is built and comprises genes in a sequence table SEQ ID NO:1. Antigen protein of the haemophilus parasuis is obtained and expressed through gene transformation Escherichia coli in the SEQ ID NO:1. The invention further discloses a preparation method and application of the recombination Escherichia coli. The haemophilus parasuis subunit vaccine has good safety, and an immune protection effect reaches 83%.

Description

A kind of haemophilus parasuis immune protective antigen

Technical field

The invention belongs to zoonosis subunit vaccine preparing technical field, be specifically related to a kind of haemophilus parasuis protective antigen preparation and application.For the anti-system of Haemophilus parasuis, the present invention has designed clonal expression, functional verification and the vaccine applied research of a kind of protective antigen protein gene Hbp B that encodes.The antigen protein of described genetic expression can improve the ability of pig opposing Haemophilus parasuis.

Background technology

Haemophilus parasuis (Haemophilus parasuis, HPS) can cause the Ge Lazeshi of pig sick (Glasser ' s disease), take fiber disposition polyserositis, sacroiliitis and meningitis as principal character.Haemophilus parasuis can be divided into 15 standard serum types (Kieletein etc., 1992) according to agar diffusion serotype method at present, about clinical separation strain more than 20% can't be finalized the design in addition.The serotype that wherein virulence is stronger is serum 4 types, 5 types and 13 types, and the main serotype that China swinery is separated to is 4 types, 5 types, 13 types (Cai Xuwang etc., 2005).This bacterium is the pig upper respiratory tract bacterium of being everlasting, and invades body under given conditions and causes serious systemic disease.In recent years because the new respiratory complication of the improper and frequent burst of adjustment of breeding technology so that the increasingly popular harm of Haemophilus parasuis is day by day serious, causes huge harm to the pig industry in the global range.Haemophilus parasuis affect 2 ages in week to 4 the monthly age swinery, mainly before and after wean and child care stage morbidity, sickness rate is generally at 10%-15%, mortality ratio can reach 50% in the time of seriously.At present research has found that HPS can cause polyinfection or secondary infection with dyspnoea syndrome virus and PCV-II etc. with streptococcus suis 2-type, actinobacillus pleuropneumoniae, swine influenza virus, pig breeding, makes the state of an illness complicated.Since in June, 2006, " unknown high fever " is popular in the several provinces and cities of south China and middle part big area, already caused huge financial loss for pig-breeding, there are some researches show that haemophilus parasuis is one of important secondary infection or cause of disease of polyinfection, increase the weight of the symptom of hyperpyrexia disease, increased mortality ratio.

Paid much attention at present the research to Haemophilus parasuis abroad, the states such as the U.S., Spain, Australia, Denmark have all carried out epidemiology survey to the Haemophilus parasuis of this country.China is less to the understanding of this disease, just attracts great attention in recent years.Because lack enough epidemiologic datas, pathogenesis is unclear, there is no effective general commodity seedling and responsive diagnostic method, this disease fails to be effectively controlled in many countries always.

The Haemophilus parasuis fashion trend in recent years that is caused by HPS is the rising state, and current main anti-means processed are medicine control and vaccine inoculation.Swinery improves constantly the dependency of antibacterials in recent years, and Ampicillin Trihydrate, cynnematin, spectinomycin and fluoroquinolones etc. can be used for preventing and the medicine for the treatment of Haemophilus parasuis has caused the appearance of a large amount of Resistant strain.Antibiotic long-term, extensive and unreasonable use will certainly cause being on the increase of Resistant strain, Antibiotic Resistance expanding day and serious drug residue, and food safety and human health are caused serious threat.Although current pathogenesis to haemophilus parasuis is still located conceptual phase, still obtained certain progress for this sick vaccine research now.Commercial deactivation vaccine and oneself seedling can be controlled the infection of haemophilus parasuis to a certain extent, however serotype diversity and the effect of control that can not the somatotype bacterial strain have affected commercial seedling that accounts for significant proportion, therefore usually failure when extensive immunity.The protection of oneself seedling may be more targeted, but a lot of failed examples are also arranged, and mainly is because the haemophilus parasuis that is separated to is not real pathogenic strains or is not the serotype of mainly causing a disease.The advantages such as the immune efficacy that subunit vaccine has living vaccine simultaneously is high, cross-protection strong and the security of deactivation vaccine is good have a good application prospect.

Given this; the present invention gets down to the research of a kind of Novel immune protective antigen of haemophilus parasuis; the evaluation immune protective effect is good; and the immunogenic outer membrane that in 15 kinds of haemophilus parasuis standard serum types, extensively exists, and then be prepared into effective haemophilus parasuis subunit vaccine.

The haemophilus parasuis subunit vaccine of the present invention's preparation has good security, and immune protective effect reaches more than 83%.

Summary of the invention

The object of the invention is to overcome the defective of prior art; its first purpose is to obtain the recombination bacillus coli that a strain can be secreted haemophilus parasuis immune protective antigen albumen, utilizes the good protectiveness outer membrane protein of this recombination bacillus coli adaptive immune originality.

Second purpose of the present invention is to utilize above-mentioned outer membrane protein to prepare the haemophilus parasuis subunit vaccine.

The present invention is achieved through the following technical solutions:

The applicant passes through gene clone method, separating clone obtains a kind of outer membrane protein H bp B gene fragment from haemophilus parasuis SH0165 bacterial strain, its nucleotide sequence is shown in sequence table SEQ ID NO:1, sequence length is 1695bp, the encoding sequence of this gene shown in sequence table SEQ ID NO:2, its 564 amino acid of encoding.Described Hbp B gene fragment is transformed intestinal bacteria, obtain a kind of recombination bacillus coli (Escherichia coli) BL21/PET28a-Hbp B that comprises haemophilus parasuis SH0165 bacterial strain outer membrane protein H bp B gene and can express this gene.

In order to realize purpose of the present invention; the applicant obtains a strain can secrete the recombination bacillus coli of haemophilus parasuis immune protective antigen (Escherichia coli) BL21/pET-28a-Hbp B; this bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on June 30th, 2011, and its deposit number is CCTCC NO:M 2011228.

