CN104651376A - Application of acinetobacter baumannii membrane protein A1S_0851 as antigen - Google Patents

Abstract

The invention discloses application of an acinetobacter baumannii membrane protein A1S_0851 as an antigen. The gene information of acinetobacter baumannii is analyzed, the acinetobacter baumannii membrane protein A1S_0851 is selected and used as a candidate vaccine antigen, the acinetobacter baumannii membrane protein A1S_0851 antigen is cloned and expressed by a pEGX-6P-2 fusion protein expression vector by means of genetic engineering, and the expressed protein immuned mice is used to perform antigenicity evaluation. The results show that the recombinant protein encoded by the gene of acinetobacter baumannii has the effect of resisting acinetobacter baumannii infection and is an ideal acinetobacter baumannii vaccine antigen.

Description

Acinetobacter bauamnnii membranin A1S_0851 is as the application of antigen

Technical field

The invention belongs to biological technical field, be specifically related to the application of a kind of Acinetobacter bauamnnii membranin A1S_0851 as antigen.

Background technology

Acinetobacter bauamnnii (Acinetobacter baumannii) belongs to Neisseriaceae, acinetobacter, is a kind of nonfermented carbohydrate, oxidase negative, the gram negative bacillus that can not move.Acinetobacter bauamnnii is extensively present in natural water and soil, hospital environment and human body skin, respiratory tract, digestive tube and urogenital tract, is conditioned pathogen.Acinetobacter bauamnnii all has stronger resistibility to temperature, heat, ultraviolet and chemostefilant, conventional sterilizing agent can only suppress it to grow, can not be killed, therefore, Acinetobacter bauamnnii is often concealed in various medicine equipment and hospital ward corner, for patient with severe symptoms, and patient as weak in gerontal patient, critical illness and Abwehrkraft des Koepers, and using the patient of various store period and life-time service broad-spectrum antibiotic therapy, Acinetobacter bauamnnii can become important pathogenic bacterium.Clinically, it mainly causes pulmonary infection, also can cause septicemia, the serious symptom such as soft tissue or wound infection, catheter related infection, urinary tract infections, microbemia and Secondary cases meningitis.Section office's distribution of hospital infection is at most with intensive care unit, is secondly Respiratory Medicine.

Acinetobacter bauamnnii infects long history, from 20th century the eighties, just there is Acinetobacter bauamnnii to infect the report of outburst, and after the U.S. reports Carbapenem-resistant Acinetobacter bauamnnii (CRAB) bacterial strain for 1991, various countries report Carbapenem-resistant Acinetobacter bauamnnii all successively, and data according to statistics, its ratio raises year by year.The infection of Acinetobacter bauamnnii and resistance have become a global problem, normal in multidrug resistant and general resistance, and resistant organism often causes large-scale outbreak popular in specific crowd, the case fatality rate Chang Gaoda 75% of infected patient.The monitoring data display acinetobacter calcoaceticus of Chinese Drug Resistance Detection in 2010 is 30.7% to cefoperazone/Sulbactam resistant rate, be 31.2% to Minocycline HCl resistant rate, other drug is if the resistant rate such as imipenum, meropenem, cefepime, ceftazime, cefoxitin, piperacillin/Tazobactam Sodium, ammonia benzyl west/Sulbactam, amikacin, gentamicin, Ciprofloxacin are all more than 50%.Due to resistance and resistance mechanism complexity, the infection that the Resistant strain of Acinetobacter bauamnnii causes has become clinically very stubborn problem.Normal antibiotics is not obvious to its therapeutic action, Acinetobacter bauamnnii is about to face the condition that antibiotic-free can be controlled, adopt vaccine to carry out the infection of prevention and corntrol Acinetobacter bauamnnii, particularly the infection of antagonism height resistance Acinetobacter bauamnnii has become instant task.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of Acinetobacter bauamnnii membranin A1S_0851 as the application of antigen.

Technical scheme of the present invention is as follows:

1, Acinetobacter bauamnnii membranin A1S_0851 is as the application of antigen, and the aminoacid sequence of described membranin A1S_0851 is as shown in SEQ ID NO.2.

2, Acinetobacter bauamnnii recombinant protein A 1S_0851 is preparing the application in vaccine, and the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

3, the Acinetobacter bauamnnii vaccine prepared by Acinetobacter bauamnnii recombinant protein A 1S_0851, the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

Preferably, described vaccine also comprises adjuvant, and described adjuvant is the aluminium hydroxide of final concentration 13.5mg/mL.

Preferably, described vaccine also comprises diluent, and described diluent pH value is 6.0, by concentration be the Histidine of 10mM, the PLURONICS F87 of the NaCl of massfraction 0.9% and massfraction 0.01% forms.

4, the preparation method of above-mentioned vaccine, comprises the steps:

(1) be the aluminum hydroxide adjuvant of 30mg/mL by the vaccine diluent concentration of ordinary dissolution that pH value is 6.0, obtain solution A; The volume ratio of described aluminum hydroxide adjuvant and vaccine diluent is 9:1;

(2) with step (1) described vaccine diluent dilution recombinant protein A 1S_0851, the solution B that protein concentration is 0.3 μ g/ μ L is obtained;

(3) get isopyknic solution A and solution B is adsorbed under 4 ~ 32 DEG C of conditions, obtain vaccine.

5, the application of above-mentioned vaccine in the medicine that preparation prevents or treatment Acinetobacter bauamnnii infects.

6, the antibody that obtains as antigen-immunized animal of Acinetobacter bauamnnii recombinant protein A 1S_0851, the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

Preferably, described antibody is the IgG antibody that immune mouse or rabbit obtain.

