CN105732817A

CN105732817A - PA (pseudomonas aeruginosa) recombinant protein Vac33, as well as preparation method and application thereof

Info

Publication number: CN105732817A
Application number: CN201610117615.1A
Authority: CN
Inventors: 顾江; 章金勇; 邹全明; 赵莉群; 杨茜; 杨峰; 彭六生; 吴翼; 王逸麟
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU; Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2016-03-02
Filing date: 2016-03-02
Publication date: 2016-07-06
Anticipated expiration: 2036-03-02
Also published as: CN105732817B

Abstract

The invention provides recombinant protein. The recombinant protein is formed by fusion of a PA III type secretory system component protein (PcrV) fragment and a PA outer membrane lipoprotein (OprI) fragment through a Linker, and has a general formula: PcrV fragment-(Linker)n-Oprl fragment, wherein the sequence of the Linker is selected from one of GGGGS, GGSGG and YAPVDV, and preferentially GGGGS; n is 1, 2, 3 or 4, and preferentially 1. The invention also provides a preparation method and an application of the recombinant protein. Subunit vaccine prepared from the recombinant protein can stimulate the organism to generate high-titer IgG antibodies; the recombinant protein can be used for preparing diagnostic reagents, and preventive or therapeutic drugs.

Description

A kind of Pseudomonas aeruginosa recombiant protein Vac33 and preparation method and application

Technical field

The invention belongs to biotechnological pharmaceutics field, be specifically related to a kind of Pseudomonas aeruginosa recombiant protein Vac33 (PcrV-OprI), the invention further relates to the preparation method and applications of this recombiant protein.

Background technology

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) it is commonly called as bacillus pyocyaneus, for the Rhodopseudomonas in non-zymocyte, it is distributed widely in nature, normal human skin, intestinal, respiratory tract, hospital ward and medical apparatus and instruments the most generally exist, is one of the most most commonly seen conditioned pathogen.The most in the world, this bacterium has become ICU ward, burn, War injury infection, one of pathogen that ventilator-associated pneumonia (VAP) separation rate is the highest.PA infects and can occur at any position of human body and tissue, such as burn or wound site, middle ear, cornea, urethra and respiratory tract etc., it is possible to causing the systemic infection such as endocarditis, gastroenteritis, pneumonia even septicemia, systemic infection mortality rate is more than 20%.

Additionally, due to reasons such as the abuses of antibiotic, the resistance problems of PA gradually highlights, and occurs in that general tolerant Pseudomonas aeruginosa (PDR-PA) and multi-resistant Pseudomonas aeruginosa (MDR-PA), and the separation rate of drug resistance PA rises year by year.China's continuous monitoring materials of CHINET2005--2012 shows, PA is maintained at higher level to the resistant rate of Common Antibiotics, but the most on a declining curve.Annual resistant rate such as imipenum is respectively 31.0%, 42.8%, 35.8%, 30.5%, 30.5%, 30.8%, 29.1% and 29.1%, the annual resistant rate of meropenem is respectively 32.0%, 34.1%, 28.5%, 24.5%, 25.2%, 25.8%, 25.0% and 27.1%, but the most general drug resistance (PDR-PA) bacterial strain quantity significantly increases, respectively reached 1.8% and 1.5% (pneumonia caused by Pseudomonas aeruginosa infected specialist's common recognition at 2011 and 2012, China's tuberculosis and breathing magazine, volume 37 1 phase in January, 2014).The most soaring due to PA high infection rate clinically and resistant rate, finds new " non-antibiotic therapy " extremely urgent, starts with from immunology angle, develop the selection that safely and effectively vaccine becomes ideal.

Genetic engineering subunit vaccine is expressed in engineering bacteria on a large scale by engineered means, the protein expression vector virulence factor of pathogen or the gene of its mutant being cloned into, recon, through being prepared as genetic engineering subunit vaccine after purification.This vaccine has that technique is simple, low cost, workable etc. a little, it has also become the important directions of vaccine research and development.Research and development recombinant vaccine it is crucial that screen good protective antigen molecule.