In an embodiment of the present invention, the applicant provides the detailed preparation method of this haemophilus parasuis Novel immune protective antigen Hbp B, and has described the possible application prospect of this protective antigen albumen.

More detailed technical scheme is as described in " embodiment ".

Description of drawings

Fig. 1: be basic research route of the present invention.

Fig. 2: be that recombinant plasmid pET-28a of the present invention (+)-Hbp B double digestion is identified figure (swimming lane 1) and superhelix recombinant plasmid collection of illustrative plates (swimming lane 2) thereof.

Fig. 3: be SDS-PAGE (A) and Western Blot (B) figure behind the recombinant antigen protein Hbp B purifying of the present invention.

Fig. 4: be that recombinant antigen gene Hbp B of the present invention is in the expression versatility test-results (swimming lane 1-15 is expressed as HPS 1-15 serotype reference culture successively) of 15 standard serum types of haemophilus parasuis.

Fig. 5: the antibody horizontal that is the test group of inducing behind the recombinant antigen protein Hbp B immune mouse of the present invention and control group detects.

Fig. 6: be to attack poison (5LD behind the recombinant antigen protein Hbp B immune mouse of the present invention ₅₀) the protection effect.

Fig. 7: the collection of illustrative plates that is the used vector plasmid pET-28a (+) of the present invention.

Embodiment

Embodiment 1: the Cloning and Expression of haemophilus parasuis recombinant antigen protein Hbp B

One material

1 plasmid and bacterial strain

PET-28a (+) plasmid that the present invention adopts is available from German Novagen company, and this pET-28a (+) plasmid map as shown in Figure 7.Competent cell bacillus coli DH 5 alpha and BL 21 (DE 3) are Beijing Quanshijin Biotechnology Co., Ltd's product.

The used haemophilus parasuis bacterial strain of the present invention is that HPS SH0165 bacterial strain is that Hua Zhong Agriculture University agricultural microorganism National Key Laboratory screening is (referring to Cai Xuwang, the research of the isolation identification of haemophilus parasuis and diagnostic method and inactivated vaccine, in June, 2006, Hua Zhong Agriculture University's Ph D dissertation, China National Library, the http://res4.nlc.gov.cn/home/search.trs of China national digital library? method=showDetail﹠amp; Channelid=3﹠amp; Id=003448570), 15 standard serum types of haemophilus parasuis bacterial strain is so kind as to give by Australian Blackall doctor PJ.

2 haemophilus parasuis antigen gene Hbp B (as described below)

Gene source: Hbp B (＞gi| 2198702792142475-2144169, Haemophilus parasuis SH0165 chromosome, complete genome)

The accession number of the antigen gene sequences that the present invention uses and (what be shown as underscore in the bracket is the accession number of described antigen gene Hbp B in Genebank in residing position in haemophilus parasuis SH0165 bacterial strain, thereafter the numeral behind the branch is the position of gene order shown in this accession number in haemophilus parasuis SH0165 strain gene group, and the overstriking italics that shows behind this position is the bacterial strain number of described haemophilus parasuis.

3 main agents and damping fluid

Sodium-chlor, yellow soda ash, glycerine, ethanol, acetic acid etc. are Shanghai Chemical Reagent Co., Ltd., Sinopharm Group products; Tris alkali, dithiothreitol (DTT) (DTT), sodium laurylsulfonate (SDS), acrylamide, ammonium persulphate, Tetramethyl Ethylene Diamine (TEMED) etc. are given birth to worker's biotechnology company limited available from Shanghai; Glycine, coomassie brilliant blue R_250, Xylene Brilliant Cyanine G G-250 are available from AMRESCO company; Kantlex (Kanamycin, Kana), inductor IPTG (isopropyl-β-D-thiogalactoside(IPTG)) are Invitrogen company product; Suction nozzle and centrifuge tube are AxyGen company products; The endonucleases such as Taq enzyme and 10 * Taq enzyme Buffer, BamH I, Xho I and relevant Buffer, T4 ligase enzyme and 10 * T4 ligase enzyme Buffer, 0.1% (W/V) BSA standardized solution etc. are precious biotechnology (Dalian) company limited product; Proteinase K (stock solution concentration is 20mg/ml, and working concentration is 1mg/ml), benzidine (TMB) are available from the suitable biotechnology company limited of Shanghai China; The sheep anti-mouse igg of bovine serum albumin (BSA), proteinase inhibitor (PMSF), Tween-20, horseradish peroxidase (HRP) mark, Imidazole (imidazoles), NAD (Reduced nicotinamide-adenine dinucleotide), DAB colour developing diagnostic kit are Sigma company product; XOM's white-oil adjuvant is available from Dongguan branch office of the living emerging row petrochemical industry in Heyuan City company limited; Bacterial genomes DNA extraction test kit is available from Tianjin biochemical technology (Beijing) company limited; UNIQ-10 pillar DNA glue reclaims test kit and gives birth to worker's biotechnology company limited available from Shanghai; DNA Marker (2K Plus), EasyPure Plasmid MiniPrep Kit are Beijing Quanshijin Biotechnology Co., Ltd's product.

Ordinary culture medium: every liter of LB liquid nutrient medium contains yeast extract 5g, Tryptones 10g, and NaCl 10g adjusts pH value to 7.5 with 10mmol/L NaOH, 121 ℃ of autoclaving 20min, 4 ℃ save backup.In per 100 milliliters of LB liquid nutrient mediums, add 1.5g agar and be solid LB substratum, 121 ℃ of autoclaving 20min, 4 ℃ save backup.

TSB (Tryptic Soy Broth, Trypsin soybean broth) liquid nutrient medium: accurately take by weighing TSB (Difco ^TM) 30g is dissolved in the 1000ml distilled water, fully shakes up rear 121 ℃ of high pressure steam sterilization 15min.Used TSB liquid culture is based on needing to add 10% NAD (storage concentration is 10mg/ml) of deactivation calf serum (Hangzhou folium ilicis chinensis biological products company limited product) and 0.1% (V/V) filtration sterilization before using among the present invention.

