CN105732818B

CN105732818B - A kind of pseudomonas aeruginosa recombinant protein POP and its preparation method and application

Info

Publication number: CN105732818B
Application number: CN201610118248.7A
Authority: CN
Inventors: 赵莉群; 顾江; 章金勇; 邹全明; 杨峰; 杨茜; 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: 2019-06-07
Anticipated expiration: 2036-03-02
Also published as: CN105732818A

Abstract

The present invention provides a kind of pseudomonas aeruginosa recombinant protein POP and its preparation method and application, link peptide (linker) is passed sequentially through by pseudomonas aeruginosa type III excretory system component albumen PcrV segment, pseudomonas aeruginosa outer membrane lipoprotein OprI segment and pseudomonas aeruginosa IV type pilin PilA segment and connects the recombinant protein formed, the recombinant protein has general formula: PcrV segment-(Linker A)_m- OprI segment-(Linker B)_n- PilA segment；Wherein, the Linker A and Linker B sequence separately one in GGGGS, GGSGG, YAPVDV；Separately value is 1,2,3 or 4 to m and n.The present invention also provides the preparation method and application of above-mentioned recombinant protein.Body can be excited to generate high titre IgG antibody using subunit vaccine prepared by recombinant protein of the invention, can be used for diagnosing, prevent or the preparation of therapeutic agent.

Description

A kind of pseudomonas aeruginosa recombinant protein POP and its preparation method and application

Technical field

The invention belongs to biotechnological pharmaceutics field, it is related to a kind of pseudomonas aeruginosa recombinant protein POP, the present invention is into one Step is related to the preparation method of the recombinant protein and its is preparing the application in vaccine and detection kit.

Background technique

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is commonly called as Pseudomonas aeruginosa, is the vacation in non-zymocyte Zygosaccharomyces, be distributed widely in nature, normal human skin, enteron aisle, in respiratory tract, it is also general in hospital ward and medical instrument Store-through exists, and is clinically most commonly seen one of conditioned pathogen.At present in the world, which has become the ward ICU, burns Wound, War injury infection, one of highest pathogen of ventilator-associated pneumonia (VAP) separation rate.PA infection can occur in human body Any position and tissue, such as burn or wound site, middle ear, cornea, urethra and respiratory tract, can also cause endocarditis, stomach General infections, the general infection death rates such as enteritis, pneumonia even septicemia are more than 20%.

Further, since the reasons such as abuse of antibiotic, the resistance problems of PA gradually protrude, it is false single to there is general drug resistance verdigris Born of the same parents bacterium (PDR-PA) and multi-resistant Pseudomonas aeruginosa (MDR-PA), and the separation rate of drug resistance PA rises year by year.China The continuous monitoring materials of CHINET2005--2012 show that PA is maintained at higher level to the resistant rate of Common Antibiotics, but It is slightly on a declining curve.As the annual resistant rate of Imipenem is respectively 31.0%, 42.8%, 35.8%, 30.5%, 30.5%, 30.8%, 29.1% and 29.1%, the annual resistant rate to Meropenem is respectively 32.0%, 34.1%, 28.5%, 24.5%, 25.2%, 25.8%, 25.0% and 27.1%, but wherein general drug resistance (PDR-PA) bacterial strain quantity significantly increases, Respectively reaching within 2011 and 2012 1.8% and 1.5%, (pneumonia caused by Pseudomonas aeruginosa infects specialist's common recognition, China Tuberculosis and breathing magazine, the phase of volume 37 1 in January, 2014).It is continuous soaring due to PA high infection rate clinically and resistant rate, It is extremely urgent to find " non-antibiotic therapy " newly, starts with from immunology angle, developing safely and effectively vaccine becomes Ideal selection.

Genetic engineering subunit vaccine is by the means of genetic engineering, by the virulence factor of pathogen or the base of its mutant Because of the protein expression vector being cloned into, recon is expressed on a large scale in engineering bacteria, by being prepared into gene work after purification Journey subunit vaccine.The vaccine has simple process, at low cost, strong operability etc. a little, it has also become vaccine is researched and developed important Direction.The key for researching and developing recombinant vaccine is to screen good protective antigens molecule.

