CN101781632A - Brucella melilitensis bp26 gene-deleted M5-90 vaccine strain - Google Patents

Abstract

The invention relates to a recombined brucella melilitensis, wherein the recombined brucella melilitensis completely deletes the bp26 gene. Safe tests prove that the brucella melilitensis is consistent with a parent vaccine strain. Virulent attack protection tests prove that a mutant strain has immune protection consistent with the parent vaccine strain. The mutant strain has significance for distinguishing vaccine immunity from wild type brucellosis infection from the standpoint of serology and controlling, monitoring and purifying brucellosis disease.

Description

The Brucella melilitensis bp 26 gene-deleted M 5-90 vaccine strain

Technical field

The present invention relates to recombinant bacteria vaccine field.Particularly, the present invention relates to its Brucella (Brucella melitensis) of a kind of reorganization horse ear, its Brucella of wherein said reorganization horse ear lacks the bp26 gene fully.The invention still further relates to method and its Brucella application in preparation vaccine and epidemiological surveillance reagent of described reorganization horse ear of its Brucella of the described reorganization of preparation horse ear.

Background technology

Brucella (Brucella) is a gram negative bacterium, and cytozoicus can infect human and multiple animal causes heating, miscarriage and sterile, chronic arthritis and nervous lesion etc., causes loss of world wide tremendous economic and serious public health problem.Brucellosis lacks effective treatment means, and existing antibiotic therapy produces effects limited ^[1]

Its Brucella (Brucella melitensis) vaccine strain M5-90 of horse ear is a little less than China separates voluntarily and causes, and has obtained good effect in clinical application, has has successfully prevented and treated the Brucella ovis disease ^[2], but conventional serology detects and its and natural infection can not be distinguished mutually with diagnostic method, and China's brucellosis is prevented and treated for this and purification is disadvantageous.The method one that solves this difficult problem is the natural rough strains of bacteria of screening ^[3], it does not cause anti-O antigen-antibody, thereby obscures when avoiding serodiagnosis, but this needs plenty of time and heavy work.Another method is to seek to have antigenic proteantigen, knocks out corresponding gene then, sets up corresponding detecting method.

Scholar [OL Rossetti is arranged, AI Arese, ML Boschiroli and SL Cravero.Cloning ofBrucella abortus gene and characterization of expressed 26-kilodaltonperiplasmic protein:potential use for diagnosis.Journal of ClinicalMicrobiology, Jan 1996,165-169, Vol 34, No.1] utilize biology techniques from brucella abortus vaccine strain S19 lambda gt11 library, to be separated to the gene of coding 26-kDa protein B P26.Bp26 albumen is a kind of periplasm protein in S19, expresses justacrine to pericentral siphon with precursor forms in the intestinal bacteria that transform, and contains 28 amino acid whose signal peptides, altogether 29KDa.

Summary of the invention

Studies show that BP26 albumen has immunocompetence preferably, is used as SD target protein.The deletion mutantion of bp26 does not change the biological characteristics and the immunoprotection originality of vaccine strain; adopting recombinant expressed BP26 albumen is the indirect ELISA method that antigen is set up; with Brucella LPS extract is that antigenic conventional indirect ELISA is compared, and has enough susceptibility and specificity.This research has made up its type M5-90 bp26 genetically deficient mutant strain of horse ear first, and for distinguishing this vaccine strain immunity and natural infection in conjunction with the relevant diagnostic method of BP26, prevention and control and purification brucellosis provide new way.

Particularly, the invention provides and the following:

1. its Brucella (Brucella melitensis) of reorganization horse ear, its Brucella of wherein said reorganization horse ear lacks the bp26 gene fully.

2. according to its Brucella of above 1 described reorganization horse ear, wherein in its Brucella of described reorganization horse ear fully the bp26 gene of disappearance replaced by foreign gene (for example, antibiotic resistance gene).

3. according to its Brucella of above 2 described reorganization horse ears, wherein said antibiotic resistance gene is kalamycin resistance gene (Kan ^r).