Beneficial effect of the present invention is: the present invention is by analyzing Acinetobacter bauamnnii gene information, choose the candidate antigens of membranin A1S_0851 as vaccine of Acinetobacter bauamnnii, by engineered means, pEGX-6P-2 fusion protein expression vector is selected to clone A1S_0851 antigen, express, and the immune protective evaluation of antigenicity evaluation and the infection of anti-Acinetobacter bauamnnii is carried out with the recombinant protein immune mouse of expressing, result shows that the albumen of Acinetobacter bauamnnii A1S_0851 genes encoding has the effect of anti-Acinetobacter bauamnnii infection, it is desirable Acinetobacter bauamnnii vaccine antigen.Compared with full bacterium inactivated vaccine; because full bacterium inactivated vaccine outer membrane complex comparison of ingredients is complicated; effective protective antigen composition may be contained; also may exist and have nothing to do with immunoprotection or the composition of side effect, and preparation process is difficult to stdn, and genetic engineering subunit vaccine antigenic component is clear and definite; definite effect; side effect is little, and security is high, is a kind of desirable vaccine form.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 Acinetobacter bauamnnii A1S_0851 gene PCR amplification Gel electrophoresis results; Wherein, M swimming lane is marker, and 1 swimming lane is A1S_0851 gene PCR amplified production.

Fig. 2 pGEX-6p-2/A1S_0851 recombinant plasmid gel electrophoresis qualification result; Wherein, 1 ~ 4 swimming lane is recombinant plasmid, and M swimming lane is marker, and wherein swimming lane 3 is the recombinant plasmid that qualification is correct.

Fig. 3 recombinant bacterial strain pGEX-6P-2-A1S_0851/XL-1blue abduction delivering recombinant protein A 1S_0851 result; Wherein, M swimming lane is marker, and 1 ~ 3 swimming lane is followed successively by under 16,25 and 30 DEG C of conditions after IPTG induction process, broken thalline recombinant protein A 1S_0851 in centrifugal gained supernatant liquor; 4 ~ 6 are followed successively by under 16,25 and 30 DEG C of conditions after IPTG induction process, broken thalline recombinant protein A 1S_0851 in centrifugal gained precipitation.

The growth curve chart of bacterium in Fig. 4 recombinant bacterial strain pGEX-6P-2-A1S_0851/XL-1blue fermenting process; Whole growth curve standard of comparison, early stage, bacterial growth was very fast, and during later stage induction, curve is more steady.

The target protein qualification result that Fig. 5 recombinant bacterial strain pGEX-6P-2-A1S_0851/XL-1blue fermentation obtains; Each swimming lane is from left to right followed successively by maker, IPTG and induces 1h, 2h, 3h, 4h; As seen from the figure, restructuring A1S_0851 albumen is successfully obtained through engineering bacterium fermentation.

Fig. 6 is to the purification result figure of the recombinant protein A 1S_0851 of fermentation acquisition through GST sepharose 4B affinity chromatography, and result shows, and target protein purity is more than 95%, and target protein molecular weight, at about 33kDa, is consistent with expection size.M: protein standard substance, swimming lane 1: recombinant protein A 1S_0851 and gst fusion protein, swimming lane 2 is the recombinant protein A 1S_0851 after purifying after enzyme is cut.

Fig. 7 recombinant protein A 1S_0851Q HP tomographic map.

Fig. 8 is recombinant protein A1S_0851 SDS-PAGE result figure after Q HP chromatography, in figure, and swimming lane 1: standard protein; Swimming lane 2:GST is affine, and PP enzyme cuts rear elution samples; Swimming lane 3:Q HP stream wears sample; Sample after swimming lane 4:Q HP chromatography.

Fig. 9 is that recombinant protein A1S_0851Q HP chromatography removes SDS-PAGE result figure after intracellular toxin, in figure, and swimming lane 1: standard protein; Swimming lane 2:Q HP chromatography removes sample after intracellular toxin.

After Figure 10 Acinetobacter bauamnnii attacks poison, the mouse survival rate figure of different concns bacterium liquid.

After Figure 11 recombinant protein A 1S_0851 immunity the 14th day, vaccine antigen specific IgG humoral response level after ELISA detection mouse immune, experimental group titre is compared with aluminum hydroxide adjuvant control group and vaccine diluent control group titre, and all there were significant differences (P<0.01).

Embodiment

Below the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.

Acinetobacter bauamnnii ATCC international standard strain ATCC 17978, is provided by U.S. ATCC; Primer is synthesized by TaKaRa company; Host strain XL1-blue intestinal bacteria are purchased from Agilent company of the U.S.; Plasmid pGEX-6p-2 is purchased from GEHealthcare company of the U.S.; PrimeSTAR HS archaeal dna polymerase, DNA molecular amount standard (marker), restriction enzyme BamH I and Not I, Protein Marker (marker), DNA ligase are all purchased from Dalian TakaRa company; Bacterial genomes is extracted test kit and is purchased from Tian Gen company; Plasmid extraction kit and gel reclaim test kit and are purchased from Omega company of the U.S.; Ampicillin Trihydrate, kantlex are purchased from Huabei Pharmaceutic Co., Ltd; Glutathione Sepharose 4B purchased from American GEHealthcare company; High pressure homogenizer APV-1000 is purchased from Denmark An Invernsys Group; BALB/C mice is purchased from Beijing Fukang company of China; Aluminum hydroxide adjuvant purchased from American GENERAL CHEMICAL company; Pharmaceutical grade Histidine purchased from American Merck company; Pharmaceutical grade PLURONICS F87 purchased from American Merck company, pyrogen-free.

