CN109486736A

CN109486736A - The purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505

Info

Publication number: CN109486736A
Application number: CN201811366387.7A
Authority: CN
Inventors: 郭刚; 卢文根; 周璐; 杨念; 张娇娇
Original assignee: Chongqing Alibei Biotechnology Co Ltd
Current assignee: Chongqing Alibei Biotechnology Co Ltd
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2019-03-19
Anticipated expiration: 2038-11-16
Also published as: CN109486736B

Abstract

The invention belongs to biopharmaceutical technologies, disclose the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 a kind of, the DNA sequence dna for encoding PA5505 protein active function fragment is cloned on pGEX-6p-2 carrier by technique for gene engineering, Escherichia coli recombination engineering pGEX-6p-2-PA5505/XL-1 blue is constructed, PA5505 albumen is obtained by inducing expression.The present invention carries out height by the genetic engineering bacterium to expression PA5505 and crushes the technologies such as bacterium, GST affinity chromatography, the digestion of PP enzyme, SP HP chromatography, Q HP chromatography, obtains the vaccine candidate antigen PA5505 of high-purity.The invention purifying process is simple and direct, is easy amplification, is reproducible, obtained target protein purity is high, and animal experiment proves can the effective stimulus body higher humoral immune response of generation and good immanoprotection action.

Description

The purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505

Technical field

The invention belongs to biopharmaceutical technology more particularly to a kind of pseudomonas aeruginosa gene engineered vaccine candidate are anti- The purification process of former PA5505.

Background technique

PA is clinically most commonly seen one of conditioned pathogen, is ranked first in the gram-negative bacteria of clinical infection, mesh Before have become one of the highest pathogen of the infection rates such as the global ward ICU, burn, War injury (Horino T etc., Intern Med, 2012).PA infection can occur to be common in burn or wound site, middle ear, cornea, urine in any position of human body and tissue Road and respiratory tract can also cause endocarditis, gastroenteritis, pyothorax even septicemia.Its infection can have unicity, also there is mixing Property, mechanism is complicated.In recent years, the disease incidence of PA nosocomial infection especially pulmonary infection is continuously increased, and severe infections are especially exhaled Suction machine pneumonia patient and the septic patient death rate are high.Clinically mainly PA is infected using antibiotic and is carried out Treatment, but due to abuse of antibiotics etc., PA drug resistance is increasingly severe, and antibody-resistant bacterium continues to bring out, and brings to clinical treatment Great challenge.CHINET data shows that PA in 2013 is respectively to the annual resistant rate of Imipenem and Meropenem 29.1% and 27.1%, and HAP detection sample is up to 70.7% and 48.8% to the resistant rate of the two respectively.Based on this, vaccine It increasingly attracts people's attention with non-antibiotic treatments means such as acology antibody.PA5505 is to pass through reversed epidemic disease this room early period One of the Immunodominant Antigenic that Miao Xue technology is screened from PA full-length genome, shows good exempt from animal experiment Epidemic focus and protecting effect, can effective stimulus body generate higher humoral immune response, and lung tissue bacterium is effectively reduced Field planting, is conducive to the prevention and treatment of charrin disease, can be used as the candidate antigens of PA vaccine research and development.

Problem of the existing technology is: PA5505 is to assume albumen, and structure and function is unknown, does not also there is any document pair It is reported, more the research without being directed to the recombinant protein purification method.Solve the difficulty and meaning of above-mentioned technical problem: PA5505 is a completely new albumen, and physicochemical property is unclear, and mature purifying process process is not referred to, and needs basis The purifying process of the Experience albumen.Technology through the invention, realizes and isolates and purifies to PA5505, has obtained high-purity The destination protein of degree, so that furtheing investigate the physicochemical property of the albumen, evaluating its immunity protection function and mechanism becomes possibility, And it lays a good foundation for the foundation of protein production technique.