The big complex of the syringe-like that the type III excretory system of Pseudomonas aeruginosa is made up of more than 20 protein, some virulence factors (such as ExoU, ExoS, ExoY, ExoT etc.) and other effect protein can be directly injected into host cell, cause host cell damage thus play a significant role in bacterial infection disease.PcrV is one of significant components of Pseudomonas aeruginosa type III excretory system, it can form homology polymer, it is assembled into excretory system transport virulence factor and " pipeline " of effect protein, it it is the key component (Teiji S. etc., Critical Care 2014) of Pseudomonas aeruginosa type III excretory system.In view of PcrV Pseudomonas aeruginosa cause a disease in important function, this albumen become charrin disease therapeutic antibodies for important target.The polyclonal antibody of anti-PcrV, monoclonal antibody can suppress the secretion of type III excretory system; protection body is invaded (Sawa by Pseudomonas aeruginosa; T. .Nat Med 1999 is waited; Milla; C.E. the J Infect Dis 2002 such as Pediatr Pulmonol 2014, Frank, D.W. are waited; the Infect Immun 2009 such as Baer, M..Same PcrV is also important candidate vaccine molecule, but can form the reasons such as homology polymer due to itself, yet there are no the report of solubility this protein monomer recombinant expressed.

OprI albumen is the outer membrane lipoprotein (outer membrane lipoprotein) of Pseudomonas aeruginosa, and it is positioned at the adventitia of Pseudomonas aeruginosa, plays an important role in the physiology and pathological process of antibacterial.OrpL albumen is guarded at Pseudomonas aeruginosa camber, there is diagnosis target spot (the J Gen Microbiol.1992 such as Saint-Onge A studied as Pseudomonas aeruginosa, the J Clin Microbiol.2003 such as J Clin Microbiol.1997, Qin X such as De Vos D).Although OprI contains only 83 aminoacid, but OprI has good immunogenicity and immune protective.nullOprI individually uses or can induce effective immunne response during with other protective antigen use in conjunction，It is effective against the infection (Baumann of Pseudomonas aeruginosa，U. Vaccine 2004 is waited，Knapp，B. Vaccine 1999 is waited，Mansouri，E. Infect Immun 1999 is waited，Toth，The .Vaccine such as A 1994，von Specht，B.U etc.，Infect Immun etc. 1995，von Specht，B.U etc.，Vaccine 1996，Weimer，E.T. etc.，Vaccine 2009，Weimer，E.T. etc.，Infect Immun 2009，Westritschnig，The .Hum Vaccin Immunother 2014 such as K).In view of these character of OprI, this albumen can be as good recombinant vaccine candidate antigens.

Summary of the invention

The present inventor finds under study for action, with Pseudomonas aeruginosa outer membrane lipoprotein (OprI), Pseudomonas aeruginosa type III excretory system assembly albumen (PcrV) is connected the recombiant protein excitating organism formed by connection peptides produce the prevention of the antibody of preferably identification Pseudomonas aeruginosa, beneficially Pseudomonas aeruginosa, diagnose and treat.

For achieving the above object, the present invention provides following technical scheme:

A kind of recombiant protein, described recombiant protein is formed by the segment composition of connection peptides (Linker) with Pseudomonas aeruginosa outer membrane lipoprotein (OprI) by the fragment of Pseudomonas aeruginosa type III excretory system assembly albumen (PcrV), and described recombiant protein has below general formula: PcrV fragment-(Linker)_n-OprI fragment；

Wherein, described connection peptides sequence is selected from GGGGS, GGSGG, YAPVDV, preferably GGGGS；N is 1,2,3 or 4, preferably 1.

According in one embodiment of the invention, the aminoacid sequence of the fragment of described Pseudomonas aeruginosa type III excretory system assembly albumen (PcrV) is SEQ ID NO:3.