4. according to its Brucella of above 1 to 3 each described reorganization horse ear, its Brucella of wherein said reorganization horse ear is reorganization melitococcus M5-90.

5. method for preparing according to its Brucella of above 1 to 4 each described reorganization horse ear, this method comprise the bp26 gene that lacks its Brucella of horse ear fully.The method of disappearance (knocking out) gene is to well known to a person skilled in the art method, for example can replace the bp26 gene by the method for homologous recombination with foreign gene (for example, antibiotic resistance gene) by suicide plasmid.

6. according to above 5 described methods, wherein replace the bp26 gene of its Brucella of horse ear with antibiotic resistance gene.

7. according to above 6 described methods, wherein said antibiotic resistance gene is kalamycin resistance gene (Kan ^r).

8. according to each described method of above 5-7, its Brucella of wherein said horse ear is melitococcus M5-90.

9. treat and/or prevent application in the vaccine of the disease that its Brucella of horse ear caused according to its Brucella of any one described reorganization horse ear among the above 1-4 in preparation.The disease that its Brucella of horse ear is caused for example has the heating that causes, miscarriage and sterile, chronic arthritis and nervous lesion etc. in human and multiple animal.

10. according to its Brucella application in its Brucella epidemiological surveillance reagent of preparation horse ear of any one described reorganization horse ear among the above 1-4, described reagent is used to distinguish natural infection and vaccine immunity.In one embodiment, its the Brucella epidemiological surveillance of described horse ear comprises the step of utilizing the ELISA method to detect the proteic antibody of BP26 in the target organism, and wherein said target organism has been applied or has not used described its brucellin of reorganization horse ear.

11. an inactivated vaccine, it comprises according to any one its brucellin of reorganization horse ear among the above-mentioned 1-4.

12. a test kit that is used to distinguish its Brucella natural infection poultry of its brucellin of horse ear immunity poultry and Ma Er, it comprises according to any one its brucellin of reorganization horse ear and the proteic antibody of BP26 among the above-mentioned 1-4.

Melitococcus M5-90 is China's attenuated vaccine strain of separation and Culture voluntarily, has security preferably and immune protective efficiency, for vital role has been brought into play in the sick prevention and control of China's cloth.This research as the reorganization target site, is parent with M5-90 with the bp26 gene, utilizes bp26 gene ORF outside sequence as homology arm, with kalamycin resistance gene (Kan ^r) be incorporated in the bacterial genomes, dual crossing reorganization positive strain, show that through SDS-PAGE and Western blot test the BP26 albumen that mobility is about 29kd expresses also and can be discerned by mouse-anti BP26 hyper-immune serum in the parent bacterium, and mutant strain M5-90-26 reaction result is negative, shows the expression in sudden change vaccine strain M5-90-26 of BP26 albumen.The mouse infection model shows that residual virulence is compared with parent plant and do not taken place obviously to change, and rehabilitation duration was about for 13 weeks.The protection test of M28 strong virus attack proves that mutant strain has consistent with parent's vaccine strain immune protective, and spleen separation of C FU number is than low 3 orders of magnitude of blank.M5-90-26 possesses from serology angle differentiation M5-90-26 immunity and the infection of wild-type Brucella, brucellosis is prevented and treated, monitored and purifies having great importance.

The accompanying drawing summary

Fig. 1 .M5-90 (A) and the synoptic diagram of M5-90-26 (B) bp26 gene expression element and the synoptic diagram of transfer vector pSP-bp26-k (C).The bp26 size is 753bp among the M5-90.Left and right sides homology arm is respectively 1048bp and 1020bp.B. the Kan of 795bp in M5-90-26 ^rGene is replaced bp26, its use the bp26 upstream region of gene regulating and controlling sequence as expression regulation sequence;

Fig. 2 .M5-90bp26 homology arm and Kan ^rGene PCR amplification and transfer vector pSP-bp26-k enzyme are cut evaluation.A .M, λ-HindIII molecular weight standard .1, bp26 left side homology arm; 2, the right homology arm .B.M of bp26, DL2000marker.1, Kan ^rThe PCR product of gene.C.M, the DL2000 molecular weight standard.1, Kpn I and Pst double digestion plasmid pSP-bp26-k.2, EcoR I and Kpn I double digestion plasmid pSP-bp26-k.D, Pst I and Sph I double digestion pSP-bp26-k;