TSA (Tryptic Soy Agar, Trypsin soy agar) solid medium: accurately take by weighing TSA (Difco ^TM) 40g, add distilled water and be settled to 1000ml, 121 ℃ of high pressure steam sterilization 15min.TSA solid medium used herein need treat to add when temperature is down to 60 ℃ the NAD (storing concentration is 10mg/ml) of the aseptic calf serum of 100ml and 1ml filtration sterilization, is down flat ware after fully shaking up, and 4 ℃ of storages are for subsequent use.

PBS damping fluid (pH 7.4): NaCl 8.0g, KCl 0.2g, KH ₂PO ₄0.24g, Na ₂HPO ₄12H ₂O 3.628g is dissolved in the 800ml distilled water, is 7.4 with the hydrochloric acid adjust pH, and distilled water is settled to 1000ml, 121 ℃ of autoclaving 20min, room temperature preservation.

ELISA substrate solution preparation: coating buffer: 25mmol/L carbonate buffer solution (pH 9.6), take by weighing 1.59g yellow soda ash, the 2.93g sodium bicarbonate adds tri-distilled water and is settled to 1L and gets final product; Washings (PBST, pH 7.4): NaCl 8.0g, KCl 0.2g, Na ₂HPO.12H ₂O 2.99g, polysorbas20 0.5mL, tri-distilled water is settled to 1000.0mL; Confining liquid and serum dilution: 0.5g BSA is dissolved in the 100mL washings; Substrate solution A:0.006%H ₂O ₂Damping fluid; Substrate solution B: get Na ₂HPO ₄12H ₂O 14.2g, Trisodium Citrate 10.5g is settled to 500mL with distilled water and is made into 0.1mm phosphoric acid salt citrate buffer solution (pH 5.0), then adds TMB.During use A liquid and B liquid equal-volume are mixed, mix in rear 5 minutes and use, now with the current; Stop buffer: 2mol/L H ₂SO ₄

Agarose gel electrophoresis related solution: 50 * TAE: take by weighing Tise base 242.0g and be dissolved in a small amount of single steaming in the water, add Glacial acetic acid 5.7mL again, 0.5mol/L EDTA (pH8.0) 100.0mL adds single water that steams and is settled to 1000mL, and room temperature preservation is for subsequent use; 0.8% sepharose: 100mL 1 * TAE damping fluid, agarose 0.8 gram, heating is dissolving fully, to be cooledly adds an ethidium bromide during to 60 ℃ of left and right sides, and the glue that falls gets final product.

Western-blot damping fluid: the 2 * SDS Loading Buffer:100mmol/L Tris-Cl (pH6.8) that is correlated with, 4%SDS (electrophoresis level), 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, the time spent adds the 200mmol/L dithiothreitol (DTT) (DTT) of 10% volume; Electricity turns damping fluid: 39mmol/L glycine, 48mmol/L Tris alkali, 0.037%SDS (electrophoresis level), 20% methyl alcohol.Namely prepare 1000mL and need take by weighing the 2.9g glycine, 5.8g Tris alkali, 0.37g SDS adds 200mL methyl alcohol, add ddH2O to cumulative volume be 1000mL; TBS (pH 8.0) damping fluid: 10mmol/L Tris-HCl, 150mmol/L NaCl; The TBST damping fluid: adding final concentration in above-mentioned TBS is that 0.05%Tween-20 gets final product; Confining liquid: contain the TBST of 2%BSA, 4 ℃ of storages are for subsequent use.

Recombinant antigen protein Hbp B adopts Ni Sepharose 6 Fast Flow purification columns (GE Healthcare company product).

4 design of primers are with synthetic

Use the primer that primer-design software Primer 5.0 is designed for amplification Hbp B gene, this primer is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.Primer sequence is as shown in table 1:

The pcr amplification primer of table 1:Hbp B gene

5 experimental animals

4 the week ages female BALB/c mouse, available from Disease Prevention Control Center, Hubei Prov.

Two, antigen protein preparation method

The extraction of 1 haemophilus parasuis genomic dna

1) cultivation of haemophilus parasuis

On the TSA substratum, the single colony inoculation of picking is in 5mL TSB liquid nutrient medium with the streak inoculation of HPS SH0165 bacterial strain lyophilized powder, and 37 ℃ with 150r/min concussion cultivation 14-16h.

2) extraction of haemophilus parasuis HPS SH0165 strain gene group DNA

Adopt the genomic dna of the bacterial genomes DNA extraction test kit extraction HPS SH0165 bacterial strain of Tianjin biochemical technology (Beijing) company limited, carry out according to the operation steps of bacterial genomes DNA extraction test kit specification sheets, specific as follows:

1. get inoculum 1-5mL, and 10,000rpm (～11,500 * g) centrifugal 1 minute, exhaust supernatant as far as possible.

2. add 200 μ L damping fluid GA to bacterial sediment, shake to thalline and thoroughly suspend.

Attention: if need to remove RNA, can add 4 μ L RNase A (100mg/ml) solution, shake 15 seconds, room temperature was placed 5 minutes.

3. in pipe, add 20 μ L protein enzyme solutions, mixing.

4. add 220 μ L damping fluid GB, shook 15 seconds, placed 10 minutes for 70 ℃, the solution strain is limpid, brief centrifugally covers the globule of Nell wall to remove pipe.

Attention: may produce white precipitate when adding damping fluid GB, can disappear during general 70 ℃ of placements, can not affect subsequent experimental.Do not become limpid such as solution, illustrate that lysis is not thorough, may cause DNA amount less and the DNA that extracts impure.

5. add 220 μ L dehydrated alcohols, fully shook mixing 15 seconds, flocks may appear in this moment, brief centrifugally covers the globule of Nell wall to remove pipe.

6. previous step gained solution and flocks are all added among the adsorption column CB3 (adsorption column is put into collection tube), and 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.

7. add 500 μ L damping fluid GD (please checking first whether added dehydrated alcohol before using) in the adsorption column CB3, and 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.