Preparation of reagents:

5 × protein sample-loading buffer is composed of the following components: pH6.8,250mM Tris-HCl 10% (W/V), SDS 0.5% (W/V), tetrabromophenol sulfonphthalein 50% (V/V), glycerine 5% (W/V), all the other are beta-mercaptoethanol;

LB liquid nutrient medium: sodium-chlor 10g/L, yeast extract 5g/L, Tryptones 10g/L pH value to 7.4 ± 0.2;

LB solid medium: it is 15g/L that LB liquid nutrient medium adds agarose to final concentration; Getting about 10mL after autoclaving successively pours in sterile petri dish, closes lid mark and preserve after cooling;

1000 × Ampicillin Trihydrate sodium solution (Amp+): preparation Ampicillin Trihydrate sodium solution 100mg/mL, 0.22 μm of sterile filter, the used time adds by 1:1000;

1mol/L IPTG: preparation IPTG 238.3g/L, 0.22 μ sterile filter;

LB solid medium (Amp+): LB solid medium, is cooled to about 40 DEG C to add Amp+ to final concentration 100 μ g/mL, gets about 10mL successively and pour in sterile petri dish after autoclaving, close lid mark and preserve after cooling;

10mM PBS: get KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o 2.9g, NaCl 8.0g, KCl 0.2g, adds water to 1000mL, and now pH value is 7.4;

20mM PB damping fluid: get KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o 2.9g, KCl 0.2g, adds water to 1000mL, and now pH value is 7.0;

The vaccine diluent of pH6.0: be made up of Histidine, NaCl and PLURONICS F87, three components is water-soluble altogether; The final concentration of each component is Histidine 10mM, NaCl massfraction is 0.9%, and PLURONICS F87 massfraction is 0.01%.

Embodiment 1 expresses the structure of the pGEX-6P-2-A1S_0851/XL-1blue recombinant bacterial strain of A1S_0851 albumen

1, the Design and synthesis of A1S_0851 aligning primer

According to A1S_0851 gene (No. ID, Genbank: 4916977) information, A1S_0851 gene nucleotide series total length 975bp, totally 324 amino acid of the Acinetobacter bauamnnii that Gene Bank includes.Use SignalP 4.1Server to carry out signal peptide prediction, 1-30 amino acid is signal peptide.Adopt Primer Premier5.0 software as follows to the conservative region design primer removing signal peptide, by this primer amplification Acinetobacter bauamnnii genome, obtaining A1S_0851 mrna length is 894bp, nucleotide sequence is shown in SEQ ID NO.1, albumen is 299 amino acid, and aminoacid sequence is shown in SEQ ID NO.2.

PCR cloning primer:

PA1S0851B1：5’-cgcgg atccg atatt gactc agtcc gtgcg-3’(SEQ ID NO.3)；

PA1S0851N2：5’-ttatg cggcc gctta aattg atgtc ttttc ggc-3’(SEQ ID NO.4)。

2, the extraction of genomic dna

Picking Acinetobacter bauamnnii conservation liquid, is inoculated in antibiotic-free respectively and containing in the LB flat board of Ampicillin Trihydrate, cultivate 12h for 37 DEG C, the flat board of antibiotic-free grows a large amount of bacterium colony, and the flat board containing Ampicillin Trihydrate grows without bacterium colony.Single colony inoculation on picking non-resistant flat board is in the LB substratum of 10mL, and in 37 DEG C, 80rpm shaking culture 12h, finally adopts bacterial genomes to extract test kit, extract bacterial genomes according to the explanation of test kit specification sheets.

3, pcr amplification Acinetobacter bauamnnii A1S_0851 gene

PCR system: template is ATCC17978 genomic dna 2 μ L, primer PA1S0851B1 (10 μMs) 1 μ L, PA1S0851N2 (10 μMs) 1 μ L, primeSTAR HS archaeal dna polymerase 0.25 μ L, dNTP 4 μ L, 5 × Buffer 10 μ L, sterilizing distilled water 31.75 μ L, cumulative volume 50 μ L.Pcr amplification reaction condition: 94 DEG C of denaturation 5min; 94 DEG C of sex change 10s; 60 DEG C of annealing, 30s; 72 DEG C extend 1.5min, 30 circulations; 72 DEG C extend 10min completely.Use the agarose gel electrophoresis of 1% to detect pcr amplification result as Fig. 1 after completion of the reaction, pcr amplification goes out the gene fragment that size is about 900bp, conforms to expection.

4, gel reclaims A1S_0851PCR product

The recovery sepharose of preparation 1%: take 1.0g agarose and move in triangular flask, add 1 × TAE nucleic acid electrophoresis buffered soln 100mL, put into microwave oven after shaking up gently and heat; After agarose dissolves completely, add nucleic acid dye EB 8 μ L, shake up gently, solution is poured into glue groove, remove bubble with rifle head, plug comb, can loading electrophoresis after gelling is solid.

Goal gene applied sample amount 50 μ L, 120V electrophoresis 20min isolation of DNA fragments.Electrophoresis is complete, cuts object band and load in 1.5mL centrifuge tube under uv irradiating condition.Reclaim test kit with DNA gel and carry out DNA recovery, operation steps by specification carries out, specific as follows:

DNA reclaims: add 400 μ L PB solution, hatch 7min and melt completely to glue, DNA gel is melted thing in adsorption column, the centrifugal 1min of room temperature 10000g in 57 DEG C; Abandon filtrate, adsorption column is put back in collection tube and add 300 μ L PB solution in post, the centrifugal 1min of room temperature 10000g; Abandon filtrate, adsorption column is put back in collection tube and add the diluted Wash Buffer of 700 μ L dehydrated alcohols, the centrifugal 1min of room temperature 12000g; Abandon filtrate, again add the Wash Buffer that 700 μ L dehydrated alcohols are diluted, the centrifugal 1min of room temperature 12000g; Abandon filtrate, the centrifugal 2min of blank pipe 13000g; Adsorption column is put into clean 1.5mL centrifuge tube, room temperature places 5min, then adds 30 μ L aqua sterilisas, and the centrifugal 2min eluted dna of 13000g after placement 2min, collects elutriant, be placed in-20 DEG C of preservations.The agarose gel electrophoresis of 1% detects the A1S_0851PCR product of purifying.