The large complex for the syringe-like that the type III excretory system of pseudomonas aeruginosa is made of more than 20 a protein, Some virulence factors (such as ExoU, ExoS, ExoY, ExoT) and other effect proteins can be directly injected into host cell, drawn Host cell damage is played to play a significant role in bacterial infection disease.PcrV is the secretion of pseudomonas aeruginosa type III One of significant components of system, it is capable of forming homologous polymer, is assembled into excretory system transport virulence factor and effect protein " pipeline ", be key component (Teiii S. etc., the Critical Care of pseudomonas aeruginosa type III excretory system 2014).In view of important function of the PcrV in pseudomonas aeruginosa is caused a disease, which has become charrin disease treatment The important target that property antibody is directed to.The polyclonal antibody of anti-PcrV, monoclonal antibody can inhibit point of type III excretory system It secretes, protects invasion (.Nats such as Sawa, T. Med 1999, Milla, C.E. etc. of the body by pseudomonas aeruginosa The J Infect such as PediatrPulmonol 2014, Frank, D.W. Dis 2002, Baer, the Infect such as M. Immun 2009.Same PcrV is also important candidate vaccine molecule, but due to itself being capable of forming homologous polymer etc., at present It yet there are no the soluble report for recombinantly expressing the protein monomer.

OprI albumen is the outer membrane lipoprotein (outer membrane lipoprotein) of pseudomonas aeruginosa, is located at The outer membrane of pseudomonas aeruginosa plays an important role in the physiology of bacterium and pathologic process.OprI albumen is false single in verdigris It is highly conserved in born of the same parents bacterium, there is research as the diagnosis target spot (J such as the Saint-Onge A Gen of pseudomonas aeruginosa The J ClinMicrobiol.2003 such as the J ClinMicrobiol.1997, Qin such as Microbiol.1992, De Vos D X).Though Right OprI only contains 83 amino acid, but OprI has good immunogenicity and immune protective.OprI individually use or Effective immune response can be induced when with other protective antigens use in conjunction, is effective against the infection of pseudomonas aeruginosa (Vaccine such as Baumann, U. 2004, Knapp, B. etc. Vaccine 1999, Mansouri, the Infect such as E. Immun The such as the .Vaccine such as 1999, Toth, A 1994, von Specht, B.U, Infect Immun etc. 1995, von Specht, The such as B.U, Vaccine 1996, Weimer, E.T. etc., Vaccine 2009, Weimer, E.T. etc., Infect Immun The .Hum such as 2009, Westritschnig, K VaccinImmunother 2014).In view of these properties of OprI, which can Using as good recombinant vaccine candidate antigens.

PilA albumen is the IV type pili of pseudomonas aeruginosa, in the movement of bacterium, adherency with host cell and Play a significant role (Hahn HP.Gene 1997.) in the active invasion process of tissue.It is dynamic in pneumonia animal model and keratitis Find that the missing of PilA albumen causes its invasiveness and virulence to be decreased obviously (Hazlett LD etc., Curr Eye on object model Res 1991).Simultaneously the antibody of anti-pilA be able to suppress pseudomonas aeruginosa and epithelial cell adherency (Doig P, etc., Infect.Immun.1990).After the PilA protein immunization mouse of recombinant expression can effective activated cell immune response and Humoral immune response, and with protecting effect (Korpi F etc., J of part in infection of burn model MicrobiolBiotechnol.2015).These data show that PilA is a good protective antigens.

Summary of the invention

The present inventor has found under study for action, by pseudomonas aeruginosa type III excretory system component albumen (PcrV) The recombinant protein excitation body formed is connect with pseudomonas aeruginosa outer membrane lipoprotein (OprI) by link peptide to generate preferably The antibody for identifying pseudomonas aeruginosa, is conducive to the diagnosing and treating of pseudomonas aeruginosa.

To achieve the above object, the present invention provides the following technical solution:

A kind of recombinant protein, the recombinant protein by pseudomonas aeruginosa type III excretory system component albumen PcrV segment, Pseudomonas aeruginosa outer membrane lipoprotein OprI segment and pseudomonas aeruginosa IV type pilin PilA segment pass sequentially through company It connects peptide (linker) connection to be formed, the recombinant protein has general formula: PcrV segment-(Linker A)_m- OprI segment- (Linker B)_n-- PilA segment (POP)；

Wherein, the Linker A and Linker B sequence separately in GGGGS, GGSGG, YAPVDV one It is a, preferably GGGGS；Separately value is 1,2,3 or 4, preferably 1 to m and n.