Summary of the invention

In view of the problems of the existing technology, it is anti-that the present invention provides a kind of pseudomonas aeruginosa gene engineered vaccine candidates The purification process of former PA5505.

The invention is realized in this way a kind of recombination engineering pGEX-6p-2-PA5505/XL-1 blue, the recombination Engineering bacteria pGEX-6p-2-PA5505/XL-1 blue expresses the nucleic acid sequence of antigen PA5505 as shown in SEQ ID NO:1, ammonia Base acid sequence is as shown in SEQ ID NO:2.

Another object of the present invention is to provide a kind of application recombination engineering pGEX-6p-2-PA5505/XL-1 The purification process of the pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 of blue, the pseudomonas aeruginosa gene work The purification process of engineered vaccine candidate antigens PA5505 includes: the genetic engineering bacterium for collecting expression PA5505；According to height crush bacterium, from The heart, GST affinity purification；The sequence combination of SP HP chromatographic purifying, Q HP chromatographic purifying purifies the antigen of preparation.

Further, the purification process of the pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 specifically includes:

1) height crushes bacterium

The genetic engineering bacterium for collecting expression PA5505 is mixed with the PBS buffer solution that pH is 7.0-7.5 and is suspended, adopted after pre-cooling Bacterium is broken with high-pressure homogenization, high speed centrifugation collects supernatant；

2) GST affinity purification

GST affinity chromatography filler carries out preliminary purification, after eluting foreign protein using PBS, uses Prescission Protease enzyme digestion elutes target protein, and digestion and elution buffer are A liquid；

3) SP HP chromatographic purifying

The target protein collected through step 2), with loading after B liquid balance tomographic system and SP HP chromatographic column, the linear ladder of C Degree elution；

4) Q HP chromatographic purifying

The mark albumen that will be purified through step 3), D liquid balance loading after tomographic system and Q HP chromatographic column, remove the non-mesh of trace The impurity such as albumen, endotoxin are marked, target protein is obtained；

Wherein, A liquid is the 20mM Na of pH value 7.0-7.5₂HPO₄-NaH₂PO₄Buffer solution, B liquid are the 10mM of pH6.0 Histidine buffer solution, C liquid are 10mM Histidine, 1M the NaCl buffer solution of pH6.0, and D liquid is the 10mM of pH6.0 Histidine, 0.9%NaCl buffer solution, PBS buffer solution are that 150mM NaCl is added in A liquid.

Further, high-pressure homogenization described in the step 1) breaks bacterium and uses 60-80MPa pressure, and high speed centrifugation obtains after broken bacterium Take bacteria break supernatant.

Further, the filler that the GST affinity chromatography of the step 2) uses be Glutathione Sepharose 4B or Glutathione Sepharose 4FF or Glutathione Sepharose HP.

Further, the Prescission Protease enzyme of the step 2) has GST label.

Further, the filler of the SP HP chromatographic column of the step 3) is SP Sepharose HP or SP Sepharose FF or Capto SP.

Further, the filler of the Q HP chromatographic column of the step 4) be Q Sepharose HP or Q Sepharose FF or Capto Q。

In conclusion advantages of the present invention and good effect are as follows: obtain PA by Recombinant organism expression Vaccine candidate antigen PA5505 purifies to have obtained the destination protein of high-purity using the process flow in the present invention.The recombination Albumen proves through animal experiment, can effective stimulus body generate higher humoral immune response and good immanoprotection action, Be conducive to prevention, the diagnosing and treating of pseudomonas aeruginosa.Currently, there is not yet for recombinant protein PA5505 purification process Report, the present invention are mainly introduced the preparation process of PA5505.

Using purification process of the present invention, from expression pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 Colibacillus engineering in can obtain purity greater than 97%, the rate of recovery is greater than 40% destination protein, passes through amino acid sequence It is about 26.0kD that column prediction the present inventor, which constructs the albumen PA5505 molecular mass obtained, and isoelectric point is in pH 6.5 or so.