According in one embodiment of the invention, the aminoacid sequence of the fragment of the outer membrane lipoprotein (OprI) of described Pseudomonas aeruginosa is SEQ ID NO:4.

According in one embodiment of the invention, the aminoacid sequence of described recombiant protein is SEQ ID NO:2；Preferably, the nucleotides sequence encoding described recombiant protein is classified as SEQ ID NO:1.

Present invention also offers a kind of expression vector, described expression vector can be connected by skeleton plasmid and encodes the nucleotide sequence of above-mentioned recombiant protein and formed with modifying；Preferably, described skeleton plasmid is selected from pGex serial carrier, pET serial carrier or pQE serial carrier, more preferably pGex-6P-2.

Invention further provides the preparation method of above-mentioned recombiant protein, described method includes:

1) expression vector as claimed in claim 5 is built；

2) by step 1) expression vector that obtains converts to Host Strains, obtains the Host Strains containing expression vector；

3) induction step 2) Host Strains containing expression vector that obtains expresses recombiant protein；

4) extract and purification obtains recombiant protein.

According in one embodiment of the invention, described Host Strains is selected from the one in E.colistrain XL1 blue, BL21 or HMS174 bacterial strain, preferably E.colistrain XL1 blue bacterial strain.

Present invention also offers above-mentioned recombiant protein and prepare the application in the medicine diagnosing, prevent or treating Pseudomonas aeruginosa.

Preferably, described medicine is the test kit for diagnosing Pseudomonas aeruginosa.

Preferably, described medicine is the subunit vaccine for preventing or treat Pseudomonas aeruginosa.

Owing to have employed technique scheme, present invention have the advantage that:

1) Vac33 expression of recombinant proteins plasmid abduction delivering in prokaryotic expression system-escherichia coli；

2), when selecting pGEX carrier families, Vac33 recombiant protein is with fusion protein form expression；The glutathione-S-transferase (GST) that molecular weight is 26kDa it is connected on expression vector, expressed fusion protein just contains a GST label, this label just becomes the labelling of protein purification, make purification condition gentleness, step is simple, need not the addition of denaturant, thus albumen after purification can keep its space conformation and immunogenicity to greatest extent；The Vac33 recombiant protein purity being purified is more than 95%；

3) Vac33 recombiant protein can produce specific antibody and have immune protective effect by induced animal.

The subunit vaccine utilizing Vac33 recombiant protein of the present invention to prepare can carry out immunity inoculation by subcutaneous (muscle) injecting pathway, and excitating organism produces high titre IgG antibody.And animal experiment proves that, described genetic engineering recombinant protein vaccine has the immune protective effect that good anti-PA infects.Study for further combined vaccine and many subunits fusion bacterin and lay the first stone, for preventing and treating vaccine and the development of diagnostic kit and application, there is important effect simultaneously.

Accompanying drawing explanation

Fig. 1 is the double digestion qualification result of recombiant plasmid pGex-6P-2-Vac33.Wherein, 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: recombinant expression plasmid pGEX-6p-2-Vac33 qualification result after enzyme action, the fragment about 4000bp separated after enzyme action and about 1000bp.

Fig. 2 is that albumen Vac33 induces qualification result.Wherein, swimming lane 1: Protein Marker (Marker), size is respectively as follows: 170Kd, 130Kd, 100Kd, 70Kd, 55Kd, 40Kd, 35Kd, 25Kd, 15Kd, 10Kd from top to bottom；The GST filler of the ultrasonic supernatant that swimming lane 2&3: zygotic induction is expressed；Swimming lane 4: the ultrasonic supernatant after PP enzyme enzyme action.

Fig. 3 is albumen Vac33SDS-PAGE electrophoresis result after purification.Wherein, swimming lane 1: Protein Marker (Marker), size is respectively as follows: 170Kd, 130Kd, 100Kd, 70Kd, 55Kd, 40Kd, 35Kd, 25Kd, 15Kd, 10Kd from top to bottom；Swimming lane 2: the Vac33 albumen of purification.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is described in more detail.Should be appreciated that the specific embodiment herein saying description, only in order to explain the present invention, is not intended to limit the present invention.