8. add 700 μ L damping fluid PW (please checking first whether added dehydrated alcohol before using) in the adsorption column CB3, and 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.

9. add 500 μ L damping fluid PW in the adsorption column CB3, and 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.

10. adsorption column CB3 is put back in the collection tube, and 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid, place room temperature to place several minutes adsorption column CB3, thoroughly to dry rinsing liquid remaining in the sorbing material.

The purpose in this step is that rinsing liquid remaining in the adsorption column is removed, residual can the impact follow-up enzyme reaction of ethanol in the rinsing liquid (enzyme is cut, PCR etc.) experiment.

11. adsorption column CB3 is changed in the clean centrifuge tube, and to the unsettled dropping in the middle part of adsorption film 50-200 μ L elution buffer TE, room temperature was placed 2-5 minute, 12,000rpm (～13,400 * g) centrifugal 2 minutes, solution is collected in the centrifuge tube.

For increasing the yield of genomic dna, the centrifugal solution that obtains can be added among the adsorption column CB3 again room temperature placement 2 minutes, 12,000rpm (～13,400 * g) centrifugal 2 minutes.The elution buffer volume should not be less than 50 μ L, the too small yield that affects of volume.The pH value of elutriant has a significant impact for elution efficiency.Should guarantee that its pH value (can be transferred to this scope with the pH value of water with NaOH) in the 7.0-8.5 scope if water is cooked elutriant, the pH value is lower than 7.0 can reduce elution efficiencies; And the DNA product should be kept at-20 ℃, in case dna degradation.

The amplification of 2 goal gene and recovery

1) the HPS SH0165 strain gene group extracted with aforesaid method of the amplification of goal gene is as pcr template.Primer shown in the use table 1 carries out pcr amplification.The PCR reaction system is as shown in table 2:

Table 2 PCR reaction system (reaction cumulative volume 50 μ L)

Response procedures is: 94 ℃ of denaturation 5min, 1 circulation; 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 2min, 30 circulations; Last 72 ℃ are extended 10min.Agarose gel electrophoresis with 0.8% is identified recovery.

2) the PCR product reclaims:

The UNIQ-10 pillar DNA glue that adopts Shanghai to give birth to worker's biotechnology company limited reclaims test kit and reclaims dna fragmentation, reclaims the step of the specification sheets of test kit according to the centrifugal dna gel of UNIQ-10 pillar and carries out, and concrete operations are as follows:

1) agarose gel with 0.8% (m/V) carries out electrophoresis, target DNA fragment and other DNA are separated as far as possible, under long-wave ultra violet lamp, be used in the knife blade that burnt on the spirit lamp flame and downcut the agar block that contains target DNA fragment, put into 1.5ml sterilization centrifuge tube.

2) add 400 μ L Binding Buffer by every 100mg agarose gel, put in the 50-60 ℃ of water-bath and act on 10min, make sepharose thoroughly melt (when adding thermosol, every 2min mixing once).

3) the UNIQ-10 post is put into collection tube, the sol solution that melts is transferred in the UNIQ-10 post, room temperature leaves standstill 2min, the centrifugal 1min of room temperature 8000r/min.

4) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, add 500 μ L Wash Solution, the centrifugal 1min of room temperature 8000r/min.

5) repeating step 4, take off the UNIQ-10 post, outwell the waste liquid in the collection tube, and the UNIQ-10 post is put into same collection tube, the centrifugal 15sec of room temperature 12000r/min.

6) the UNIQ-10 post is put into the 1.5ml centrifuge tube of a sterilization, according to the DNA amount relatively what, the film central authorities in the pillar bottom add 30-50 μ L Elution Buffer or ddH ₂O, room temperature or 37 ℃ are placed 2min, then in the centrifugal 1min of room temperature 12000r/min, the liquid of collecting in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ for subsequent use.

The structure of 3 recombinant plasmids

1) linear carrier plasmid and purpose fragment is connected

Utilize restriction enzyme BamH I and Xho I simultaneously Hbp B gene fragment and the vector plasmid pET-28a (+) (seeing Fig. 7) of double digestion pcr amplification gained, reclaim purpose fragment and expression vector, enzyme is cut rear Hbp B gene fragment cut rear pET-28a (+) with enzyme and be connected, construct pET-28a (+)-Hbp B plasmid.BamH I and Xho I are single restriction enzyme site (the external source fragment is that the fragment on position shown in the SEQ ID NO:1 is between BamH I and Xho I restriction enzyme site) in used carrier plasmid pET-28a of the present invention (+).

2) conversion of connection product

Take out competent cell bacillus coli DH 5 alphas (existing with now getting) from-80 ℃ of refrigerators, sucking-off 100 μ L join in the 1.5mL sterilization EP pipe, add simultaneously gently mixing of product pET-28a (+) after the 5 μ L connection-Hbp B.After leaving standstill 45min on ice, 42 ℃ of heat shocks 90 seconds, immediately ice bath 2min.In reaction tubes, add the fresh LB of 600 μ L, make its recovery in 37 ℃ of lower 200r/min shaking culture 45min.Recombination bacillus coli suspension centrifugal 10min of 5000r/min under 4 ℃ of conditions with after the recovery discards 600 μ L supernatants, coats the LB agar plate that contains 25 μ g/mL Kana with the remaining 100 resuspended precipitations of μ L substratum and with it.37 ℃ of forwards are hatched 1h, flat board are turned over to be inverted again to cultivate 14-16h, until single bacterium colony occurs.

3) extraction of recombinant plasmid

Adopt the EasyPure Plasmid MiniPrep Kit of Beijing Quanshijin Biotechnology Co., Ltd to extract recombinant plasmid, extract on a small quantity the specification sheets operation steps of test kit according to this plasmid and carry out, details are as follows:

1) gets centrifugal 1 minute of bacterium 10, the 000 * g of 1-4mL incubated overnight, exhaust supernatant as far as possible.

2) add 250 μ L colourless solution RB (containing RNase A), concussion suspension bacterial precipitation should not leave little bacterium piece.