Purification process of the present invention mainly includes GST affinity purification, SP HP chromatography, Q HP chromatography, passes through above-mentioned side The albumen of method purifying is detected with 12%SDS-PAGE, shows simple target protein band, molecular mass is about 26kD.HPLC C3 column analyzes destination protein purity 98.5%.PA5505 after purification is through Al (OH)₃Injecting immune BalB/C is small after adjuvant absorption Mouse finds that the IgG level in immune serum is significantly higher than negative control group (PBS group) (P < 0.01), it was demonstrated that pure using the present invention The PA5505 that change method obtains can the efficient immune response of effective stimulus body generation.Use clinical strain XN-1 (CCTCC M 2015730) infect, calculate the protective rate of PA5505 anti-PA infection is 70.3%.

Detailed description of the invention

Fig. 1 is the purifying side of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 provided in an embodiment of the present invention Method flow chart.

Fig. 2 is the double digestion qualification result signal of recombinant plasmid pGEX-6p-2-PA5505 provided in an embodiment of the present invention Figure；

In figure: swimming lane 1: nucleic acid (DNA) molecular weight standard (Marker), from top to bottom size be respectively as follows: 4500,3000, 2000,1200,800,500,200bp；Swimming lane 2~6: identification knot of the recombinant expression plasmid pGEX-6p-2-PA5505 after digestion Fruit, the segment separated after digestion about 4000bp and about 720bp.

Fig. 3 is albumen PA5505 induction qualification result schematic diagram provided in an embodiment of the present invention；

In figure: swimming lane 1: Protein Marker (Marker), from top to bottom size be respectively as follows: 130kDa, 100kDa, 70kDa,55kDa,40kDa,35kDa,25kDa,15kDa,10kDa；Swimming lane 2: the GST of the ultrasonic supernatant of zygotic induction expression is filled out Material；Swimming lane 3: the supernatant after PP enzyme digestion；Swimming lane 4: the GST filler after PP enzyme digestion.

Fig. 4 is GST affinity chromatography, the digestion of PP enzyme and SP HP chromatographic purifying PA5505 provided in an embodiment of the present invention SDS-PAGE result schematic diagram；

In figure, swimming lane M: molecular weight of albumen marker；Swimming lane 1: bacteria break supernatant；Swimming lane 2: albumen and the affine filler knot of GST It closes；Eluted sample after swimming lane 3:PP enzyme digestion；Swimming lane 4:SP HP elution samples；The affine filler of GST after swimming lane 5:PP enzyme digestion.

Fig. 5 is SP HP tomographic map provided in an embodiment of the present invention.

Fig. 6 is Q HP tomographic map provided in an embodiment of the present invention.

Fig. 7 is SDS-PAGE result schematic diagram after Q HP chromatography provided in an embodiment of the present invention；

In figure, swimming lane M: molecular weight of albumen marker；Swimming lane 1 and 2 is that Q HP flows through sample.

Fig. 8 is PA5505 albumen HPLC testing result schematic diagram provided in an embodiment of the present invention.

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.

There is not yet aiming at the problem that report of recombinant protein PA5505 purification process；Purification process of the present invention obtains PA5505 can effective stimulus body generate efficient immune response.It is infected using clinical strain XN-1 (CCTCC M 2015730), Calculate the protective rate of PA5505 anti-PA infection is 70.3%.Plasmid pGEX-6p-2 is purchased from GE Healthcare Life Sciences company；Coli strain XL-1 blue is purchased from Shanghai Chao Yan Biotechnology Co., Ltd；In recombination engineering GI:15600698 of the Insert Fragment PA5505 in NCBI, protein Accession:NP_254192.1, the full length protein 260 amino acid.

Application principle of the invention is explained in detail with reference to the accompanying drawing.

Pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 provided in an embodiment of the present invention, the nucleic acid of the antigen Sequence as shown in SEQ ID NO:1, amino acid sequence is as shown in SEQ ID NO:2.