In an embodiment according to the present invention, the pseudomonas aeruginosa type III excretory system component albumen (PcrV) amino acid sequence of segment is SEQ ID NO:3.

In an embodiment according to the present invention, the segment of the outer membrane lipoprotein (OprI) of the pseudomonas aeruginosa Amino acid sequence be SEQ ID NO:4.

In an embodiment according to the present invention, the ammonia of the pseudomonas aeruginosa IV type pilin PilA segment Base acid sequence is SEQ ID NO:5.

In an embodiment according to the present invention, the amino acid sequence of the recombinant protein is SEQ ID NO:2.

In an embodiment according to the present invention, the recombinant protein is that SEQ ID NO:1 is classified as by nucleotides sequence DNA encoding.

The present invention also provides a kind of expression vector, the expression vector can connect coding right by skeleton plasmid with modifying It is required that the nucleotide sequence of recombinant protein described in 1~6 is formed；Preferably, the skeleton plasmid be selected from pGex serial carrier, PET serial carrier or pQE serial carrier；More preferably pGex-6P-2.

Further, the present invention also provides the preparation methods of above-mentioned recombinant protein, which comprises

1) above-mentioned expression vector is constructed；

2) expression vector for obtaining step 1) is converted to host strain, obtains the host strain containing expression vector；

3) induction step 2) obtain containing expression vector host strain expression recombinant protein；

4) it extracts and purifies to obtain recombinant protein.

In an embodiment according to the present invention, the host strain be selected from E.colistrain XL1 blue, BL21 or One of HMS174 bacterial strain, preferably E.colistrain XL1 blue bacterial strain.

Further, it is false for diagnosing, preventing or treating verdigris in preparation that the present invention also provides above-mentioned recombinant proteins Application in the drug of monad；

Preferably, the drug is the kit for diagnosing pseudomonas aeruginosa；

Preferably, the drug is the subunit vaccine for preventing or treating pseudomonas aeruginosa.

By adopting the above-described technical solution, the present invention has the advantage that:

1) expression plasmid of POP recombinant protein of the invention inducing expression in prokaryotic expression system-Escherichia coli；

2) when selecting pGEX carrier families, POP recombinant protein is with fusion protein form expression；One is connected on expression vector Molecular weight is the glutathione-S-transferase (GST) of 26kDa, just contains a GST label in expressed fusion protein, this Label just becomes the label of protein purification, so that purification condition is mild, step is simple, does not need the addition of denaturant, thus pure Albumen after change can keep its space conformation and immunogenicity to greatest extent；The POP recombinant protein purity being purified is greater than 95%；

3) POP recombinant protein can induce animal to generate the antibody of specificity and have immune protective effect.

The subunit vaccine prepared using POP recombinant protein of the invention can be exempted from by subcutaneous (muscle) injecting pathway Epidemic disease inoculation, excitation body generate high titre IgG antibody.And animal experiment proves that, the genetic engineering recombinant protein vaccine tool There is the immune protective effect of good anti-PA infection.Base is laid for further combined vaccine and the research of more subunit's fusion bacterins Plinth, while being played an important role for the development of prevention and treatment vaccine and diagnostic kit and application.

Detailed description of the invention

Fig. 1 is the double digestion qualification result of recombinant plasmid pGex-6P-2-POP；Wherein, swimming lane 1: nucleic acid (DNA) molecular weight Standard (Marker), size is respectively as follows: 4500,3000,2000,1200,800,500,200bp from top to bottom；Swimming lane 2: recombination Qualification result of the expression plasmid pGEX-6p-2-POP after digestion, the segment separated after digestion about 4000bp and about 1400bp.

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

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

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right The present invention is further described.It should be appreciated that saying description herein, specific examples are only used to explain the present invention, is not used to Limit the present invention.

Embodiment 1The building and identification of recombinant plasmid

1. implementation sequence encodes POP recombinant protein DNA sequence dna, the synthesis of the DNA sequence dna as shown in SEQ ID NO:1 It is synthesized with the connection of pGEX-6p-2 by Shanghai Sheng Gong bioengineering Co., Ltd with sequence.