The PCR of Fig. 3 .M5-90-26 identifies that (A and B) and Western blot detect BP26 albumen loss of expression (C) in reorganization M5-90-26.(A): M, DL2000 molecular weight standard .1, Kan among the M5-90-26 ^rThe gene PCR amplified production.(B): M, λ-HindIII molecular weight standard .1 and 2 are respectively M5-90-26bp26 gene left and right sides homology arm pcr amplification products.(C): M, protein dye Marker in advance.1, M5-90-26 and BP26 immune serum reaction result are negative.2, M5-90 and BP26 immune serum reaction result are positive;

Fig. 4 .rBP26 albumen Western blot detects BP26 antibody in M5-90-26 and the M5-90 immune serum.1, the rBP26 reaction of M5-90 mice immunized serum and escherichia coli expression is positive.2, M5-90-26 immune serum and rBP26 are negative in Western blot;

Fig. 5 .M5-90-26, M5-90 and M28 be time length (A) and spleen weight (B) in the mouse body.The result is the average of every group of 5 mouse, and error bars represents standard deviation.By the t-check, between M5-90-26 and the M5-90, p=0.88, greater than 0.05, difference is not remarkable.Between M5-90 and the M28, p=0.02, between M5-90-26 and the M28, p=0.01, all less than 0.05, significant difference.(B) M5-90-26 and M28, p=0.01 between the M5-90 and M28, less than 0.05, difference is not remarkable.And M5-90-26 and M5-90p value=0.4, difference is not remarkable.

Fig. 6 .M5-90-26, M5-90 immune mouse M28 attack malicious body and separate M28 counting (A) and spleen weight (B).(A) Median represents median, and Dispersion range is the distribution range of every group of 10 mouse.By the analysis of distribution free Kruskal-Wallis check multiple comparisons, asterisk representative and control group (PBS immunity) significant difference, but M5-90-26, difference is not remarkable between the M5-90. and (B) the table intermediate value is the average of every group of 10 mouse, error bars represents standard deviation, by t-check, M5-90-26 and M28, p value between M5-90 and the M28 is all less than 0.05, significant difference.M5-90-26 and M5-90p value are greater than 0.05, and difference is not remarkable.

Embodiment

Hereinafter describe reference example in detail the present invention, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.

The detection of structure, screening and the kanamycin gene expression stability of embodiment 1 reorganization melitococcus M5-90-26 mutant strain

1. material and method

1.1 material

Plasmid: pBluescript-II (Strategene company), pSP72 (Promega company), pIRES2-eGFP (this plasmid contains the kana gene, is the sequence in the Tn5 transposon on the plasmid) (B.D (Bioscience-Clonetech) company).Its Brucella (Brucella Melitensis) virulent strain M28 (for the parent before the M5-90 domestication) and low virulent strain M5-90 (reference: grand force of horse ear, He Chongli, Chen Naichang etc. Brucella sheep kind M5-90 vaccine is to 2 years immune period tests of Shanxi soil species ox.China's livestock and poultry transmissible disease, 1988 the 2nd phase .432-433; Lu Jingliang, Li Jietang, Wang Zhensheng etc.The foundation of sheep type Brucella studies on Monoclonal Antibody I.M281D2C-1 hybridoma cell strains.1984 the 4th phases of livestock contagious disease.134-37) all be purchased from the Harbin veterinary institute.

Liquid TSB-YE substratum is that TSB (Bioscience-Clonetech company) adds 0.1% yeast extract (Bioscience-Clonetech company), and pH is 7.2.Plate culture medium is to add 1% agar powder among the liquid TSB-YE.The concentration of kantlex (Sigma) and penbritin (Sigma) is 50 μ g/ml and 100 μ g/ml.