5, the extraction of pGEX-6p-2 plasmid

It is dull and stereotyped that pGEX-6P-2/XL1blue intestinal bacteria are inoculated in Amp LB by trilinear method, is placed in 37 DEG C and cultivates 12h.Single colony inoculation on picking flat board containing in the LB nutrient solution of Ampicillin Trihydrate, is placed in 37 DEG C, 250rpm shaking culture 8h in 10mL.Get bacterium liquid with EP pipe, 1.5mL/ props up, for extracting plasmid.Carry out pGEX-6p-2 plasmid extraction with OMEGA plasmid extraction kit, operation steps by specification carries out, specific as follows:

Plasmid extraction: get the centrifugal 2min of bacterium liquid 12000g, abandoning supernatant collects thalline, 250 μ L SolutionI (having added RNase A) are added and resuspended in thalline, then 250 μ L Solution II are added, put upside down mixing, then add 350 μ L Solution III, put upside down mixing, room temperature places the centrifugal 10min of 5min, 13000g; After centrifugal, supernatant liquor is proceeded in HiBind DNA MiniColumns collection tube, the centrifugal 1min of room temperature 10000g; Discard filtrate in collection tube, add 500 μ L Buffer HB, the centrifugal 1min of room temperature 10000g; Abandon filtrate, add the Wash Buffer of 700 μ L containing ethanol, the centrifugal 1min of room temperature 13000g; Repeat to wash with the Wash Buffer containing ethanol; Filtrate is abandoned, the centrifugal 2min of room temperature 13000g after washing.Pillar is put into 1.5mL sterilizing EP pipe, place 5min, add the EB of 80 μ L, 65 DEG C of preheatings on filter membrane in post after ethanol is evaporated completely for 65 DEG C, room temperature places the centrifugal 2min wash-out of 3min, 13000g, collects elutriant, and-80 DEG C of preservation plasmids are for subsequent use.The PGEX-6 plasmid of the agarose gel electrophoresis Detection and Extraction of 1%.

6, the screening of the structure of recombined pronucleus expression plasmid and recombinant bacterial strain, qualification

(1) double digestion and recovery: respectively BamH I and Not I double digestion are carried out to pGEX-6P-2 plasmid and A1S_0851PCR purified product, endonuclease reaction system is: BamH I 2 μ L, Not I 2 μ L, 10 × K Buffer 2 μ L, 0.1% (w/w) BSA4 μ L, plasmid or PCR purified product 30 μ L, cumulative volume 40 μ L; Under 37 DEG C of conditions, enzyme cuts 3h, and enzyme cuts the digestion products that rear glue reclaims PGEX-6P-2 plasmid and A1S_0851.

(2) connect: after recovery, PGEX-6P-2 plasmid is connected with the digestion products of A1S_0851.Ligation system is: Solution I 5 μ L, and A1S_0851 enzyme cuts back to close product 4.5 μ L, and PGEX-6P-2 enzyme cuts back to close product 0.5 μ L, cumulative volume 10 μ L.Under 16 DEG C of conditions, 2h is connected by after each component mixing.

(3) transform: get connection product 10 μ L and join in 100 μ L XL1-blue competence, after ice bath 45min, 42 DEG C of thermal shock 90s, and then rapid ice bath 2min; Add 600uL LB blank cultures after ice bath, mix and be placed in 37 DEG C, 220rpm shaking culture 1h; In the centrifugal 5min. of 4000rpm after cultivation, discard 200 μ L supernatant liquors, resuspended thalline, and get 200 μ L and coat on ampicillin resistant LB flat board, flat-plate inverted is placed in 37 DEG C of incubators and cultivates 12h.

Competent preparation (CaCl ₂method): with the mono-bacterium colony of XL1blue that transfering loop picking is newly recovered on LB flat board, be inoculated in 10mL LB liquid nutrient medium, 37 DEG C, 100rpm shaking culture 12h; Getting the bacterium liquid 0.8mL after cultivation joins in 50mLLB liquid nutrient medium, in 37 DEG C, and 220rpm shaking culture 2-3h, measuring OD600 is 0.4 ~ 0.5; By after bacterium liquid ice-water bath 10min in 4 DEG C, the centrifugal 7min of 1600g, abandoning supernatant collecting cell; 10mL is added, the CaCl of 0.1mol/L in cell ₂solution, re-suspended cell; 4 DEG C, the centrifugal 5min of 1100g, abandoning supernatant collecting cell.2mL is added again, the CaCl of 0.1mol/L in cell ₂solution, re-suspended cell, divides the competent cell prepared by 100 μ L and is filled in 1.5mL EP pipe, be stored in-80 DEG C of refrigerators.