2. the conversion of recombinant plasmid

The conversion of recombinant plasmid from -80 DEG C of refrigerators takes 3 pipe Escherichia coli XL1blue competent cells, and (Shanghai is super to grind biology Science and Technology Ltd.), pGEX-6P-2 plasmid (GE Healthcare Life Sciences) is added in the first pipe, and it is positive right to make According to；1ul POP synthetic plasmid is added in second pipe；Exogenous DNA is not added in third pipe, makees negative control.Ice bath 50min, 42 DEG C of metals Thermal shock 90s in bath, rapid ice bath 2min.600 μ l LB blank cultures are added, mix, 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, then thallus is resuspended, takes 200 μ l to be coated on, Amp Resistance LB plate.Plate is inverted in 37 DEG C of incubators and cultivates for 24 hours.

Negative control plates do not have bacterium colony appearance；Positive control plate covers with bacterium colony, illustrates that competent cell production is correct, Credible result.Separate good bacterium colony on picking conversion plate, is inoculated in Amp resistance LB culture medium, 37 DEG C of shaken cultivation mistakes Night.

2. double digestion is identified

The positive plasmid for taking 37 DEG C of shaking table cultures overnight, to specifications the step of, pass through the small extraction reagent kit of rapid plasmid The plasmid of (Tiangeng biochemical technology Co., Ltd) extraction positive colony.Using BamHI (Takara company) and Xhol, (Takara is public Department) carry out digestion, 37 DEG C of water-bath half an hour.System is as follows:

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

1.0% Ago-Gel is recorded, wherein contain EB (Shanghai Jun Sheng Biotechnology Co., Ltd) 0.5ug/ml, it will be upper It states and 1 μ l 6 × Loading buffer is respectively added in endonuclease reaction system, after gel 80V electrophoresis 20min, UV scanner is seen Examine the result of digestion.As a result the plasmid for crossing discovery positive colony is cut into 2 segments, and large fragment about 4000bp is expression vector The part pGEX-6P-2, small fragment about 1400bp, for the segment (Fig. 1) of the coding POP of insertion.

Embodiment 2Recombination fusion protein POP inducing expression, purifying and expression shape in prokaryotic expression system-Escherichia coli The identification of formula

1.POP inducing expression

Take the 100 μ L of pGEX-6P-2-POP/XL-1blue bacterium solution being incubated overnight that the LB culture of 10mL Amp+ resistance is added In base, 37 DEG C of 180rpm are incubated overnight, and take the 400 μ L of bacterium solution being incubated overnight that the LB culture medium of 20mLAmp+ resistance is added respectively In (remaining bacterium solution is stored in spare in 4 DEG C of refrigerators), 37 DEG C of culture 2~3h, revolving speed 200rpm, re-activation to OD600 is When 0.8-1.0, IPTG4 μ L is added, makes its final concentration of 200 μM, then be placed in 30 DEG C of inducing expression 3h of shaking table inducing expression.

2) bacterium solution after inducing expression is taken out, 5min is centrifuged with 12000rpm, is discarded supernatant, 1mL lysis is added Buffer (20mM PB, pH 7.2,250mMNacl) is mixed, ultrasound cracking 3min (ultrasound 6 times 30s/ times), then 4 DEG C 14000rpm is centrifuged 15min, separation supernatant precipitating.

2. handling supernatant

20 μ l of Glutathione Sepharose 4B is taken, after washing 3 times with PBS, ready supernatant is added In Glutathione Sepharose 4B, room temperature combination 1h.After being centrifuged 3min at 4 DEG C with 14000rpm, PBS- is used 0.25% polysorbas20 washs 2 times, and PBS washed once.20 μ L 2 are added to the Glutathione Sepharose 4B after combination × protein loading buffer, boils 5min, and 14000rpm is centrifuged 3min.

3. processing precipitating

500 μ LPBS are added in precipitating, thallus is resuspended, takes 80 μ L resuspended bacterium solutions that 20 μ L5 × protein loading is added Buffer, boils 5min, and 14000rpm is centrifuged 3min.