1.2 suicide plasmid homology arm bp26 gene outside sequence and amplification of Tn5 transposon kalamycin resistance gene and sequencing

Get the vaccine strain M5-90 adding 2ml phosphate buffered saline(PBS) (PBS) that ampoule is preserved, the dissolving back is streak culture in the TSB-YE flat board fully.Extract bacterial genomes with test kit (Qiagen company) after getting single clonal expansion ,-20 ℃ of preservations are standby.Utilize high-fidelity Pfu (Takara company) taq enzyme, primer is to left arm and the right arm homologous gene of bp26-l-f and bp26-l-r and bp26-r-f and bp26-r-r (table 1) difference pcr amplification bp26.With the contained Kan of transposon Tn5 among the plasmid pIRES2-eGFP ^rGene is a template, Kan ^r-f and Kan ^r-r (table 1) Kan that increases ^rGene.The PCR program, 95 ℃ of preheating 5min, 30 circulations are 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30,72 ℃ of termination reaction 10min.Each PCR product is cloned EcoRV site in pBluescript-II respectively, called after pBlue-bp26-l respectively, pBlue-bp26-r and pBlue-kana carry out sequencing to these three genes.

Table 1 makes up suicide plasmid pSP-bp26-k and identification of M 5-90-26 the primer

1.3 contain the structure of foreign gene suicide plasmid pSP-bp26-k

Downcut the bp26-l gene with EcoR I and Kpn I from pBlue-bp26-l, be connected on the same pSP72 that cuts with EcoR I and Kpn I enzyme called after pSP-bp26-l.Utilize Kpn I and Pst I to cut pSP-bp26-l, with pBlue-Kan ^r(contain Kan ^r) handle equally with KpnI and Pst I, reclaim Kan ^rGene inserts pSP-bp26-l, called after pSP-bp26-l-k.With the bp26-r on Pst I and the Sph I cutting-out pBlue-bp26-r, insert the same pSP-bp26-l-k that handles with Pst I and SphI, called after pSP-bp26-k (plasmid map is seen Fig. 1 (C)) then.Expression element is seen Fig. 1 between the bp26 homology arm.Kan wherein ^rThe gene orientation is inserted into pSP-bp26-l-k, and the controlling element of the transcription and translation of its use and reading frame be all from the bp26 gene, and the bp26 gene is replaced fully, can guarantee that like this BP26 albumen will can not express fully.Middle amount preparation plasmid pSP-bp26-k is adjusted into 1 μ g/ μ L (Qiangen test kit) with concentration.

1.4M5-90-26 the detection of mutant strain structure, screening and kanamycin gene expression stability

M5-90 vaccine strain competent cell preparation method, the 100ml substratum is cultivated 6h, OD for 37 ℃ ₆₀₀Be about at 0.4～0.6 o'clock, the centrifugal 10min of results bacterium 5000g, 10% aqueous glycerin solution is washed 3 times, is resuspended in ice-cold 10% aqueous glycerin solution of 2.5ml, and 200 μ l packing place-80 ℃ of preservations standby.Electricity transforms (Bio-rad) plasmid pSP-bp26-K to the M5-90 bacterium ^[4]Get 10 μ g pSP-bp26-k and add the M5-90 competent cell that places on ice, the electricity conversion condition is the conversion cup (Bio-rad) of 0.2cm, 25 μ F, 2.5KV, 400 Ω, 6ms, rapidly it is transferred in the SOC substratum (Invotrogen) of 800 μ l then, transferase 12 50rpm shaking table is cultivated activation 2h after 37 ℃ of preheatings, all coats on the flat board that contains kantlex TSB, observes bacterium colony behind the 72h.The bacterium colony that will contain kalamycin resistance is divided into two and is scoring to respectively on the flat board of kantlex and penbritin, further confirms the resistance of positive colony.The single cross fork should show different resistance characteristics with the bacterium colony of dual crossing reorganization, and the bacterium colony of single cross fork reorganization has kantlex and the dual resistance of penbritin, and dual crossing has only kalamycin resistance.The positive colony that obtains is gone down to posterity 20 generations (called after M5-90-26) check external source Kan on the flat board of kantlex and penbritin ^rGene can be in M5-90-26 stably express.