Embodiment 1The structure of recombiant plasmid and qualification

The connection of the synthesis of the DNA sequence of 1.Vac33 and sequence and pGEX-6p-2 is synthesized by Shanghai Sheng Gong biological engineering company limited.

2. the conversion of recombiant plasmid

Taking 3 pipe escherichia coli XL1blue competent cells (Chao Yan bio tech ltd, Shanghai) from-80 DEG C of refrigerators, the first pipe adds pGEX-6P-2 plasmid (GE Healthcare Life Sciences), makees positive control；Second pipe adds 1ul Vac33 synthetic plasmid；3rd pipe is not added with foreign DNA, makees negative control.Ice bath 50min, thermal shock 90s in 42 DEG C of metal baths, rapid ice bath 2min.Add 600 μ l LB blank cultures, mixing, it is placed in 220rp in 37 DEG C of shaking tables and shakes 1h.

Each pipe is centrifuged 3min. with 5000rpm room temperature, discards 300 μ l supernatants, more resuspended thalline, takes 200 μ l and coat, Amp resistance LB flat board.Flat-plate inverted is placed in 37 DEG C of incubators cultivation 24h.

Negative control plates does not has bacterium colony to occur；Positive control flat board covers with bacterium colony, illustrates that competent cell makes correct, credible result.Picking converts and separates good bacterium colony on flat board, is inoculated in Amp resistance LB culture medium, and 37 DEG C of shaken cultivation are overnight.

2. double digestion is identified

Take the positive plasmid of 37 DEG C of shaking table overnight incubation, step to specifications, the plasmid of positive colony is extracted by the little extraction reagent kit of rapid plasmid (Tian Gen biochemical technology company limited).BamHI (Takara company) and Xhol (Takara company) is used to carry out enzyme action, 37 DEG C of water-bath 4h.System is as follows:

Reagent	Volume (μ l)
		plasmid DNA o	3
NdeI	0.5
		XhoI	0.5
10×buffer(H)	1
		ddH₂O	5
total volume	10

Record 1.0% agarose gel, wherein containing EB (Jun Sheng bio tech ltd, Shanghai) 0.5ug/ml, adding 1 μ l 6 × Loading buffer by each in above-mentioned endonuclease reaction system, after gel 80V electrophoresis 20min, UV scanner observes the result of enzyme action.Result is crossed and is found that the plasmid of positive colony is cut into 2 fragments, and large fragment about 4000bp is expression vector pGEX-6P-2 part, small fragment about 1000bp, the fragment (Fig. 1) of the coding Vac33 for inserting.

Embodiment 2Recombination fusion protein PcrV-OprI is the qualification of abduction delivering, purification and expression-form in prokaryotic expression system-escherichia coli

1.Vac33 abduction delivering

Take in the LB culture medium of pGEX-6P-2-Vac33/XL-1blue bacterium solution 100 μ L addition 10mLAmp+ resistance of incubated overnight, 37 DEG C of incubated overnight of 180rpm, in the LB culture medium of the bacterium solution 400 μ L addition 20mLAmp+ resistance taking incubated overnight respectively (remaining bacterium solution is saved in 4 DEG C of refrigerators standby), cultivate 2～3h for 37 DEG C, rotating speed 200rpm, re-activation to OD600 be 0.8-1.0 time, add IPTG 4 μ L, make its final concentration of 200 μMs, then be placed in 30 DEG C of abduction delivering 3h of shaking table abduction delivering.

2) bacterium solution after abduction delivering is taken out, it is centrifuged 5min with 12000rpm, supernatant discarded, add 1mL lysis buffer (20mM PB, pH 7.2,250mM Nacl) mixing, ultrasonic degradation 3min (ultrasonic 6 times 30s/ time), 4 DEG C of 14000rpm are centrifuged 15min again, cleer and peaceful precipitation in separation.