4.SDS-PAGE electrophoresis

5% concentration glue is poured into glue version, is equalled glue laminated distilled water is added, being placed at room temperature for 30min makes its solidification, will The distilled water on upper layer falls to do, then pours into 10% separation gel, plugs comb immediately, being placed at room temperature for 30min keeps its solidification spare.It will place The supernatant precipitating managed takes 10 μ L loadings respectively, carries out SDS-PAGE electrophoresis.Voltage elder generation 80v electrophoresis 30min, then be adjusted to After 180v, 1~2h of electrophoresis, glue is taken out, is placed in coomassie brilliant blue staining liquid and vibrates dyeing, then is placed in vibrate in destainer and take off After color, under imaging system observation as a result, PGEX-6P-2-POP/XL-1blue 30 DEG C be soluble protein (Fig. 2).

The preparation of 3 POP antigen of embodiment

1. amplification culture obtains albumen

Going bail for, there are 400 μ L of pGEX-6P-2-POP/XL-1blue bacterium solution spare in 4 DEG C of refrigerators to be added to 20mL containing Amp It is once activated in the LB culture medium of resistance, after 37 DEG C of 5~6h of culture of 200rpm, the bacterium solution for taking 8mL once to activate is added to Re-activation is carried out in the LB culture medium of 400mL resistance containing Amp, and 80 μ L are added when 37 DEG C of culture 3~4h to OD600 are 1.0 After IPTG (final concentration of 200 μM) is placed in 16 DEG C of shaking tables induction overnight, 12000rpm centrifugation 15min collection thallus, then plus After thallus is resuspended in 20mL lysis buffer (with embodiment 2), bacterium solution is subjected to ultrasound cracking 3min (200V), collects supernatant It is used to combine in conjunction with Glutathione Sepharose 4B (GE company) gel beads (beads) of gst fusion protein with 800 μ L Processing；PAGE gel electrophoresis is carried out again.

2. using enzymatic cleavage methods, destination protein and GST label are separated, obtain the POP purpose that sequence is SEQ ID NO:2 Albumen

800 μ L PBS and 120 are added into about 800 μ L of remainder protein-bonded Glutathione Sepharose 4B After μ L PreScission protease (PP enzyme, GE company), room temperature vertical rotary digestion 5h, after supernatant is drawn in centrifugation, respectively It is washed 3 times with 800 μ L PBS, after 10 μ L sample denaturation treatments are taken after each, 5 μ L of loading carries out protein electrophoresis (method is same as above), In observation under phase system as a result, the POP molecular weight of albumen that gets off of digestion is between 55-40kDa, with expected molecular weight of albumen size It is consistent, sees Fig. 3.

The building of 4 charrin disease Pneumonia Mice model of embodiment

1. prepared by pseudomonas aeruginosa bacterium solution

Take freeze P. aeruginosa clinical strain XN-1 (deposit number be CCTCC NO:M 2015730, classification naming are as follows: Pseudomonas aeruginosa XN-1, Latin title: Pseudomonas aeruginosa XN-1, preservation time: 2015.12.9；It protects Hide unit: China typical culture collection center (CCTCC)；Preservation place: Wuhan City, Hubei Province Wuchang District Bayi Road Luo Ka Mountain), it is recovered using LB nutrient agar panel, 37 DEG C of aerobic overnight incubations.Picking single bacterium colony is inoculated with 20mL LB Liquid Culture Base after 37 DEG C of aerobic 180rpm shaken cultivation 15h, takes 0.2mL to be inoculated in 20mL LB liquid medium, 37 DEG C aerobic 230rpm shaken cultivation 6h.Thalline were collected by centrifugation by 6000g, and brine uses physiological saline resuspension thallus afterwards twice.It adopts With the OD of spectrophotometric determination bacterium solution₆₀₀Value, and according to 1OD=6.0 × 10⁹CFU/ml is converted into bacterial concentration.