3) add 250 μ L blue solution LB, leniently counter-rotating mixes 4-6 time up and down, makes the abundant cracking of thalline, forms blue bright solution, and color becomes bright blueness by partly bright, indicates complete cracking (should not above 5 minutes).

4) add 350 μ L yellow solution NB, mix gently 5-6 time (color is by the complete yellowing of blueness, and indication mixes, and neutralization fully), until form the yellow aggegation piece of consolidation, room temperature left standstill 2 minutes.

5) the centrifugal 5min of 15,000 * g carefully draws supernatant and adds in the adsorption column.

6) the centrifugal 1min of 15,000 * g, the abandoned stream fluid.

7) add 650 μ L solution W B, the centrifugal 1min of 15,000 * g, abandoned stream fluid.

8) the centrifugal 1-2min of 15,000 * g thoroughly removes residual WB.

9) adsorption column is placed a clean centrifuge tube, add 30-50 μ L EB or deionized water (PH＞7.0) room temperature leaves standstill 1 minute (EB or deionized water are 60-70 ℃ of water-bath preheating, and result of use is better) in the central authorities of post.

10) in the centrifugal 1min of 10,000 * g, eluted dna, the DNA that elutes and-20 ℃ of preservations.

4) double digestion of recombinant plasmid is identified

Utilize BamH I and Xho I double digestion pET-28a-Hbp B recombinant expression plasmid, enzyme is cut the big or small purpose fragment of expection and carrier segments appear in product through agarose gel electrophoresis the correct recombinant plasmid that is.

The structure of 4 recombinant strains

Get 100 μ L competent cell intestinal bacteria BL-21 (DE3) and join in the 1.5mL sterilization EP pipe, other gets 5 μ L recombinant plasmid pET-28a-Hbp B and adds in the pipe and mixing gently.Leave standstill on ice behind the 45min in 42 ℃ of heat shocks 90 seconds, immediately ice bath 2min.In reaction tubes, add 600 μ L LB substratum, make its recovery in 37 ℃ of 200r/min shaking culture 45min.Recombination bacillus coli suspension after the recovery prior under 4 ℃ of conditions with the centrifugal 10min of 5000r/min, discard 600 μ L supernatants, with the remaining resuspended precipitation of 100 μ L and coat the LB agar plate that contains 25 μ g/mL Kana.37 ℃ of forwards are hatched 1h, flat board are turned over again, and are inverted and cultivate 14h-16h, until single bacterium colony occurs.

5 goal gene Expression in Escherichia colis

1) abduction delivering of goal gene: picking contains the expression strain list bacterium colony of restructuring goal gene Hbp B, is inoculated in the 3mL LB liquid nutrient medium that contains 25 μ g/mL Kana, and 37 ℃ of concussions are cultivated.Get 100 μ L and be inoculated in the fresh LB liquid nutrient medium that 10mL contains 25 μ g/mL Kana from cultured bacterium liquid, 37 ℃ of shaking culture to muddy degree of bacterium liquid reach OD ₆₀₀0.8-1.0 the time, adding IPTG is 0.8mM to final concentration, continuation is collected thalline after cultivating 3h.

2) the SDS-PAGE electrophoretic analysis of expression product

The preparation of SDS-PAGE electrophoresis sample:

In the centrifugal 15min of 8000r/min, PBS washes 2 times with the recombination bacillus coli bacterium liquid after inducing.Precipitate resuspendedly with the 50mM Tris-HCl (pH 8.0) of 1/10 volume, adding simultaneously N,O-Diacetylmuramidase to final concentration is 1mg/mL, ice bath 30min.Carry out ultrasonic broken broken under the condition of ice bath, until the limpid no longer thickness of bacterium liquid, the centrifugal 30min of 10000r/min, the upper cleer and peaceful precipitation after the cracking that takes a morsel respectively, wherein supernatant directly adds the DTT of equal-volume 2 * protein electrophoresis sample-loading buffer and 1/10 volume, and fully mixing is in order to loading; Precipitation is then resuspended with an amount of 50mM Tris-HCl (pH 8.0) in addition, then the DTT that adds equal-volume 2 * protein electrophoresis sample-loading buffer and 1/10 volume, the concussion mixing, ice bath 5min again behind the boiling water boiling 10min, the centrifugal 5min of 12000r/min gets supernatant and carries out the SDS-PAGE electrophoretic analysis.

The preparation of SDS-PAGE and electrophoresis:

The preparation of 10% separation gel: ddH ₂O 1.9mL, 30% acrylamide soln 1.7mL, 1.5mol/L Tris-Base solution (pH 8.8) 1.3mL, 10%SDS 0.05mL, 10% ammonium persulphate 0.05mL and TEMED 0.003mL.Each composition is added rear rapidly mixing, add at once in the glue plate, slowly spread a small amount of ddH on it ₂O.The preparation of 5% concentrated glue: purified water 0.68mL, 30% acrylamide soln 0.17mL, 1.0mol/L Tris-Base solution (pH 6.8) 0.13mL, 10%SDS 0.01mL, 10% ammonium persulphate 0.01mL, TEMED 0.001mL adds behind above-mentioned each composition and mixes rapidly, add above the separation gel of glue plate, fill rear insertion application of sample comb (adding the front thoroughly exhaustion of concentrated glue upper water).Take off comb after gelling to be concentrated is solid, gel is fixed on the electrophoresis apparatus, add the Tris-glycine electrophoretic buffer of q.s, in well, add respectively each sample; It is 80V that voltage is set first during electrophoresis, and electric current remains in the 20mA-40mA scope, behind the about 30min of electrophoresis, enters to all samples and to adjust voltage to 120V after separating glue-line, until stop electrophoresis during tetrabromophenol sulfonphthalein swimming plastic emitting bottom surface.

The dyeing of SDS-PAGE and decolouring:

Unload gel, (contain 50% methyl alcohol, 45% purified water with the coomassie brilliant blue R250 staining fluid, 5% glacial acetic acid, 0.25% coomassie brilliant blue R250) dyeing 4-6h, and then (contain 50% methyl alcohol with destainer, 45% purified water, 5% glacial acetic acid) decolour observations.