2. process supernatant

Taking Glutathione Sepharose 4B 20 μ l, after washing 3 times with PBS, added in Glutathione Sepharose 4B by ready supernatant, room temperature combines 1h.After being centrifuged 3min with 14000rpm at 4 DEG C, using PBS-0.25% polysorbas20 to wash 2 times, PBS washed once.Glutathione Sepharose 4B after combining adds 20 μ L 2 × protein loadings buffer, boils 5min, 14000rpm and is centrifuged 3min.

3. process precipitation

In precipitation, add the resuspended thalline of 500Ml PBS, take the 80 resuspended bacterium solution of μ L and add 20 μ L5 × protein loading buffer, boil 5min, 14000rpm and be centrifuged 3min.

4.SDS-PAGE electrophoresis

Concentrating glue by 5% and pour in glue version, glue laminated put down adding distilled water, room temperature places 30min makes it solidify, and is fallen by the distilled water on upper strata dry, then pours into 10% separation gel, plugs comb immediately, and it is standby that room temperature placement 30min makes it solidify.The upper cleer and peaceful precipitation handled well is taken respectively 10 μ L loadings, carries out SDS-PAGE electrophoresis.Voltage elder generation 80v electrophoresis 30min, it is adjusted to 180v again, after electrophoresis 1～2h, glue is taken out, it is placed in coomassie brilliant blue staining liquid vibration dyeing, after being placed in destaining solution vibration decolouring again, observed result under imaging system, PGEX-6P-2-Vac33/XL-1blue is soluble protein (Fig. 2) at 30 DEG C.

The preparation of embodiment 3 Vac33 antigen

1. amplification culture obtains albumen

nullGo bail for and there is standby pGEX-6P-2-Vac33/XL-1blue bacterium solution 400 μ L in 4 DEG C of refrigerators and join in the 20mL LB culture medium containing Amp resistance and once activate，After 200rpm 37 DEG C cultivates 5～6h，Take the bacterium solution that 8mL once activates to join in the 400mL LB culture medium containing Amp resistance and carry out re-activation，When 37 DEG C of cultivations 3～4h to OD600 are 1.0，Add 80 μ L IPTG (final concentration of 200 μMs) be placed in 16 DEG C of shaking tables overnight induction after，12000rpm is centrifuged 15min and collects thalline，After adding 20mL lysis buffer (with embodiment 2) resuspended thalline again，Bacterium solution is carried out ultrasonic degradation 3min (200V)，Collect supernatant and 800 μ L to process for Glutathione Sepharose 4B (GE company) gel beads (beads) combination combining gst fusion protein；Carry out PAGE gel electrophoresis again.

2. use enzymatic cleavage methods, destination protein and GST label are separated, obtains Vac33 destination protein

800 μ L PBS and 120 μ L PreScission protease (PP enzymes are added in remainder about 800 μ L the most protein-bonded Glutathione Sepharose 4B, GE company), after room temperature vertical rotary enzyme action 5h, after centrifugal absorption supernatant, wash 3 times with 800 μ L PBS respectively, after taking 10 μ L sample degenerative treatments after each, loading 5 μ L carries out protein electrophoresis (method is ibid), observed result under in phase system, the Vac33 molecular weight of albumen that enzyme action gets off is between 95-130kDa, it is consistent with expection molecular weight of albumen size, sees Fig. 3.