2. the foundation of mouse anesthesia and collunarium scheme

In the infection experiment of pseudomonas aeruginosa pneumonia model, to guarantee going on smoothly for collunarium process, use first Isoflurane is by mouse anesthesia.Anesthesia: carrying out sucking induced anesthesia using oxygen as delivery vehicles with the isoflurane of 5% concentration, After mouse enters surgery stage of deep narcosis, anesthesia is maintained with 3% concentration, this mode can achieve preferable anaesthetic effect. The control of collunarium dosage is the guarantee of model stability, in order to preferably control collunarium dosage, while preventing bacterium during collunarium Liquid enters oral cavity and generates bubble, and take following measures: (1) since pharynx nasalis has a physiologic radian, head is faced upward more severe, Nasal drops is more easily accessible oral cavity, so mouse head is lain on the back physiologic radian of the amplitude no more than pharynx nasalis during collunarium； (2) medicine is dripped along lateral wall of nasal cavity, the generation of bubble as far as possible during reduction collunarium；(3) rhythm for getting hold of collunarium, in mouse The moment that air-breathing starts is added dropwise 4 μ L or less and measures；(4) collunarium amount is 20 μ L in the present embodiment, and single-nostril one-time continuous instills.

3. the determination of infective dose

The PAXN-1 bacterium solution that embodiment 1 obtains is adjusted to by five kinds of various concentrations using physiological saline, experimental animal is female Property BALB/c mouse is randomly divided into 5 groups, with being infected by the way of collunarium after isoflurane anesthetized mice, every mouse infection dosage For 20 μ L, blank control is used as using same dose of physiological saline (NS).The grouping of animal and infection conditions are as shown in table 1. Every 1 day observation dead mouse situation after infection, observing the period is 7 days, and remaining animal is after the observation period with CO₂Sucking Method 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*10⁸	10	3*10¹⁰	20
2	PAXN-1	3*10⁸	10	1.5*10¹⁰	20
						3	PAXN-1	1.5*10⁸	10	7.5*10⁹	20
4	PAXN-1	7.5*10⁷	10	3.75*10⁹	20
						5	PAXN-1	3.75*10⁷	10	1.88*10⁹	20
6	Physiological saline	-	10	-	20

When infecting BALB/c mouse using the pseudomonas aeruginosa strains PA XN-1 of various concentration (2 times of gradient dilutions), warp Observation in 7 days is crossed, as a result infective dose is 1.5 × 10 as shown in Figure 1:⁸When CFU mouse death rate be 40%, infective dose be 3.0 × 10⁸Mouse death rate is 80% when CFU, and infective dose reaches 6.0 × 10⁸Mouse death rate 100% when CFU.Determine mouse infection Preferred dose is 3.0 × 10⁸CFU。

5 animal of embodiment is immunized

1) first immunisation dilutes POP antigen with PBS, and the Al (OH) that concentration is 1mg/mL is added₃；With No. 5 half mould syringe needles, To an injection, every BALB/C mice injection volume is 100 μ L, and positive control is arranged for bilateral inguinal, vola and dorsal sc Group, negative control group and blank control group；

2) it is immunized for second, carries out within the 7th day being immunized for second, immune component is same as above, and injection volume proteantigen amount is for the first time Immune 1/2, immunization route is same as above；

3) third time is immune, and the third time of progress in the 14th day is immune, and immune component is same as above, injection volume proteantigen amount and second Secondary to be immunized identical, immunization route is same as above.

Embodiment 6: the detection of antibody

7th and 14 day after third time is immune, the blood of BALB/C mice is acquired, after detecting mouse immune with ELISA, IgG, IgG1、IgG2_aHumoral response is horizontal.

1. preparing liquid

1) preparation of coating buffer: Na is weighed₂CO₃1.6g, NaHCO₃2.9g is dissolved in 1L ddH₂O is counted with PH and is adjusted to pH 9.6；

2) preparation of confining liquid: 1g cow's serum V is dissolved in 100mL antibody diluent (1: 100)；

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

4) preparation of cleaning solution: synantibody dilution

5) developing solution (TMB) is Tiangeng Products；

6) terminate liquid (2M H₂SO₄) preparation: the 22.2mL concentrated sulfuric acid is poured into 177.8mL ddH₂In O.