The great expression of 6 recombinant proteins and purifying

Picking is identified the single bacterium colony of correct recombinant expressed bacterium, is inoculated in the LB liquid nutrient medium that 10mL contains 25 μ g/mLKana, and 37 ℃ of concussions are cultivated 12h as seed liquor.Second day takes out 8mL and is inoculated in the fresh LB substratum that 800mL contains 25 μ g/ml Kana, and 37 ℃ of shaking culture are until bacterium liquid OD ₆₀₀Reach about 0.8, add IPTG to final concentration be 0.8mM, continue at 37 ℃ and collect thalline after inducing 3h.

The recombinant bacterium thalline washes twice with PBS (pH 7.4) first, and it is resuspended to re-use Binding buffer (containing 20mM Tris-HCl (pH 7.9), 5mM imidazoles, 0.5M NaCl), and ultrasonication is until mixture is limpid.The centrifugal 15min of 12000g abandons supernatant under 4 ℃ of conditions.Inclusion body precipitation with PBS (pH 7.4) washing 2 times, adds 19.6mL BufferA and 0.4ml 20% (V/V) SKL (dodecyl creatine sodium) first again, and concuss is with dissolution precipitation, 4 ℃ of reaction overnight.Second day with the centrifugal 30min of 15000g, is collected it supernatant and is added respectively 210 μ L 20%PEG, 4000,210 μ L 50mM Sleep-promoting factor B and 420 μ L 100mM reduced glutathions, fully leaves standstill 2h in 4 ℃ behind the mixing.Solution is transferred in the dialysis membrane, dialysed with the TE (PH 8.0) of 20-30 times of volume, every 4-6h changes dialyzate, takes out behind the 72h.Then the recombinant protein that dialysis is good uses the Ni-NTA affinity column to carry out affinity chromatography through the 220nm membrane filtration.Recombinant protein is further purified and is operated in AKTA-FPLC (GE Healthcare, USA) carry out on, A pump prepackage Binding buffer, use at first respectively Binding buffer and Washing buffer to wash post before the B pump prepackage Washing buffer. albumen loading, until OD reaches near the baseline, use the recombinant protein of Elution buffer elution of bound, collect the crest Partial Protein and analyze for further as the recombinant antigen protein of purifying ,-80 ℃ of storages are for subsequent use.

Recombinant plasmid pET-28a (+) in the present embodiment-Hbp B double digestion evaluation figure and superhelix recombinant plasmid collection of illustrative plates thereof are seen Fig. 2, and recombinant antigen protein Hbp B mainly is present in the inclusion body precipitation, and its expression and purification effect is seen shown in Fig. 3 (A).

Embodiment 2: the specificity analysis of recombinant antigen protein

The characteristic of 1 usefulness Western-blot methods analyst recombinant antigen protein

The recombinant antigen protein Hbp B of Purification among the embodiment 2 is carried out the SDS-PAGE gel electrophoresis according to ordinary method.Thereafter step is as follows:

1) transferring film: cut out 6 Whatman 3M filter paper and 1 nitrocellulose filter (NC film), the size of filter paper and NC film will equate fully with gel or be slightly less than gel, marks one jiao of NC film with pencil, determines the relative direction of transfer printing caudacoria and gel; The NC film is soaked 5min in purified water; In another shallow pallet, add a small amount of transfering buffering liquid, 6 layers of Whatman 3M filter paper are soaked in wherein.Then the electrophoretic blotting groove is installed in accordance with the following steps: keep flat the base (anode) of Graphite Electrodes, put successively 3 layers of 3M filter paper, NC film, SDS-PAGE gel and 3 layers of 3M filter paper.The bubble of thoroughly getting rid of each interlayer; The loam cake of electrophoretic blotting groove is anchored on the Graphite Electrodes one transfer film glue complex body; Connect power supply, according to the gel slab area according to 0.65mA/cm ²-1.0mA/cm ²Parameter electric current is set, electrophoretic transfer 0.5h-2h.

2) sealing: take off the NC film after transferring film is complete, place confining liquid, room temperature sealing 2h;

3) washing: abandon confining liquid, wash the NC film 3 times with TBST, each 5min;

4) primary antibodie is hatched: the mouse-anti HPS SH0165 that the NC film is put into 1: 100 times of dilution infects serum, hatches 2h for 37 ℃;

5) washing: take out the NC film, wash film 3 times with TBST, each 5min;

6) two anti-hatching: the NC film is changed in the sheep anti-mouse igg antibody (HRP mark) of using 1: 5000 times of dilution of confining liquid, hatch 2h for 37 ℃;

7) washing: take out the NC film, wash film 5 times with TBST, each 5min;

8) colour developing: the NC film is placed the DAB nitrite ion of new configuration, and the lucifuge colour developing after purpose band color depth reaches requirement, is washed with termination reaction with TBST rapidly.

The sero-fast preparation of 2 recombinant proteins

1) mensuration of recombinant protein concentration

With reference to the Pehanorm Brooker, Huang Peitang translates, " molecular cloning experiment guide " third edition, Science Press, the method for version introduction in 2002, and slightly improvement.At first carry out the making of typical curve: with 0.1% (W/V) BSA solution as standard protein solution.Get 6 1.5mL EP pipe number consecutivelies, every pipe all accurately adds Xylene Brilliant Cyanine G G-250 staining fluid 1mL, adds respectively successively 0.1%BSA solution 0 μ L again, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, add tri-distilled water and be settled to cumulative volume and reach 1.1mL, mixing joins in the elisa plate gently, every hole 180 μ L, each concentration is done 5 repetitions.Behind the room temperature reaction 10min, on micro ELISA reader, read OD ₅₉₅Value.Take protein concn as X-coordinate, OD ₅₉₅Value is ordinate zou, and the production standard curve is determined OD ₅₉₅Relation between value and the protein concentration.After finishing, Specification Curve of Increasing carries out the concentration determination of protein sample: after getting 3 1.5mL EP pipe and finishing sequence number successively, add respectively sample protein solution 1 μ L, and 2 μ L, then 5 μ L measure separately light absorption value by upper method in wavelength 595nm place.The reference standard working curve is determined the concentration of protein example, gets its mean value as the sample protein ultimate density.