1.5 the PCR of mutant strain M5-90-26 identifies

Get the M5-90-26 bacterium liquid 12000g of 1ml fresh culture and discard supernatant after centrifugal.Be resuspended in 67% methyl alcohol PBS (pH 7.2) solution, hatch 2h for 70 ℃, test kit extracts bacterial genomes.Utilize primer Kan ^r-f and Kan ^r-r PCR identifies Kan ^rWhether gene is present in M5-90-26.Next utilizes jd-26-l-q-f and jd-kana-l-r (table 1) to identify the existence of left homology arm.Utilize jd-26-r-h-r and jd-kana-r-f to identify the existence of right homology arm.Utilize jd-26-l-q-f and jd-26-r-h-r at last.Carry out sequencing.

1.6 the Western blot of mutant strain M5-90-26 identifies

SDS-PAGE and Western blot identify, cultivate M5-90 and M5-90-26, work as OD ₆₀₀Get 1ml bacterium liquid during=0.4-0.6, discard supernatant after 12000g is centrifugal.Be resuspended in 67% methyl alcohol PBS (pH 7.2) solution, hatch deactivation 2h for 70 ℃.The centrifugal supernatant that goes, be resuspended among the 100 μ l PBS, add 80 μ l 2 * SDS sample loading buffers and 20 μ l DTT (dithiothreitol (DTT)), boiling water cracking 30min, cell lysate is carried out SDS-PAGE at 10% separation gel, coomassie brilliant blue R250 (Amresco) dyeing.Simultaneously, to nitrocellulose filter (Amresco), after 4 ℃ of sealings of 10% skimming milk were spent the night, PBST (the PBS solution that contains 0.05%Tween 20) washed 3 times cell lysate through SDS-PAGE, electrotransfer, each 3min.The mouse-anti BP26 albumen hyper-immune serum (preparation method's of mouse-anti BP26 albumen hyper-immune serum the reference: Hu Sen that adds 1: 50 times of dilution, Wang Qinghua, Wang Xijun etc., recombinant poxvirus is expressed sars coronavirus spike protein and immunogenicity analysis thereof. viral journal .2007 (4): 287-291.), bp26 gene ORF is cloned on the pET30a (Novagen) carries out prokaryotic expression, gained albumen is with 100 a μ g/ dosage immune mouse 3 times, in each 3 weeks at interval, three exempt from back 3 all separating obtained serum).Behind 37 ℃ of effect 1h, the PBST washing is the same.Add and to release horseradish peroxidase (HRP) mark sheep anti-mouse igg (Sigma, 1: 2000 times of 10% skimming milk is rare), behind 37 ℃ of effect 1h, PBST washing, DAB (3, the 3-diaminobenzidine) 3-5min that develop the color, deionized water termination.

2 results

2.1pSP-bp26-k transfer vector makes up and identifies

The amplified production of bp26 dna homolog arm (shown in Fig. 2 A arrow), homology arm size in the left and right sides conforms to 1020bp with theoretical value 1048bp.Kan ^rThe amplified production of gene big or small conform to the 795bp theoretical value (shown in Fig. 2 B arrow).The sequencing result shows bp26 dna homolog arm gene from M5-90, Kan ^rGene conforms to expection from the Tn5 transposon.The last bp26 gene of pSP-bp26-k left and right sides homology arm is cut evaluation, Kan with EcoR I, KpnI and Pst I, SphI enzyme ^rGene is cut with KpnI and Pst I enzyme.(Fig. 2 C and 2D) conforms to theoretical value.Proof pSP-bp26-k transfer vector successfully constructs.

2.2 mutant strain M5-90-26 PCR and Western blot identify

Get the M5-90-26 bacterium liquid of fresh culture, utilize the genome of corresponding primer PCR identification of M 5-90-26 after the test kit extraction bacterial genomes.Kan as a result ^rThe gene bp26 position of correctly recombinating, and its sequence of wholly replace are as Fig. 3 (A).Penbritin gene amplification is negative.The suicide vector framework is because of intersecting the reorganization disappearance for the second time, and amplification left and right sides homology arm size conforms to theoretical value, as Fig. 3 (B).Proof reorganization M5-90-26 successfully constructs.