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

1) POP recombinant protein after purification is diluted with coating buffer are as follows: 1ug/mL, 5ug/mL, 10ug/mL；

2) be coated with: by recombinant protein dilution be added ELISA Plate, 100 holes μ L/, 4 DEG C overnight after washed 3 times with cleaning solution, It is wrapped, is placed in spare in 4 DEG C of refrigerators with preservative film after sky is dry；

3) close: ELISA Plate adds 200 hole μ L/ of confining liquid, is placed in 37 DEG C of incubators 2 hours, washs 3 times；

4) serum is carried out 1: 100,1: 500,1: 1000,1: 2000,1: 4000,1: 8000 and waits doubling dilutions；

5) ELISA Plate closed is taken, dilute serum is sequentially added, 100 holes μ L/ are placed in 37 DEG C of incubator 1h, washing 3 Time, sky is dry；

6) goat anti-mouse igg for adding HRP to mark, IgG1, IgG2a antibody are saved into liquid, antibody work is made in dilution 1: 5000 Make liquid；

7) dilution antibody working solution is added, 100 holes μ L/ are placed in 37 DEG C of incubator 40min, wash three times, and sky is dry；

8) 100 hole μ L/ substrate developing solution (TMB) is added, room temperature is protected from light 5min；

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

10) result judges: A_Sample/A_{It is negative}Value >=2.1 be the positive (negative control be before mouse immune 1: 1000 times of serum it is dilute It releases).

As a result: detection POP proteantigen is immunized the antibody titer that mouse generates and reaches 1: 1024000；7th day after immune Antibody positive rate reaches 100%, illustrates that the POP recombinant protein that the present invention constructs can make to generate antibody in immune Mice Body.

Embodiment 7:POP recombinant protein animal immune attacks poison protection evaluation

10~14 days after POP final immunization, using method same as Example 5, 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, every mouse with after isoflurane anesthetized mice 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 it is shown in table 2.

Malicious protecting effect is attacked after the immune mouse of 2 POP recombinant protein of table

Table 2 is shown: the average immune protective rate of negative control group and blank control group is respectively 10% and 20%, and recombination is melted Hop protein POP adds Al (OH)₃The average immune protective rate of adjuvant group is 85%.Therefore, POP recombinant protein of the invention has good Good immunogenicity can induce body to generate immune response, and can play a protective role to the infection of PAXN-1, can be with It is aided with aluminium adjuvant and prepares subunit vaccine for preventing the infection of pseudomonas aeruginosa.

By above embodiments, those skilled in the art can apparently be applied using ordinary skill knowledge The systems such as recombinant protein prepared by the present invention and other related reagents, such as coating reagent, detection antibody, color developing agent, terminator Whether standby related kit, such as detection kit, infect pseudomonas aeruginosa, determine prognosis etc. for diagnosing.

POP recombinant protein of the invention can be used for other any applicable purposes by those skilled in the art.

Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right It is required that range comprising the equivalent replacement of each factor.

Claims

1. a kind of recombinant protein, which is characterized in that the recombinant protein is by pseudomonas aeruginosa type III excretory system component albumen PcrV segment, pseudomonas aeruginosa outer membrane lipoprotein OprI segment and pseudomonas aeruginosa IV type pilin PilA segment according to Secondary connected by link peptide (linker) is formed, and the recombinant protein has general formula: PcrV segment-(Linker A)_m -OprI Segment-(Linker B)_n-- PilA segment；

The amino acid sequence of the recombinant protein is SEQ ID NO:2.

2. recombinant protein as described in claim 1, which is characterized in that the recombinant protein is to be classified as SEQ ID by nucleotides sequence The DNA encoding of NO:1.

3. a kind of expression vector, which is characterized in that the expression vector can connect coding claim 1 by skeleton plasmid with modifying The nucleotide sequence of the recombinant protein is formed.

4. expression vector as claimed in claim 3, which is characterized in that the skeleton plasmid is selected from pGex serial carrier, pET system It lists body or pQE serial carrier.

5. expression vector as claimed in claim 4, which is characterized in that the skeleton plasmid is pGex-6P-2.

6. a kind of preparation method of recombinant protein as described in claim 1 characterized by comprising

1) expression vector as claimed in claim 3 is constructed；

4) it extracts and purifies to obtain recombinant protein.

7. method as claimed in claim 6, which is characterized in that the host strain be selected from E.colistrain XL1 blue, BL21 or One of HMS174 bacterial strain.

8. the method for claim 7, which is characterized in that the host strain is E.colistrain XL1 blue bacterial strain.

9. the recombinant protein as described in claim 1-2 is any is in preparing the drug for preventing or treating pseudomonas aeruginosa Application.

10. application as claimed in claim 9, which is characterized in that the drug is for preventing or treating pseudomonas aeruginosa Subunit vaccine.