(4) screening of the positive recombinant plasmid of pGEX-6p-2/A1S_0851 and recombinant bacterial strain, qualification: picking transformation plate is separated 4 good bacterium colonies, be inoculated in 10mL ampicillin resistant LB substratum, 37 DEG C, 100rpm shaking culture 12h; Get bacterium liquid plasmid extraction kit and carry out plasmid extraction, then 37 DEG C of enzymes cut 2h.Double digestion reaction system is: BamH I 0.5 μ L, Not I 0.5 μ L, 10 × K Buffer 0.5 μ L, 0.1%BSA 1 μ L, recombinant plasmid 8 μ L, cumulative volume 12.5 μ L.The agarose gel electrophoresis detected result of 1%, the results are shown in Figure 2.As shown in Figure 2, the mono-clonal bacterium corresponding to No. 3 swimming lanes is pGEX-6p-2/A1S_0851 recombinant vectors engineering bacteria.The plasmid of positive colony bacterial strain is sent to Hua Da gene sequencing, the consensus nucleic acid sequence that the A1S_0851 gene order in the expression plasmid constructed by sequencing result display and GenBank announce.By this recombinant bacterium called after pGEX-6P-2-A1S_0851/XL-1blue, prepare for follow-up vaccine.

The abduction delivering of embodiment 2 recombinant bacterial strain pGEX-6P-2-A1S_0851/XL-1blue

The target recombinant strain 20 μ L that Example 1 obtains adds in the LB substratum of 10mL ampicillin resistant, cultivates 12h for 37 DEG C; Getting 1mL bacterium liquid joins in the LB substratum of 19mL ampicillin resistant, 37 DEG C, 250rpm shaking culture 3-4h; Measure bacterium liquid OD ₆₀₀when being about 1.0, in bacterium liquid, adding 1M IPTG 4 μ L, be placed in shaking table abduction delivering, rotating speed 220rpm, 30 DEG C of abduction delivering 3h, 25 DEG C of abduction delivering 5h, 16 DEG C of abduction delivering 12h; After inducing, bacterium liquid takes out, with the centrifugal 2min of 12000rpm, with the resuspended thalline of 1mL PBS after abandoning supernatant, and ultrasonic degradation 2min (15% power, super 5s, stops 3s); In 4 DEG C after ultrasonic, the centrifugal 15min of 14000rpm, separation of supernatant and precipitation; SDS-PAGE electrophoresis is carried out by after supernatant liquor and precipitation process.

Supernatant liquor process: get Glutathione Sepharose 4B 20 μ L, after washing 3 times with PBS, ready supernatant liquor is added in Glutathione Sepharose 4B, in conjunction with 1-2h under 20 ~ 25 DEG C of conditions, then the centrifugal 3min of 14000rpm under 4 DEG C of conditions, collects filler after combining.After using PBS to wash a filler, the Glutathione Sepharose 4B after combining adds 20uL PBS, 5uL 5 × protein sample-loading buffer, heat treated.

SDS-PAGE prepares:

10mL, 10% separation gel: distilled water 4.0mL, 30% acrylamide soln 3.3mL, the Tris-HCl2.5ml of 1.5M, pH8.8,10%SDS 0.1mL, 10% ammonium persulphate 0.1mL, TEMED 0.004mL;

6mL, 5% concentrated glue: distilled water 4.1mL, 30% acrylamide soln 1.0mL, the Tris-HCl0.75mL of 1.0M, pH6.8,10%SDS 0.06mL, 10% ammonium persulphate 0.06mL, TEMED 0.006mL.

Glue: 5% concentrated glue is poured in glue version, then add distilled water glue laminated is put down, 20 ~ 25 DEG C of conditions place 30min makes gelling solid; After gelling is solid, the dry doubling that fallen by the distilled water on upper strata pours into separation gel, plugs comb immediately, and 20 ~ 25 DEG C of conditions place 30min, and that separation gel is solidified is rear for subsequent use.

Electrophoresis: the upper cleer and peaceful precipitation handled well is got respectively 20 μ L loadings, when carrying out SDS-PAGE electrophoresis, first 80V electrophoresis 30min, then be adjusted to 180V, electrophoresis 1 ~ 2h.Electrophoresis is complete, is taken out by glue, is placed in coomassie brilliant blue staining liquid vibration dyeing, then after being placed in destainer vibration decolouring, observations under gel imaging system.

Result: under 30 DEG C, 25 DEG C, 16 DEG C conditions, abduction delivering is carried out to PGEX-6P-2-A1S_0851/XL-1 respectively with IPTG, induction the results are shown in Figure 3, PGEX-6P-2-A1S_0851/XL-1blue all can give expression under 30 DEG C, 25 DEG C, 16 DEG C conditions molecular size range be about 55kDa containing the recombinant protein A 1S_0851 of GST label.

The fermentation of embodiment 3 recombinant bacterial strain pGEX-6P-2-A1S_0851/XL-1blue

Prepared by 1, bacterial strain recovery, activation and kind daughter bacteria

(1) recovery of bacterial strain: be inoculated in by pGEX-6P-2-A1S_0851/XL-1blue bacterium in the LB solid medium containing Ampicillin Trihydrate, is inverted into 37 DEG C of cultivation 16h in electro-heating standing-temperature cultivator after carrying out mark.

(2) activation of daughter bacteria is planted: the uniform single colony inoculation of picking form, in containing in the LB liquid nutrient medium of 10mL Ampicillin Trihydrate, is put into constant-temperature table 37 DEG C and cultivated 8h, horizontal rotating speed 220rpm after mark.

(3) preparation of daughter bacteria is planted: get activation bacterium and be inoculated in containing in the LB substratum of Ampicillin Trihydrate of 1L in the ratio of 1:2000, put into large-scale isothermal vibration shaking table 37 DEG C after mark and cultivate 8h, horizontal rotating speed 200rpm.

(4) plant daughter bacteria to detect: sampling 2mL, OD surveyed by ultraviolet spectrophotometer ₆₀₀, and carry out sediments microscope inspection.