2) preparation of vaccine and immunity test

The preparation of recombinant antigen protein vaccine: add 1% (V/V) Tween-80 first in recombinant antigen protein Hbp B, carry out emulsification with white-oil adjuvant take volume ratio as 1: 1.5 ratio again behind the mixing, the final concentration that makes antigen protein in the vaccine is 200 μ g/mL.

Immunization method: to the recombinant protein vaccine of test mice intraperitoneal inoculation through white-oil adjuvant emulsification, dosage of inoculation is 200 μ L/; 2 weeks were carried out again immunity by the abdominal cavity; Interval afterwards docking blood sampling in 10 days, separation of serum is also for subsequent use in 4 ℃ of storages.

3) distribution of Western-blot test detectable antigens albumen Hbp B in 15 standard serum types of haemophilus parasuis

The preparation of electrophoresis sample

Get 15 standard serum types of haemophilus parasuis lyophilized powder respectively streak inoculation on the TSA plate that contains 10% (V/V) deactivation calf serum and 0.1 μ g/mL NAD, the single colony inoculation of picking is in 5mL TSB (containing 10% (V/V) deactivation calf serum and 0.1 μ g/mL NAD) liquid nutrient medium, and 37 ℃ with 150r/min concussion cultivation 14-16h.12, the centrifugal 2min of 000g collects thalline, first with PBS (pH 7.4) washing 2 times, again with the resuspended bacterial sediment of certain volume PBS (pH 7.4), the DTT that adds equal-volume 2 * protein electrophoresis sample-loading buffer and 1/10 volume, boiling water boiling 10min behind the abundant mixing, cooled on ice 5min, the centrifugal 10min of 12000r/min gets supernatant in order to the SDS-PAGE electrophoretic analysis.

The preparation of SDS-PAGE gel, electrophoresis

Embodiment 2 is seen in concrete operations.

The Western-blot test

Concrete operations reference example 2.Wherein primary antibodie is used the anti-Hbp B of the mouse albumen serum of above-mentioned preparation, and all the other steps are with embodiment 2.

Adopt reactionogenicity and the distribution of this albumen in 15 standard serum types of haemophilus parasuis of Western-blot method validation recombinant antigen protein Hbp B in the present embodiment, the invention effect is seen Fig. 4.The result shows that outer membrane protein H bp B all can express, and has verified the versatility of subunit vaccine of the present invention in haemophilus parasuis standard serum type 1 type, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, 11 types, 13 types, 14 types, 15 type bacterial strains (being so kind as to give by Australian Pat doctor Blackall).

Embodiment 3: the test of recombinant antigen protein mouse immune protection

The great expression of 1 recombinant protein and purifying

To contain recombinant strains list colony inoculation and contain the LB liquid nutrient medium of 0.25 μ g/mL kantlex in 10mL, 37 ℃ of shaking tables concussions are cultivated.Cultured bacterium liquid is seeded in the fresh LB liquid nutrient medium that 1L contains 2.5 μ g/mL kantlex, in 37 ℃ of about 3h of shaking culture, to OD ₆₀₀When reaching 0.6-0.8, add IPTG to final concentration be 0.8mmol/L, collect thalline after continuing to induce 3h.The concrete purge process of recombinant protein is seen embodiment 2.

The mensuration of 2 recombinant protein concentration (conventional Bradford method)

Embodiment 2 is seen in concrete operations.

The preparation of 3 recombinant antigen protein vaccines and immunization method

1) preparation of recombinant antigen protein vaccine

The preparation of recombinant antigen protein vaccine: in recombinant antigen protein Hbp B, add 1% (V/V) Tween-80 first, fully carry out emulsification with white-oil adjuvant take volume ratio as 1: 1.5 ratio again behind the mixing, the final concentration that makes antigen protein in the vaccine is 200 μ g/mL.

The inactivated vaccine preparation: after the cultivation of haemophilus parasuis SH0165 bacterial strain purifying, picking list colony inoculation is in 5mLTSB (NAD that contains 10% deactivation new-born calf serum and 10 μ g/mL) substratum.Behind 37 ℃ of cultivation 16h-18h, add the about 48h of 3% (V/V) formalin-inactivated, line check bacterium liquid deactivation situation.The centrifugal 2min of 12 000g collects thalline, and with PBS (pH 7.4) washing 2 times, then resuspended bacterial sediment is in certain volume PBS first.First in bacterium liquid, add 1% (V/V) Tween-80, carry out emulsification with white-oil adjuvant take volume ratio as 1: 1.5 ratio again behind the mixing, with white-oil adjuvant be 1: 1.5 mixing and emulsifying by volume, make the final bacteria containing amount of deactivation vaccine reach 3.0 * 10 ⁹CFU/ml.

2) immunization method is to the recombinant protein vaccine of test mice intraperitoneal inoculation through white-oil adjuvant emulsification, and dosage of inoculation is 200 μ L/; 2 weeks were carried out again immunity by the abdominal cavity; Interval afterwards docking blood sampling in 10 days, separation of serum is also for subsequent use in 4 ℃ of storages.

3) medium lethal dose (LD ₅₀) mensuration

(1) trial test: in trial test, try to achieve 4 ages in week female BALB/c mouse all dead or 90% or more the dead toxic agent amounts of attacking and animal is not dead or 10% below the toxic agent amount of attacking of death, respectively as the highest and lowest dose level of official test.

(2) attack malicious mode: abdominal injection.

(3) animal grouping: set up 4 dosage groups, every group of 6 BALB/c mouse.

(4) attack the toxic agent amount: will all be scaled denary logarithm by the highest, the lowest dose level that trial test draws, then will be the highest, the logarithmic difference of lowest dose level, by needed group of number, be divided into the dosage group of several logarithms equidistant (or not equidistant).

(5) calculating of test-results and statistics.