As shown in Figure 1, pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 provided in an embodiment of the present invention Purification process the following steps are included:

S101: the genetic engineering bacterium of expression PA5505 is collected；

S102: bacterium, centrifugation are crushed according to height；

S103:GST affinity purification；

S104:SP HP chromatographic purifying；

The sequence combination of S105:Q HP chromatographic purifying purifies the antigen of preparation.

Application principle of the invention is further described combined with specific embodiments below.

Bacterial strain is as follows with various reagents:

1. pseudomonas aeruginosa strains

Pseudomonas aeruginosa international standard strain PAO1 from U.S. ATCC buy (BAA-47^TM)；

2. reagent

Plasmid pGEX-6p-2, Glutathione Sepharose 4B, SP Sepharose HP, Sephadex G-25 The fillers such as Medium, Q Sepharose HP are purchased from GE Healthcare Life Sciences company, and applicant saves；

Coli strain XL-1blue is purchased from Shanghai Chao Yan Biotechnology Co., Ltd, and applicant saves；

Na₂HPO₄.12H₂O、NaH₂PO₄.2H₂O、NaCl、NaOH、Tween-20、NaHCO₃, Na₂CO₃Purchased from Chinese medicines group Chemical reagent Co., Ltd；

The goat anti-mouse igg antibody of PBS, HRP label is purchased from Bioisystech Co., Ltd, Beijing Zhong Shan Golden Bridge；

Tryptone, Yeast extract are purchased from OXOID company, Britain；

Ampicilline, 0.9% sodium chloride injection are purchased from Taiji Group southwest medicine company；

Agar powder, L-Histidine, is purchased from Sangon Biotech (Shanghai) Co., Ltd. at IPTG；

Albumen sample-loading buffer is purchased from green skies biotechnology；Albumen Marker is purchased from Thermo company；

TFA, acetonitrile are purchased from TEDIA company；

Al(OH)₃Purchased from Brenntage company；

Tris is purchased from ANGUS company；

Sulfuric acid, hydrochloric acid are purchased from Chengdu Ke Long chemical reagent factory；

Bovine serum albumin(BSA) V is purchased from BIOSHARP company；

Isoflurane is purchased from RWD Life Science Co., Ltd. Shenzhen.

Embodiment 1: the building of recombination engineering pGEX-6p-2-PA5505/XL-1blue

Upstream and downstream primer is designed according to the coded sequence of PA5505, aim sequence is obtained using PCR amplification, by aim sequence It is attached after BamH1 and Xho1 double digestion using T4 ligase with pGEX-6p-2 carrier, obtains recombinant plasmid pGEX-6p- 2-PA5505 (Fig. 2 is recombinant plasmid double digestion qualification result).By above-mentioned recombinant plasmid transformed competence colibacillus Escherichia coli XL-1 Blue obtains recombination engineering pGEX-6p-2-PA5505/XL-1 blue.

The expression and digestion of embodiment 2:PA5505 is identified

Going bail for, there are 200 μ L of pGEX-6p-2-PA5505/XL-1 blue bacterium solution spare in 4 DEG C of refrigerators to be added to 20mL It is once activated in the LB culture medium of the resistance containing Amp, after 37 DEG C of 5~6h of culture of 200rpm, is added final concentration of 200 μM IPTG is placed in overnight induction in 16 DEG C of shaking tables, and 5000rpm is centrifuged 10min and collects thallus, then plus 1.5mLPBS weight after induction After outstanding thallus, bacterium solution is subjected to ultrasound cracking 2min (200V), supernatant is collected and 40 μ L is used in conjunction with gst fusion protein Glutathione Sepharose 4B (GE company) gel beads (beads) combination processing, conjugation condition are 4 DEG C and combine 3h；Knot Foreign protein 3 times be not associated with after conjunction using PBS elution take 40 μ L to be used for electricity after then filler being resuspended with 40 μ L PBS Swimming.5 μ L PreScission are added into about 40 μ L of remainder protein-bonded Glutathione Sepharose 4B After protease (PP enzyme, GE company), room temperature digestion 2h, after supernatant is drawn in centrifugation, is washed filler 3 times with PBS, respectively take 20 μ L samples Protein electrophoresis (method is same as above) is carried out after product denaturation treatment, the PA5505 albumen as a result, under digestion is observed under in phase system Molecular weight~26kDa is consistent with expected molecular weight of albumen size, sees Fig. 3.