The structure of embodiment 4 charrin disease Pneumonia Mice model

1. prepared by Pseudomonas aeruginosa bacterium solution

(preserving number is CCTCC NO:M 2015730, and Classification And Nomenclature is: Pseudomonas aeruginosa XN-1, Latin title: Pseudomonas aeruginosa XN-1, preservation time: 2015.12.9 to take frozen P. aeruginosa clinical strain XN-1；Depositary institution: China typical culture collection center (CCTCC)；Preservation place: Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province), use the recovery of LB nutrient agar panel, 37 DEG C of aerobic overnight incubation.Picking single colony inoculation 20mL LB fluid medium, after 37 DEG C of aerobic 180rpm shaken cultivation 15h, takes 0.2mL and is inoculated in 20mL LB fluid medium, 37 DEG C of aerobic 230rpm shaken cultivation 6h.The centrifugal thalline of collecting of 6000g, with normal saline resuspension thalline after brine twice.Use the OD of spectrophotometric determination bacterium solution₆₀₀Value, and according to 1OD=6.0 × 10⁹CFU/ml is converted into bacterial concentration.

2. mouse anesthesia and the foundation of collunarium scheme

In the infection experiment of pseudomonas aeruginosa pneumonia model, for being smoothed out, initially with isoflurane by mouse anesthesia of guarantee collunarium process.Anesthesia: carry out sucking induced anesthesia for delivery vehicles with the isoflurane of 5% concentration with oxygen, after mice enters surgery stage of deep narcosis, the concentration with 3% maintains anesthesia, and this mode can reach preferable anaesthetic effect.The control of collunarium dosage is the guarantee of model stability, in order to preferably control collunarium dosage, during preventing collunarium, bacterium solution enters oral cavity and produces bubble simultaneously, take following measures: (1) has a physiologic radian due to nasopharynx part, head is faced upward the most severe, nasal drop is more easily accessible oral cavity, so mouse head amplitude of lying on the back not can exceed that the physiologic radian of nasopharynx part during collunarium；(2) medicine is dripped along lateral wall of nasal cavity, the generation of bubble during minimizing collunarium as far as possible；(3) get hold of the rhythm of collunarium, drip 4 below μ L amounts in the moment that mice air-breathing starts；(4) in the present embodiment, collunarium amount is 20 μ L, and single-nostril one-time continuous instills.

3. the determination of infective dose

The PAXN-1 bacterium solution using normal saline embodiment 1 to be obtained regulates to five kinds of variable concentrations, laboratory animal female BAl BIc/c mice is randomly divided into 5 groups, infect with using the mode of collunarium after isoflurane anesthesia mice, every mouse infection dosage is 20 μ L, uses same dose of normal saline (NS) as blank.Packet and the infection conditions of animal are as shown in table 1.Infection terminate after every 1 day observe dead mouse situation, the observation cycle is 7 days, the observation period terminate after remain animal with CO₂Inhalation euthanasia.

Table 1: the determination of pseudomonas aeruginosa pneumonia model infective dose

Group	Collunarium	Dosage (CFU)	Quantity	Concentration (CFU/ml)	Infusion volume (μ l)
						1	PAXN-1	6.00×10⁸	10	3.00×10¹⁰	20
2	PAXN-1	3.00×10⁸	10	1.50×10¹⁰	20
						3	PAXN-1	1.50×10⁸	10	7.50×10⁹	20
4	PAXN-1	7.50×10⁷	10	3.75×10⁹	20
						5	PAXN-1	3.75×10⁷	10	1.88×10⁹	20
6	Normal saline	-	10	-	20

Use variable concentrations (2 times of gradient dilutions) pseudomonas aeruginosa strains PA XN-1 infect BALB/c mouse time, through the observation of 7 days, result as shown in Figure 1: infective dose is 1.5 × 10⁸During CFU, mouse death rate is 40%, and infective dose is 3.0 × 10⁸During CFU, mouse death rate is 80%, and infective dose reaches 6.0 × 10⁸Mouse death rate 100% during CFU.The preferred dose determining mouse infection is 3.0 × 10⁸CFU。

The immunity of embodiment 5 animal

1) first immunisation, dilutes Vac33 antigen with PBS, adds the Al (OH) that concentration is 1mg/mL₃；With No. 5 half mould syringe needles, bilateral inguinal, vola and dorsal sc are to an injection, and every BALB/C mice injection volume is 100 μ L, and arranges positive controls, negative control group and blank group；

2) second time immunity, carries out second time immunity on the 7th day, and ibid, injection volume proteantigen amount is the 1/2 of first immunisation to immune component, and immunization route is ibid；

3) third time immunity, carries out third time immunity on the 14th day, and ibid, injection volume proteantigen amount is identical with second time immunity, and immunization route is ibid for immune component.