M5-90 and M5-90-26 cell lysate increase respectively, behind SDS-PAGE, electrotransfer is to nitrocellulose filter, one anti-be 1: 50 times of dilution (10% skimming milk) mouse-anti BP26 albumen hyper-immune serum, the result shows in M5-90 the BP26 protein expression and can be discerned by mouse-anti BP26 albumen serum, and this band does not appear in the corresponding position in mutant strain M5-90-26, and consistent with expected results (Fig. 3 C) further proved the expression of BP26 protein delation.

The bp26 gene of China's attenuated vaccine strain M5-90 success is by the foreign gene wholly replace in this research, thereby guaranteed that its product do not express fully, got rid of in theory because of ORF not produced its possibility because of expression product of part by wholly replace.For setting up with BP26 is that diagnostic antigen ELISA method is laid a good foundation.

The biologic activity of embodiment 2 mutant strain M5-90-26 detects

1. material and method

1.1 material

Material is the same.

1.2 mutant strain M5-90-26 and M5-90 immune mouse produce the Westernblot of anti--bp26 antibody situation and detect

With M5-90 and M5-90-26 with 10 ⁵The dosage of CFU/ mouse is the female BALB/c mouse (Beijing dimension tonneau China laboratory animal company limited) in 3 4-5 of immunity age in week respectively, and immunization route is an abdominal injection.Separation of serum after 4 weeks.With the rBP26 of expression in escherichia coli (preparation method: Old Great achievementDeng, reorganization Brucella BP26 albumen and OMP31 albumen are as the research of indirect ELISA diagnostic antigen. China Amphixenosis journal.2006, (6): 518-521)) be antigen, utilize Westernblot to detect the anti--BP26 antibody situation that produces behind M5-90 and the M5-90-26 immune mouse.The rBP26 of purifying is carried out electrophoresis at 10% separation gel, and 4 ℃ of sealings are spent the night after the transfer printing.One anti-is M5-90 and M5-90-26 Brucella immune serum (1: 50 times of dilution).Second antibody is for releasing horseradish peroxidase (HRP) mark sheep anti mouse IgG (Sigma), and 1: 2000 times of 10% skimming milk is rare, and the DAB 3-5min that develops the color stops.

1.3 rehabilitation (Recover Time, RT) mensuration of time in the mouse body

150 4-5 age in week, female BALB/c mouse was divided 3 groups, 50 every group.Difference peritoneal immunity M28, M5-90, M5-90-26, immunizing dose is 1 * 10 ⁸CFU (colony-forming unit).Respectively after immunity the 1st, 3,5,7,9,12,13 when week neck break and put to death 5 of mouse.The aseptic spleen of getting is weighed, and adds the pH 7.2PBS homogenate (Qiagen Tissue lyser) of 1ml then.10 times of serial dilution homogenates are got 100 μ l and are coated with TA substratum (B.D company) flat board, and bacterium colony can occur behind 37 ℃ of cultivation 4d.Calculate the RT time

1.4 mouse immune protection test

45 4-5 age in week, female BALB/c mouse was divided 3 groups, 15 every group.Difference immune M5-90, M5-90-26 and PBS (negative control), immunizing dose is 1 * 10 ⁵CFU.Immunity is all attacked poison with the strain of M28 intensity after 25 weeks, and (attacking the toxic agent amount is 5 * 10 ⁴CFU, approach is intraperitoneal injection), attack poison and put to death the mouse spleen after 15 days and weigh separation of bacterial.Get 100 μ l and be coated with the TA culture medium flat plate, cultivate after 4 days enumeration for 37 ℃.