The purifying process of 3 PA5505 of embodiment is studied

Groped by the purification condition to PA5505, the process flow of the protein purification has finally been determined, such as Fig. 1 institute Show, concrete operations are as follows.

1. height crushes bacterium, centrifugation

By the colibacillus engineering of above-mentioned expression PA5505, by high density fermentation, it is spare that thalline were collected by centrifugation.

Thallus 300g or so is taken, by weight: volume ratio 1:5 ratio is mixed with PBS buffer solution and suspended, 4 DEG C of pre-coolings.

High pressure homogenizer: use distilled water flushing high pressure homogenizer pipeline, low-temperature circulating system open in advance be cooled to 1-4 DEG C it is standby With.

High pressure homogenizer is added in the suspension bacteria liquid of pre-cooling, and pressure maintains 60-80Mpa and breaks bacterium 3-5 times, takes brokenly bacterium solution smear Violet staining, unbroken bacterium is less than 1-2 to be considered as brokenly bacterium complete under each visual field of oil mirror.

High speed centrifugation: the liquid after broken bacterium is packed into centrifugal barrel, and 4 DEG C, 10,000-15,000g is centrifuged 15-30min, in collection It is clear spare.

2.GST affinity purification

GST affinity chromatography filler is selected to carry out preliminary purification, GST is affine, and filler is Glutathione Sepharose One of 4B, Glutathione Sepharose 4FF, Glutathione Sepharose HP break the every 100g of bacterium thallus weight in wet base Amount of filler is 100ml.

Prescission Protease enzyme (PP enzyme) carries out digestion elution: used PP enzyme has GST label, with benefit In removal PP enzyme, destination protein is obtained, enzyme cutting buffering liquid is A liquid (20mM Na₂HPO₄-NaH₂PO₄, pH pH7.0-7.5).Electricity Result of swimming is as shown in Figure 4.By pure at the beginning of GST, the purity of destination protein has reached 90% or more, but still to be improved, removes trace Measure impurity.

3.SP HP chromatographic purifying

The sample for collecting GST affinity chromatography balances tomographic system and SP using B liquid (10mM Histidine, pH6.0) HP chromatographic column, C liquid (10mM Histidine, 1M NaCl, pH6.0) linear gradient elution set elution flow rate 10ml/min, Gradient be B% from 0 to 100%, elution volume 500ml, collect the destination protein eluted be stored in 4 DEG C it is spare. Tomographic map is as shown in figure 5, electrophoresis result is as shown in Figure 4.

4.Q HP chromatographic purifying

The sample that above-mentioned SP HP purifying is obtained, is balanced using D liquid (10mM Histidine, 0.9%NaCl, pH6.0) Tomographic system and Q HP chromatographic column, remove the non-targeted albumen of trace, and the impurity such as endotoxin are collected the destination protein for flowing through and protected It is spare there are 4 DEG C.Tomographic map is as shown in fig. 6, electrophoresis result is as shown in Figure 7.

6.HPLC detection

Purity detecting is carried out to PA5505 albumen using C3 (being purchased from Agilent company), with 0.1%TFA aqueous equilibrium Pillar, loading 10ul sample, the elution of 0.1%TFA acetonitrile solution set 60 DEG C of column temperature, flow velocity 0.5ml/min.Elution program are as follows: 10%~100%B, 30min are 98.5% by the purity that curve measures PA5505 albumen, and tomographic map is as shown in Figure 8.