Embodiment 7: the detection of antibody

After immunity the 7th and 14 day for the third time, gather the blood of BALB/C mice, after ELISA detection mouse immune, IgG, IgG1, IgG2_aHumoral response level.

1. prepare liquid

1) preparation of liquid it is coated: weigh NaHCO₃1.6g, Na₂CO₃2.9g, is dissolved in 1L ddH₂O, is adjusted to 9.6 with PH meter by pH；

2) preparation of confining liquid: 1g Ox blood serum V, is dissolved in 100mL antibody diluent (1: 100)；

3) preparation of antibody diluent: phosphate is dissolved in 1L ddH₂O, adds 500 μ L Tween 20, then with PH meter, pH is adjusted to 7.4；

4) preparation of cleaning mixture: weigh 2.42g Tris and be dissolved in 1L ddH₂O, adds 500 μ L Tween 20, then with PH meter, pH is adjusted to 7.4；

5) nitrite ion (TMB), for sky root Products；

6) stop buffer (2M H₂SO₄) preparation: 22.2mL concentrated sulphuric acid is poured into 177.8mL ddH₂In O.

The antibody titer that 2.ELISA detection Vac33 recombiant protein immune mouse produces

1) Vac33 recombiant protein after purification is diluted to being coated liquid: 1ug/mL, 5ug/mL, 10ug/mL；

2) it is coated: recombiant protein diluent adds ELISA Plate, 200 μ L/ holes, and 4 DEG C are washed 3 times with cleaning mixture the most afterwards, wrap with preservative film, be placed in 4 DEG C of refrigerators standby after sky is dry；

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

4) serum is carried out the doubling dilution such as 1: 100,1: 500,1: 1000,1: 2000,1: 4000,1: 8000；

5) take the ELISA Plate closed, be sequentially added into dilute serum, 100 μ L/ holes, it is placed in 37 DEG C of incubator 30min, washs 3 times, empty dry；

6) by adding the goat anti-mouse igg of HRP labelling, IgG1, IgG2a antibody preservation liquid, dilute 1: 5000, make antibody working solution；

7) add dilution antibody working solution, 100 μ L/ holes, be placed in 37 DEG C of incubator 1h, wash three times, empty dry；

8) substrate nitrite ion (TMB) 100 μ L/ hole is added, room temperature lucifuge reaction 5min；

9) stop buffer (2M H is added₂SO₄), it is immediately placed in microplate reader at 450nm wavelength, to measure OD value；

10) result judges: A_Sample/A_NegativeValue >=2.1 are positive (negative control is serum 1: 1000 times dilution before mouse immune).

Result: the antibody titer that detection Vac33 proteantigen immune mouse produces reaches 1: 512000；After immunity, the antibody positive rate of the 7th day reaches 95%, illustrates that the Vac33 recombiant protein that the present invention builds produces antibody in can making immune mouse body.

The counteracting toxic substances protection of embodiment 8:Vac33 recombiant protein animal immune is evaluated

After Vac33 final immunization 10～14 days, use method same as in Example 5, by preparation PA XN-1 bacterium solution and regulate concentration to 1.5 × 10 with normal saline¹⁰CFU/mL, with using the mode of collunarium to infect after isoflurane anesthesia mice, every mouse infection amount is 20 μ L, uses same dose of normal saline (NS) as blank.Infection terminate after every 1 day observe dead mouse situation, the observation cycle is 7 days, the observation period terminate after remain animal with CO₂Inhalation euthanasia.Add up the survival rate of each group of mice.Result is shown in table 2.