4) test grouping and immunity

Got for 4 ages in week, 24 of the female BALB/c small white mouses of body weight 18 ± 2g are equally divided into 4 groups, Hbp B vaccine group, positive controls, white-oil adjuvant control group and PBS control group, 6 every group.Abdominal injection, per 2 week immunity 1 time, immunity is 2 times altogether, and immunizing dose is 0.2mL/, and blood is got in docking weekly therebetween, is used for the monitoring of serum specific antibody.

5) detection of serum specific antibody

Use the recombinant antigen protein Hbp B of purifying of the present invention with the coated elisa plate of 250ng/100 μ L, 4 ℃ of night incubation.37 ℃ of 1%BSA sealing 1h after then washings is washed plate 1 time, is packaged in-20 ℃ of preservations.With the mouse blood sampling separation of serum of booster immunization after one week, get 100 μ L behind the doubling dilution and add elisa plate, establish simultaneously white-oil adjuvant contrast and blank.37 ℃ of reaction 30min.Added volume ratio 1: 5 after washing plate 3 times, the sheep anti-mouse igg (H+L) of 000 dilution-HRP, 37 ℃ of reaction 1h.Add successively substrate solution A and each 50 μ L of substrate solution B after washing plate 5 times, add 0.25% hydrofluoric acid termination reaction behind the lucifuge colour developing 10min, in wavelength 630nm reading.Get the OD value greater than 0.3 serum maximum dilution multiple as serum antibody titer.

4 immune mouses are attacked malicious protection situation

The mouse of booster immunization after two weeks carried out challenge test.Challenge test is divided into two batches to carry out, and 6 every group, the mouse inoculation 5LD of every protein immunization ₅₀HPS SH0165 strain bacterium.Continuous Observation 10 days, Clinical symptoms and the death condition of record mouse.

Recombinant antigen protein mainly is present in the inclusion body precipitation in the present embodiment, and its expression and purification effect is seen Fig. 3 (A); Medium lethal dose (the LD of HPS SH0165 bacterial strain ₅₀) be 8 * 10 ⁸CFU; Recombinant antigen protein Hbp B is carried out the detection of serum specific antibody and find to induce very high antibody horizontal, effect is seen Fig. 5; With the recombinant antigen protein HbpB immune mouse of purifying, with 5LD ₅₀Attack poison and estimate the immune protective efficiency of recombinant antigen protein, the result as shown in Figure 6.

The present invention is by clonal expression haemophilus parasuis outer membrane protein gene Hbp B, and the immunological characteristic of Hbp B is carried out analysis of experiments, proved that this antigen protein has preferably immunogenicity, can induce than High antibody level behind the immune mouse.Mouse is attacked malicious protection test and shows that this albumen can obviously strengthen mouse to the resistibility of HPS, is a kind of good immune protective antigen, can be used as the candidate antigens of haemophilus parasuis subunit vaccine.

Reference

1. Si Telao etc. hyoiatrics (the 8th edition) [M]. Zhao Deming etc. translate. Beijing: China Agricultyre University Press .1998,407-416.

2. Cai Xu is prosperous etc. the separation and Culture of haemophilus parasuis and Serotype Identification [J]. and Hua Zhong Agriculture University's journal, 2005,24 (1): 55-58.

3.Kielstein?P，Rapp-Gabrielson?VJ.Designation?of?15?serovars?of?Haemophilus?parasuis?on?the?basis?of?immunodiffusion?using?heat-stable?antigen?extracts.J?Clin?Microbiol?1992；30(4)：862-865.

4.Nielsen?R.Pathogenicity?and?immunity?studies?of?Haemophilus?parasuis?serotypes.Acta?Vet?Scand?1993；34(2)：193-198.

5.Zhou?M，Guo?Y，Zhao?J，Hu?Q，Hu?Y，Zhang?A，et?al.Identification?and?characterization?of?novel?immunogenic?outer?membrane?proteins?of?Haemophilus?parasuis?serovar?5?Vaccine?2009；27(38)：5271-5277.

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Claims

1. the outer membrane protein H bp B gene of a separating clone, its nucleotide sequence is shown in SEQ ID NO:1.

2. the outer membrane protein H bp B gene of a separating clone, its encoding sequence is shown in SEQ ID NO:2.

3. a recombination bacillus coli (Escherichia coli) BL21/pET-28a-Hbp B, its preserving number is CCTCC NO:M 2011228, it comprises the described gene of sequence table SEQ ID NO:1.

4. the antigen protein of a haemophilus parasuis, it is by the described gene transformation intestinal bacteria of sequence table SEQ ID NO:1, and it is expressed to obtain the recombination bacillus coli that preserving number is CCTCC NO.M 2011228 (Escherichia coli) BL21/pET-28a-Hbp B.

5. the preparation method of a haemophilus parasuis envelope antigen albumen, its step comprises:

1) design primer pair, by the pcr amplification method, the clone obtains a kind of outer membrane protein H bp B gene from haemophilus parasuis SH0165 bacterial strain, and its nucleotide sequence is shown in sequence table SEQ ID NO:1;

2) with step 1) haemophilus parasuis SH0165 bacterial strain outer membrane protein gene Hbp B be inserted between the EcoR I and Xho I multiple clone site of prokaryotic expression carrier pET-28a (+), obtain middle interstitial granules pET28a-Hbp B;

3) with step 2) described middle interstitial granules pET28a-Hbp B conversion e. coli bl21 (DE3) competent cell, obtain positive recombination bacillus coli BL21/pET-28a-Hbp B through resistance screening, its preserving number is CCTCC NO:M2011228;

4) with step 3) described recombination bacillus coli cultivates in the LB liquid nutrient medium, after isopropylthio-β-D-galactoside (IPTG) is induced, carry out Western blot and analyze, obtain having the haemophilus parasuis Hbp B albumen of immunologic competence.

6. claim 1 or the 2 described genes application in preparation haemophilus parasuis antigen protein.

7. the application of recombination bacillus coli claimed in claim 3 in preparation haemophilus parasuis subunit vaccine.

8. the application of antigen protein claimed in claim 4 in preparation haemophilus parasuis subunit vaccine.