2. result

2.1Western blot detect mutant strain M5-90-26 and M5-90 in the mouse body to the BP26 immunne response

The rBP26 of expression in escherichia coli as antigen in Western blot respectively with the sero-reaction of mutant strain, parent's bacterial immunity mouse, band with this albumen test appears as a result shown in arrow among Fig. 3 A, about 37KD in the M5-90 serum.Because natural B P26 albumen is about 29KD, in expression vector pET30, merged the polypeptide about one section 8KD, size is 38KD.And the serum of mutant strain immunity not with the reaction of this recombinant protein, proved that further mutant strain fails to produce anti-BP26 protein antibodies.

Western blot (Fig. 3 C) show M5-90-26 not with the polyclonal antibody reaction of anti-BP26, but the band of specific reaction has appearred in wild-type M5-90, from theoretical and actual proof BP26 expression deletion mutant strain.Western blot (Fig. 4) shows that the rBP26 that expresses among intravital antibody of immune M5-90 mouse and the E.coli reacts, and the M5-90-26 immune serum does not react with rBP26.Prove that from the another one angle M5-90-26 can not cause the antibody of anti-BP26 in the mouse body, this result is for establishing theoretical with actual feasibility based on the diagnostic method of BP26.The position that it may be noted that band among Fig. 3 C and Fig. 4 is in different size, is 29KD because BP26 albumen size occurs with precursor forms usually in thalline, is 26KD after the cracking.RBP26 in this research has merged one section 8KD left and right sides albumen in prokaryotic expression carrier pET30a, so little of about 38KD.

2.2 the mensuration of residual virulence in the mutant strain M5-90-26 mouse body

In order to assess the residual virulence of M5-90-26, survival time and propagation situation in the body.M5-90-26 and parent's vaccine strain M5-90 and M28 are compared spleen separation of bacterial situation.Studies show that the sudden change of Bp26 genetically deficient can not change the growth characteristics of bacterial strain.Fig. 5 shows mutant strain M5-90-26 and parent's vaccine strain M5-90 low 0.7log when first week, and it is lower slightly to seem residual virulence, slightly is better than the former again in 7-12 week virulence, but increment and rehabilitation duration aspect do not have considerable change as a whole.Substantially separate less than bacterium during to the 13rd week, whole spleen isolated bacterial number is 0-4.Strong malicious M28 exceeds the two about 2log all the time.Virulence is obviously very strong, proves that further it attacks malicious feasibility as virulent strain.

In the above-mentioned malicious results from residue tests result of (also claiming rehabilitation duration experiment RT), the trend of M5-90 and M5-90-26 integral body is roughly consistent, presents the trend of successively decreasing gradually in 13 weeks, and the 1st all CFU numbers are between 4.5 and 5, in the almost full scale clearance of the 13rd week.Different with S19 (crest appears at 1-15 days, and CFU maintains 8Log10) and S2 (time length is very short, drops to 0 about 6 weeks) ^[6]Closely similar with Rev1.More weak but its protection of very short virulence of S2 time length also relatively a little less than.Though but M5-90-26 BP26 disappearance its duplication characteristic and just residual virulence of time length are in vivo compared with the parent not have obviously and are changed in a word.Compare about low 3log10 with M28 (virulent strain is the parent before the M50-90 domestication) simultaneously.Heavy M5-90 of spleen and M5-90-26 compare and also do not have significant difference, but obvious lower than M28, inflammatory reaction is the most serious to occur in the 21st day, but swelling all the time.The weight of average spleen about 70-80mg, even but separate less than bacterium in the body, spleen is swelling still, proves that inflammatory reaction still exists.

2.3 mouse immune protection test

The World Health Organization (WHO) ^[5]Determined the standard of assessment mutant strain immune protection in 1997, be in time specific behind strong virus attack from mouse spleen or/and liver separates the minimizing of Brucella quantity weighs.Whether consistent for measuring M5-90-26 with the M5-90 immune protection, the PBS dissolved 10 of 3 groups of 15 mouse peritoneals injection 0.2ml ⁵Individual CFU M5-90-26 and M5-90.Attack poison after 15 days, the spleen separation of bacterial.Table 2 shows that M5-90-26 separates the M28 bacterial count with the M5-90 mice immunized and is starkly lower than very much non-immune negative control.M5-90-26 compares with M5-90 and does not see significant difference.The result points out to compare with M5-90, and the anti-microbial activity of M5-90-26 is that immune protective does not change.