(5) the good kind daughter bacteria liquid of growth conditions is got as ferment-seeded bacterium.

2, strain fermentation

The instruments such as feed supplement bottle, soda acid bottle, funnel are carried out autoclaving, then fermentor tank pH electrode is calibrated, by TB basic medium (potassium primary phosphate 2.3g/L, the disodium hydrogen phosphate 13g/L of pH 7.4, glycerine 10mL/L, yeast extract 24g/L, Tryptones 12g/L, magnesium sulfate 1g/L) proceed in fermentor tank, drive agitator, add defoamer to non-foam, 121 DEG C of sterilizing 30min, when 121 DEG C, calibrate PO ₂electrode 0%.After autoclaving EP (end of program), initial parameter is set, is specially leavening temperature 37 DEG C, rotating speed 300rpm, pH value 7.4, ventilation flow rate 10L/min, calibration PO ₂electrode 100%.Initial parameter is fermented after setting completed, and 1L is activated bacterium and add 15L fermentor tank and ferment, along with the increase of cell density, the rising of oxygen consumption, when after turn up 800rpm, change pure oxygen and dissolved oxygen control association into, namely pure oxygen controller is servo controller, PO ₂all the time control 45%.Monitoring pH, PO ₂value, when two values rise all fast, instruction glycerine runs out of, and starts induction, and adds feed supplement (i.e. glycerine (100ml/L)) to make pH value to steady state.IPTG concentration is 0.2mmol/L, induction time 4h, and inducing temperature is 30 DEG C, in fermenting process, continuous sampling and measuring bacteria concentration, and result is as shown in table 1:

Different time bacterial concentration in table 1 fermenting process

Sample time	OD 600
		Induction 0h	41.1
Induction 1h	42.75
		Induction 3h	45.65
Induction 4h	45.9

Induction 4h secondary fermentation terminates, and as shown in Figure 4, as shown in Figure 4, whole growth curve standard of comparison, early stage, bacterial growth was very fast, and during later stage induction, curve is more steady for growth curve of bacteria.Final acquisition bacteria concentration OD after fermentation ends ₆₀₀reach 45.9, unit bacterium weight in wet base 35g/L.

3, fermentation purposes protein expression qualification

(1) sample pretreatment: by sample centrifugal and with PBS dilution after carrying out ultrasonic bacteria breaking, ultrasound condition is: power 20%, 10min, 9s is opened, and 8s stops; The centrifugal 10min of 12000rpm after broken, gets supernatant liquor 1mL and joins in the 60 μ L Glutathione Sepharose 4B handled well, put vertical mixed instrument room temperature in conjunction with 1h; In conjunction with after in the centrifugal 1min of 14000rpm, abandon supernatant liquor, PBS washes twice, centrifugal abandon supernatant liquor after add 18 μ L PBS and 12 μ L 5 × sample-loading buffers, place 5min under 100 DEG C of conditions; After the process of Glutathione Sepharose 4B associated proteins terminates, carry out 12%SDS-PAGE electrophoresis;

(2) 12%SDS-PAGE electrophoresis: sample applied sample amount 10 μ L, albumen Marker applied sample amount 5 μ L; Deposition condition is: concentrated glue voltage 60V, separation gel voltage 120V; After electrophoresis terminates, with the dyeing of coomassie brilliant blue staining liquid, destainer decolours, gel imaging instrument scanning protein electrophoresis figure (Fig. 5).

Result: as shown in Figure 5, unit target protein expression amount prolongation in time obviously increases, and 4h expression amount is the highest.PGEX-6P-2-A1S_0851/XL-1blue engineering bacterium fermentation technique meets expected results completely, and zymotechnique of the present invention is applicable to the application of large-scale industrial production.

The preparation of embodiment 4 recombinant protein A 1S_0851 and purifying

1, broken bacterium prepares supernatant

Bacterium liquid collected by centrifugation thalline embodiment 3 fermented, gets thalline 200 ~ 500g and 10mmol/L, and the ratio mixing of the PB damping fluid of pH7.0 volume ratio 1:10 by weight suspends, 4 DEG C of precoolings;

High-pressure homogenization: use distilled water flushing high pressure homogenizer (high pressure homogenizer AH100B, ATS company) pipeline, cold cycle system open be chilled in advance 1-4 DEG C for subsequent use.The suspension bacteria liquid of precooling is added high pressure homogenizer, and pressure maintains 60-80Mpa and breaks bacterium 3-5 time, gets brokenly bacterium liquid smear and carries out violet staining, and under oily mirror, under each visual field, not broken bacterium is less than 2 and is considered as brokenly bacterium completely (broken bacterium rate is greater than 90%).

High speed centrifugation: the liquid after broken bacterium loads centrifugal barrel (Beckman, the U.S.), 4 DEG C, the centrifugal 15-30min of 10,000-15,000g, collects supernatant.

2, GST-sepharose 4B affinitive layer purification

Add 200mL GST filler to often going up clearly, in conjunction with more than 4h when 20 ~ 25 DEG C, cohesive process adopts the method for vertical rotary or stirring to promote the combination of target protein and GST filler.PBS is adopted by the GST filler of above-mentioned combining target albumen to wash 5 volumes to remove the foreign protein be not combined with GST filler.Then the Prescission protease enzyme (PP enzyme) of 20mL is added by every 100mLGST filler, suction filtration after enzyme cuts 6 hours under 20 ~ 25 DEG C of conditions also collects filtrate, obtain the target protein after enzyme excision GST label, carry out 10%SDS-PAGE analysis, result as shown in Figure 6.Shown in Fig. 6 is the purification result figure of recombinant protein A 1S_0851 through GST sepharose 4B affinity chromatography, and result shows, and target protein purity is more than 90%, and target protein molecular weight, at about 33kDa, is consistent with expection size.