Counteracting toxic substances protected effect after table 2 Vac33 recombiant protein immune mouse

Table 2 shows: the average immune protective rate of negative control group and blank group is respectively 10% and 20%, and recombination fusion protein Vc33 adds Al (OH)₃The average immune protective rate of adjuvant group is 75%.Therefore; the Vac33 recombiant protein of the present invention has good immunogenicity; body can be induced to produce immunne response, and the infection of PA XN-1 can be played a protective role, aluminium adjuvant can be aided with and prepare subunit vaccine for preventing the infection of Pseudomonas aeruginosa.

Pass through above example, those skilled in the art utilize ordinary skill knowledge can apply the recombiant protein prepared by the present invention and other related reagents apparently, such as it is coated reagent, detection antibody, developer, terminator etc. and prepares related kit, whether such as detection kit, infect Pseudomonas aeruginosa for diagnosis, determine prognosis etc..

Vac33 recombiant protein of the present invention can be used for other any applicable purposes by those skilled in the art.

Although present invention has been a certain degree of description, it will be apparent that, without departing from the spirit and scope of the present invention, can carry out the suitable change of each condition.Being appreciated that and the invention is not restricted to described embodiment, and be attributed to the scope of claim, it includes the equivalent of described each factor.

Claims

1. a recombiant protein, it is characterised in that described recombiant protein is divided by Pseudomonas aeruginosa type III Secrete the fragment of system component albumen (PcrV) by connection peptides (Linker) and Pseudomonas aeruginosa adventitia The segment composition of lipoprotein (OprI) forms, and described recombiant protein has a formula: PcrV fragment -(Linker)_n-OprI fragment；

Wherein, described connection peptides sequence is selected from GGGGS, GGSGG, YAPVDV, It is preferably GGGGS；N is 1,2,3 or 4, preferably 1.

2. recombiant protein as claimed in claim 1, it is characterised in that described Pseudomonas aeruginosa III The aminoacid sequence of the fragment of type excretory system assembly albumen (PcrV) is SEQ ID NO:3.

3. recombiant protein as claimed in claim 1 or 2, it is characterised in that described Pseudomonas aeruginosa The aminoacid sequence of fragment of outer membrane lipoprotein (OprI) be SEQ ID NO:4.

4. the recombiant protein as according to any one of claims 1 to 3, it is characterised in that described restructuring egg White aminoacid sequence is SEQ ID NO:2；Preferably, the nucleotide sequence of described recombiant protein is encoded For SEQ ID NO:1.

5. an expression vector, it is characterised in that described expression vector can be connected by skeleton plasmid with modifying The nucleotide sequence of coding recombiant protein described in Claims 1 to 4 is formed；Preferably, described skeleton matter Grain-by-grain seed selection is from pGex serial carrier, pET serial carrier or pQE serial carrier, more preferably pGex-6P-2.

6. a preparation method for the recombiant protein as according to any one of Claims 1 to 4, its feature exists In, including:

1) expression vector as claimed in claim 5 is built；

2) by step 1) expression vector that obtains converts to Host Strains, obtains the host containing expression vector Bacterium；

4) extract and purification obtains recombiant protein.

7. method as claimed in claim 6, it is characterised in that described Host Strains is selected from escherichia coli One in XL1-blue, BL21 or HMS174 bacterial strain, preferably E.colistrain XL1 blue bacterial strain.

8. the recombiant protein as according to any one of Claims 1 to 4 is used for diagnosing, prevent or controlling in preparation Treat the application in the medicine of Pseudomonas aeruginosa.

Apply the most as claimed in claim 8, it is characterised in that described medicine is for being used for diagnosing Aerugo vacation The test kit of Zymomonas mobilis.

Apply the most as claimed in claim 8, it is characterised in that described medicine is for being used for preventing or controlling Treat the subunit vaccine of Pseudomonas aeruginosa.