Wherein, the preparation of 0.1%TFA aqueous solution: I grades of water of 1L add 1ml TFA to mix, 0.22 μm of membrane filtration.

The preparation of 0.1%TFA acetonitrile solution: 1L acetonitrile adds 1ml TFA to mix.

Antigen after purification does following experiment:

Animal is immunized in embodiment 5

The preparation of vaccine: PA5505 proteantigen is diluted using PBS, addition concentration is Al (OH)₃Adjuvant carries out antigen It adsorbs, antigen concentration is 0.5mg/ml, aluminium content 0.7mg/ml in final vaccine finished product；

Immunization method: female BAl BIc/c mouse is randomly divided into 3 groups, every group 30, experimental group is passed through using above-mentioned vaccine Mouse is immunized in the mode of intramuscular injection (quadriceps muscle of thigh), and every mouse injection volume is 100 μ L, and control group uses 100 μ l PBS and Al (OH)₃Adjuvant is immunized respectively, and immunization protocol D0, D14, D21 are immunized three times.

The detection of 6 antibody of embodiment

7th day and the 14th day after third time is immune, the tail vein of BALB/c mouse is acquired, after being immunized with ELISA detection Specific IgG antibodies are horizontal in mice serum.

1. preparing liquid

(1) coating buffer: Na is weighed on an electronic balance₂CO₃1.6g, NaHCO₃2.9g, NaN₃0.2g adds distilled water 500ml is adjusted to pH 9.6, and distilled water is settled to 1000ml, and batch is set as preparation time on the same day, mark: coating buffer.

(2) antibody diluent: NaCl 8g, KH2PO4 0.2g, Na are weighed on an electronic balance₂HPO₄·12H₂O 2.9g, 20 0.5ml of KCl 0.2g, Tween, is adjusted to pH 7.4, adds distilled water to be settled to 1000ml, when batch is set as same day preparation Between, mark: antibody diluent.

(3) cleaning solution: 0.05%Tween 20-PBS (pH 7.4) takes specification to be 1000ml/ bags 1 bag of PBS and is dissolved in In 1000ml pure water, 0.5ml Tween 20 is added.

(4) confining liquid: it is ready-to-use, the antibody diluent of appropriate volume is measured, BSA is added in 1% ratio, 4 DEG C put It purchases use.

(5) terminate liquid: 2mol/L sulfuric acid draws (18mol/L) H with liquid-transfering gun₂SO₄111ml to 889ml ddH₂In O.

2.ELISA detects PA5505 recombinant protein and the antibody titer that mouse generates is immunized

1) it is coated with: PA5505 recombinant protein after purification being diluted to 2 μ g/mL with coating buffer.Enzyme mark is added in 200 holes μ L/ Plate, 4 DEG C overnight after wash 3 times with cleaning solution, it is empty do after wrapped with preservative film, be placed in spare in 4 DEG C of refrigerators；

2) close: ELISA Plate adds 100 hole μ L/ of confining liquid, is placed in 37 DEG C of incubators 2 hours, washs 3 times；

3) serum is subjected to the doubling dilutions such as 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000；It takes The ELISA Plate closed, sequentially adds dilute serum, 100 holes μ L/, 37 DEG C of incubator 30min are placed in, are washed 3 times, sky is dry；

4) goat anti-mouse igg antibody (1:5000 dilution) of HRP label is added in 100 holes μ L/, 37 DEG C of incubator 1h are placed in, Washing three times, sky are dry；

5) 100 hole μ L/ substrate developing solution (TMB) is added, room temperature is protected from light 5min；

6) terminate liquid (2M H is added₂SO₄), it is immediately placed in microplate reader to measure OD value at 450nm wavelength；

7) result judges: (negative control is 1:1000 times of serum before mouse immune to be positive for A sample/A feminine gender Zhi≤2.1 Dilution).