3, anion-exchange chromatography purifying

The theoretical iso-electric point of recombinant protein A 1S_0851 is 4.91.Therefore, the anion-exchange chromatography filler (strong anion exchange) first having selected GE company common carry out consummateization, re-uses G 25 to the displacement damping fluid after consummateization, finally uses QHP to remove intracellular toxin.Concrete steps are as follows successively:

(1) AKTA-Avant25 liquid chromatographic system (GE), QHP filler proceeds purifying to the albumen after GST-sepharose 4B affinitive layer purification, post specification is (Φ) 2.6cm × (H) 20cm, dress column volume 50mL, damping fluid is: buffer A: the PBS of 20mM, pH 7.0; The buffer B of pH 7.0: 20mM PB+1M NaCl; Loading flow velocity 15mL/min, elution flow rate 15mL/min; With 0-100% buffer B wash-out 5 column volumes (in each elution program, the damping fluid of all the other shares is corresponding buffer A) during wash-out.After wash-out, target protein is respectively walked elution samples and carry out SDS-PAGE purity check, evaluate purification effect.

Fig. 7 is the QHP tomographic map of recombinant protein A 1S_0851, and Fig. 8 is the electrophorogram of recombinant protein A 1S_0851 after QHP chromatography, and can find out, purity reaches more than 95%.

(2) AKTA-Avant25 liquid chromatographic system (GE), G25 chromatographic stuffing displacement damping fluid, post specification is (Φ) 5.0cm × (H) 30cm, dress column volume 500mL, damping fluid is: the damping fluid C:10mM His+0.3M NaCl of pH 6.0, loading flow velocity 20mL/min, elution flow rate 20mL/min.

(3) AKTA-Avant25 liquid chromatographic system (GE Healthcare) QHP chromatography removes intracellular toxin, post specification (Φ) 1.6cm × (H) 2.5cm, dress column volume 5mL, damping fluid is: the damping fluid C:10mM His+0.3M NaCl of pH 6.0, damping fluid D:1M NaOH; Loading flow velocity 5mL/min, elution flow rate 5mL/min; Elution program is: first to sterilize 5 column volumes with damping fluid D incumbent firms, after placing half an hour, is 6.0 by damping fluid C equilibrium system to pH, then loading.Finally collect and penetrate peak and target protein peak.

The protein peak of collection is carried out SDS-PAGE electrophoresis, and detect purity of protein, result such as Fig. 9 shows, and can find out, by displacement damping fluid and after removing intracellular toxin, purity of protein still remains on more than 95%.

The preparation of embodiment 5 recombinant protein A 1S_0851 vaccine

Acinetobacter bauamnnii restructuring A1S_0851 vaccine formulation: measure aluminum hydroxide adjuvant (30mg/mL) 90 μ L, with the vaccine diluent 10 μ L of pH6.0, fully mix, obtain solution A; By the vaccine diluent of pH6.0, the recombinant protein A 1S_0851 after purifying obtained for embodiment 4 is diluted to 30 μ g/100 μ L, obtains solution B; After getting isopyknic solution A vertical suspendible adsorbing 1 hour under 4 DEG C of conditions with solution B and get final product.Obtaining aluminum hydroxide adjuvant final concentration in vaccine is 13.5mg/mL, restructuring A1S_0851 final concentration of protein 150 μ g/mL.

The foundation of embodiment 6 Acinetobacter bauamnnii pyemia model

Bacterium solution preparation: Acinetobacter bauamnnii type strain ATCC-17978 trilinear method be inoculated on LB solid culture flat board, cultivates 12h in 37 DEG C of incubators after carrying out mark.After cultivation, single colony inoculation of picking growth is in the shaking flask that 20mL LB substratum is housed, and put into temperature control shaking table after carrying out mark, at 37 DEG C, rotating speed is cultivate 6h under 220rpm condition.After cultivation, bacterium liquid is proceeded to 5000g in 50mL centrifuge tube, 4 DEG C of centrifugal 5min, and with the resuspended washing of physiological saline 2 times.Wash complete, 5000g, 4 DEG C of centrifugal 5min, it is resuspended then to add stroke-physiological saline solution.Getting resuspended rear bacterium liquid 50 μ L adds in the EP pipe filling 950 μ L stroke-physiological saline solution, measures OD after vortex mixing ₆₀₀, calculate total bacterium amount.

Animal Model: getting table 2 each dosage bacterium liquid by 6 ~ 8 week age of abdominal injection, body weight is the BALB/C mice of 18 ~ 20g, every mouse 200 μ L often organizes 5 mouse.Control group physiological saline replaces.Bacterium attacks the rear observed and recorded dead mouse situation of poison, continuous observation 10 days.Observation period terminates rear residue animal with CO ₂inhalation euthanasia.Adopt Kaplan-Meier survival analysis method to carry out statistical procedures to the survival time of each group of mouse and survival rate, between more different bacterial concentration, whether the existence of mouse exists significant difference, the results are shown in Table 2 and Figure 10.

Table 2 respectively organizes mouse death rate

Above result has carried out the evaluation of animal model for the survival rate of mouse, and the pathogenetic research that successful development and Acinetobacter bauamnnii for Acinetobacter bauamnnii subunit vaccine and many subunits fusion bacterin infect is laid a good foundation.