In attacking malicious protection test, immune M5-90-26 carried out challenge test on the 150th day behind the M5-90, separated the M28CFU number and was consistent, and separated the CFU number of M28 about 3log10 in body.And M28CFU is 6log10 in the contrast PBS immune group.Difference is extremely remarkable.Proof M5-90-26, M5-90 has kept identical immunoprotection level.The spleen weight in average also significantly is lower than blank PBS immunized mice, is about 1/3 of its weight.

This research has successfully made up Maltese vaccine mutant strain M5-90-26; and its residual and immunoprotection situation of virulence in the body that with the mouse is animal model of formulate, prove that this mutant strain is that security or validity have all kept the height consistence with the parent.Also be first in the residual and immunoprotection situation of mouse interior evaluating M5-90 virulence simultaneously.This mutant strain is that new way is opened up in the brucellosis prevention and control of China.

Reference

[1]Alton?GG，Jones?LM，Angus?RD.Techniques?for?the?brucellosis?laboratory.INRA(Institut?National?de?la?Recherche?Agronomique)，Paris，France，1988.

[2] Shang Deqiu. Chinese brucellosis control scientific research 50 years. Chinese epidemiology magazine (ChineseJournal of Epidemiology), 2000,21 (1): 55-57.

[3]Gerhardt?GS，Nammalwar?S，Michael?JC.Brucelosis?vaccines：past，present?and?future.Vet?Microbiol，1990，20(20)：479-496.

[4]E.Susan?Drazek，Huo-shu?H.Houng，Robert?M.Crawford.Deletion?ofpurE?Attenuates?Brucella?melitensis?16M?for?Growth?in?HumanMonocyte-Derived?Macrophages.Infection?and?Immunity，1995，63：3297-3301.

[5]World?Health?Organization(WHO).1997.Report?of?the?WHO?WorkingGroup?Meeting?on?Brucellosis?Control?and?Research：The?development?ofNew/Improved?Brucellosis?Vaccine. http://www.who.int/emc.

[6]Nicole?Bosseray，M?Plommet.Brucella?suis?S2，Brucella?melitensis?Rev.1and?Brucella?abortus?S19?living?vaccines：residual?virulence?and?immunityinduced?against?three?Brucella?species?challenge?strains?in?mice.Vaccine，1990(8)：462-468。

Claims (10)

1. its Brucella (Brucella melitensis) of reorganization horse ear, its Brucella of wherein said reorganization horse ear lacks the bp26 gene fully.

2. its Brucella of reorganization horse ear according to claim 1, wherein in its Brucella of described reorganization horse ear fully the bp26 gene of disappearance replaced by antibiotic resistance gene.

3. its Brucella of reorganization horse ear according to claim 2, wherein said antibiotic resistance gene is kalamycin resistance gene (Kan ^r).

4. according to its Brucella of each described reorganization horse ear of claim 1 to 3, its Brucella of wherein said reorganization horse ear is reorganization melitococcus M5-90.

5. method for preparing according to its Brucella of each described reorganization horse ear of claim 1 to 4, this method comprise the bp26 gene that lacks its Brucella of horse ear fully.

6. method according to claim 5 is wherein replaced the bp26 gene of its Brucella of horse ear with antibiotic resistance gene.

7. method according to claim 6, wherein said antibiotic resistance gene are kalamycin resistance gene (Kan ^r).

8. according to each described method of claim 5-7, its Brucella of wherein said horse ear is melitococcus M5-90.

9. treat and/or prevent application in the vaccine of the disease that its Brucella of horse ear caused according to its Brucella of any one described reorganization horse ear among the claim 1-4 in preparation.

10. according to its Brucella application in its Brucella epidemiological surveillance reagent of preparation horse ear of any one described reorganization horse ear among the claim 1-4, described reagent is used to distinguish natural infection and vaccine immunity.

Images