As a result: it is 1:47200 that the antibody titer geometric mean titer that mouse generates, which is immunized, in PA5505 proteantigen；Last is exempted from The 7th day antibody positive rate reaches 100% after epidemic disease, illustrates that the SBP recombinant protein that the present invention constructs has good immunogenicity.

Malicious protecting effect evaluation is attacked after 7 PA5505 of embodiment is immune

PA5505 recombinant protein animal immune attacks poison protection evaluation according to bibliography: the Clin such as Gao Chen Immunol 2017,183,354-363 is carried out.10~14 days after PA5505 final immunization, PA XN-1 will be prepared with physiological saline Bacterium solution simultaneously adjusts concentration to 1.5 × 1010CFU/mL, is infected by the way of collunarium with after isoflurane anesthetized mice, every mouse Infective dose is 20 μ L, is used as blank control using same dose of physiological saline (NS).Every 1 day observation mouse after infection Death condition, observation period are 7 days, and remaining animal is after the observation period with CO₂Inhalation euthanasia.Count each group mouse Survival rate.As a result shown in table 1.

Malicious protecting effect is attacked after the immune mouse of 1 PA5505 recombinant protein of table

Table 1 is shown: the survival rate of negative control group and blank control group is respectively 16.7% and 10.0%, recombination fusion Albumen PA5505 adds Al (OH)₃The survival rate of adjuvant group is 73.3%, is by the protective rate that PA5505 is calculated in formula 70.3%.Therefore, PA5505 recombinant protein of the invention has good immunogenicity, and body can be induced to generate immune answer It answers, and can play a protective role to the infection of PA XN-1, aluminium adjuvant can be aided with and prepare subunit vaccine for preventing copper The infection of green pseudomonad.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Sequence table

<110>Chongqing Ai Libi Biotechnology Co., Ltd

<120>purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 717

<212> DNA

<213>pseudomonas aeruginosa type strain PAO1 (Pseudomonas aeruginosa PAO1)

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aagccgctgc tggccaagga aggcgtggac ctgaagatca aggagttcac cgactacgtg 120

cagccgaacg tgcaggtctc ggaaaagcgc ctggacgcca acttcttcca gcaccagccg 180

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atcgagccgc tgggcgccta ctcgagcaag tacaagaagc tcgacgaact gccttccggc 300

gctaccgtgg tgattcccaa cgacgccacc aacggcggcc gcgccctgct cctgctggac 360

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gtcgacaatc cgaagaacat caagatccgc gaactggaag ccgcgaccct gccgcgcgtg 480

ctgacccagg tcgacatggc gctgatcaat accaactacg ccctggaagc caagctgaac 540

ccaaccaagg atgcgctggc catcgaaggc agcgactcgc cctacgtgaa catcctcgtc 600

gcgcggccgg acaacaagga cagcgacgcc atgcagaagc tggccaaggc cctgcacagc 660

gccgagatca agcagttcat ccaggagaag tacaaaggcg cggtggtacc ggcgttc 717

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<213>pseudomonas aeruginosa type strain PAO1 (Pseudomonas aeruginosa PAO1)