Embodiment 7 is recombinated the evaluation of A1S_0851 proteantigen immunity

The vaccine on mouse that Example 5 is made carries out immunity, and immunization is BALB/C mice in female 6 week age, and experiment is divided into following three groups: vaccine diluent group, aluminum hydroxide adjuvant control group, recombinant protein A 1S_0851 immune group, often organizes 30.Vaccine diluent is identical with embodiment 5 with aluminum hydroxide adjuvant, and recombinant protein A 1S_0851 system is identical with embodiment 5 with concentration.Respectively at 0 day, 14 days and 21 days quadriceps muscle of thigh intramuscular injection immune mouses, per injection amount total amount 200 μ L, antigenic content is a 30 μ g/ mouse.14th day tail vein blood after immunity, vaccine specific IgG humoral response level after ELISA detection mouse immune.

ELISA detection method is as follows:

(1) adopt recombinant protein A 1S_0851 bag by elisa plate, protein concentration is 10 μ g/mL, and every hole adds 100 μ L, under 4 DEG C of conditions, place 12h;

(2) the 200 closed 12h in 4 DEG C, μ L/ hole are pressed with 5%BSA confining liquid after PBST washing;

(3) after PBST washing, recombinant protein A 1S_0851 immune group serum antibody diluent is diluted, initial dilution 1:500; Compare with adjuvant immunity group and vaccine diluent immune serum simultaneously, dilute by 1:500; Plate is positioned over 37 DEG C of incubation 40min after shaking up;

(4) after hatching end, PBST washs, and doubly diluted by the two anti-igg antibody diluent 1:5000 that HRP marks after washing, then get two anti-igg after dilution and add by 100 μ L/ holes, vibration shakes up, 37 DEG C of incubation 40min;

(5) PBST adds TMB nitrite ion by 100 μ L/ holes after washing plate 4 times, in dark place colour developing 5 ~ 10min;

(6) colour developing adds 2mol/L H by 50 μ L/ holes after terminating ₂sO ₄termination reaction, microplate reader 490nm measures each hole OD value.

With feminine gender value >2.1 corresponding to sample/sample for positive criteria, when diluting with serum 1:500, detected result is for the positive, calculating antibody Conversion rate, obtain the most high dilution of each strain specific antibodies in each serum simultaneously, the geometric mean titer (GMT) of each experimental group antibody is adopted to make histogram, group difference compares employing t inspection, and concrete operations are carried out in SPSS 13.0 software.

Result: after recombinant protein A 1S_0851 immunity the 14th day, by antigen-specific IgG humoral response level after ELISA detection mouse immune, result as shown in figure 11, recombinant protein A 1S_0851 immune group geometric mean titer is 42224, aluminum hydroxide adjuvant control group titre is 500, and vaccine dilution group titre is 200.Through statistical analysis, recombinant protein A 1S_0851 immune group antibody titers, compared with aluminum hydroxide adjuvant control group and vaccine diluent control group titre, all has and significantly increases (P<0.01).

Embodiment 8 recombinant protein A 1S_0851 antigen immune protected effect is evaluated

Carry out immune balb/c mice by method described in embodiment 7, often organize 30, within the 14th day after final immunization, adopt lethal dose Acinetobacter bauamnnii ATCC-17978 viable bacteria abdominal injection to carry out challenge viral dosage, every BALB/C mice injection bacterium liquid measure is 4 × 10 ⁸cFU, observes 10 days, adds up the survival rate of each group of mouse.

Attack malicious Protection result: as shown in Table 3, compared with vaccine diluent and Tai-Ace S 150 vehicle control group, after A1S-0851 vaccine immunity, have good protection of animal effect.

Malicious protective capability is attacked after table 3. recombinant protein A 1S_0851 immune mouse

What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims (9)

1. Acinetobacter bauamnnii membranin A1S_0851 is as the application of antigen, it is characterized in that, the aminoacid sequence of described membranin A1S_0851 is as shown in SEQ ID NO.2.

2. Acinetobacter bauamnnii recombinant protein A 1S_0851 is preparing the application in vaccine, it is characterized in that, the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

3. the Acinetobacter bauamnnii vaccine prepared by Acinetobacter bauamnnii recombinant protein A 1S_0851, is characterized in that, the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

4. vaccine according to claim 3, is characterized in that, described vaccine also comprises adjuvant, and described adjuvant is the aluminium hydroxide of final concentration 13.5mg/mL.

5. the vaccine according to claim 3 or 4, is characterized in that, described vaccine also comprises diluent, and described diluent pH value is 6.0, by concentration be the Histidine of 10mM, the PLURONICS F87 of the NaCl of massfraction 0.9% and massfraction 0.01% forms.

6. the preparation method of vaccine described in claim 5, is characterized in that, comprises the steps:

(1) be the aluminum hydroxide adjuvant of 30mg/mL by the vaccine diluent concentration of ordinary dissolution that pH value is 6.0, obtain solution A; The volume ratio of described aluminum hydroxide adjuvant and vaccine diluent is 9:1;

(2) with step (1) described vaccine diluent dilution recombinant protein A 1S_0851, the solution B that protein concentration is 0.3 μ g/ μ L is obtained;

(3) get isopyknic solution A and solution B is adsorbed under 4 ~ 32 DEG C of conditions, obtain vaccine.

7. the application of vaccine described in any one of claim 3-5 in the medicine that preparation prevents or treatment Acinetobacter bauamnnii infects.

8. the antibody that obtains as antigen-immunized animal of Acinetobacter bauamnnii recombinant protein A 1S_0851, it is characterized in that, the aminoacid sequence of described recombinant protein A 1S_0851 is as shown in SEQ ID NO.2.

9. antibody according to claim 8, is characterized in that, described antibody is the IgG antibody that immune mouse or rabbit obtain.