<400> 2

Ala Glu Ser Leu Thr Val Ala Ala Thr Pro Val Pro His Ala Glu Ile

1 5 10 15

Leu Asn Val Val Lys Pro Leu Leu Ala Lys Glu Gly Val Asp Leu Lys

20 25 30

Ile Lys Glu Phe Thr Asp Tyr Val Gln Pro Asn Val Gln Val Ser Glu

35 40 45

Lys Arg Leu Asp Ala Asn Phe Phe Gln His Gln Pro Tyr Leu Asp Glu

50 55 60

Phe Asn Lys Ala Lys Gly Thr Asp Leu Val Ala Val Thr Gly Val His

65 70 75 80

Ile Glu Pro Leu Gly Ala Tyr Ser Ser Lys Tyr Lys Lys Leu Asp Glu

85 90 95

Leu Pro Ser Gly Ala Thr Val Val Ile Pro Asn Asp Ala Thr Asn Gly

100 105 110

Gly Arg Ala Leu Leu Leu Leu Asp Lys Ala Gly Val Ile Lys Leu Lys

115 120 125

Asp Asn Lys Ser Ile Thr Ala Thr Pro Lys Asp Ile Val Asp Asn Pro

130 135 140

Lys Asn Ile Lys Ile Arg Glu Leu Glu Ala Ala Thr Leu Pro Arg Val

145 150 155 160

Leu Thr Gln Val Asp Met Ala Leu Ile Asn Thr Asn Tyr Ala Leu Glu

165 170 175

Ala Lys Leu Asn Pro Thr Lys Asp Ala Leu Ala Ile Glu Gly Ser Asp

180 185 190

Ser Pro Tyr Val Asn Ile Leu Val Ala Arg Pro Asp Asn Lys Asp Ser

195 200 205

Asp Ala Met Gln Lys Leu Ala Lys Ala Leu His Ser Ala Glu Ile Lys

210 215 220

Gln Phe Ile Gln Glu Lys Tyr Lys Gly Ala Val Val Pro Ala Phe

225 230 235

Claims

1. a kind of recombination engineering pGEX-6p-2-PA5505/XL-1 blue, which is characterized in that the recombination engineering pGEX- 6p-2-PA5505/XL-1 blue express antigen PA5505 nucleic acid sequence as shown in SEQ ID NO:1, amino acid sequence such as Shown in SEQ ID NO:2.

2. a kind of pseudomonas aeruginosa using recombination engineering pGEX-6p-2-PA5505/XL-1 blue described in claim 1 The purification process of recombinant vaccine candidate antigens PA5505, which is characterized in that the pseudomonas aeruginosa gene engineered vaccine The purification process of candidate antigens PA5505 includes: the genetic engineering bacterium for collecting expression PA5505；Bacterium, centrifugation, GST are crushed according to height Affinity purification；The sequence combination of SP HP chromatographic purifying, Q HP chromatographic purifying purifies the antigen of preparation.

3. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 2, special Sign is that the purification process of the pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 specifically includes:

1) height crushes bacterium

The genetic engineering bacterium for collecting expression PA5505 is mixed with the PBS buffer solution that pH is 7.0-7.5 and is suspended, using height after pre-cooling Pressure is homogenized broken bacterium, and high speed centrifugation collects supernatant；

2) GST affinity purification

GST affinity chromatography filler carries out preliminary purification, after eluting foreign protein using PBS, with Prescission Protease enzyme Digestion elutes target protein, and digestion and elution buffer are A liquid；

3) SP HP chromatographic purifying

The target protein collected through step 2), with loading after B liquid balance tomographic system and SP HP chromatographic column, C linear gradient is washed It is de-；

4) Q HP chromatographic purifying

The mark albumen that will be purified through step 3), D liquid balance loading after tomographic system and Q HP chromatographic column, remove the non-targeted egg of trace The impurity such as white, endotoxin obtain target protein；

4. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 3, special Sign is that high-pressure homogenization described in the step 1) breaks bacterium and uses 60-80MPa pressure, and high speed centrifugation obtains on broken bacterium after breaking bacterium Clearly.

5. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 3, special Sign is, the filler that the GST affinity chromatography of the step 2) uses be Glutathione Sepharose 4B or Glutathione Sepharose 4FF or Glutathione Sepharose HP.

6. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 2, special Sign is that the Prescission Protease enzyme of the step 2) has GST label.

7. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 2, special Sign is that the filler of the SP HP chromatographic column of the step 3) is SP Sepharose HP or SP Sepharose FF or Capto SP。

8. the purification process of pseudomonas aeruginosa gene engineered vaccine candidate antigens PA5505 as claimed in claim 2, special Sign is that the filler of the Q HP chromatographic column of the step 4) is Q Sepharose HP or Q Sepharose FF or Capto Q.