CN103865859A - Staphylococcus sciuri, vaccine and preparation method thereof - Google Patents
Staphylococcus sciuri, vaccine and preparation method thereof Download PDFInfo
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- CN103865859A CN103865859A CN201410130127.5A CN201410130127A CN103865859A CN 103865859 A CN103865859 A CN 103865859A CN 201410130127 A CN201410130127 A CN 201410130127A CN 103865859 A CN103865859 A CN 103865859A
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- staphylococcus sciuri
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Abstract
The invention provides a staphylococcus sciuri, a vaccine and a preparation method thereof, belonging to the technical field of biotechnology. The collection number of the staphylococcus sciuri NJ1306 provided by the invention is CCTCC NO: M2013702. Experiments prove that the staphylococcus sciuri NJ1306 has pathogenicity on BALB/c mice and piglets. The invention also provides a staphylococcus sciuri inactivated vaccine containing an inactivated NJ1306 strain. A staphylococcus sciuri inactivated oil emulsion vaccine is prepared by uniformly mixing an inactivated staphylococcus sciuri culture solution and an adjuvant. By adopting the inactivated oil emulsion vaccine prepared by adopting the staphylococcus sciuri NJ1306 disclosed by the invention, good protection capacity can be provided for the BALB/c mouse, so that the strain is proved to have good immunogenicity.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a strain Staphylococcus sciuri (
staphylococcus sciuri) separation, evaluation, and the application of this bacterium in the development of the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri.
Background technology
In recent years, staphylococcus (
staphylococci) become the important pathogen of hospital infection and Community Acquired Infections, the research in country's Surveillance of antibiotic resistance in bacterial isolates 2002 years of center is found, the microbemia proportion that G+ bacterium (gram positive organism) causes is 51.1%, staphylococcemia has just accounted for 37%, wherein coagulase negative staphylococcus (coagulase negative
staphylococci, CNS) and accounting example is 57.21%, streptococcus aureus accounting example is 15.28%, coagulase negative staphylococcus (the methicillin-resistant coagulase negative of the resistance to Oxazacillin that blood preparation separates
staphylococci, MRCNS) and account for 74.7% of coagulase negative staphylococcus, the infection of coagulase negative staphylococcus is more and more outstanding, and is usually resistant organism, becomes the difficult point of clinical treatment, should strengthen correlative study.
Meng Huarong is gathered into 2001～2003 years 484 strain coagulase negative staphylococcus in Xindu District, city the People's Hospital and carries out evaluation and 11 kinds of antibiotic drug resistance analysis of planting, find in 484 strain staphylococcuses, account for before 4 be staphylococcus epidermidis 165 strains, staphylococcus xylosus 122 strains, 78 strains of Hua Nashi staphylococcus and Staphylococcus sciuri 48 strains, these bacterium, to vancomycin, rifampin, furadantin sensitivity, reach more than 80% the resistant rate such as penbritin, erythromycin.
Ji Ruoxu, Zhu Xiaodong, Di Hua, Dan Jiongcong year February in April, 2000 to 2004, children's in Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.'s hospital care is carried out to bacteriology checking, the relevant bacterial strain of finding is carried out to antibiotics sensitivity detection, to the analysis of making comparisons of obtained positive findings simultaneously.They find altogether 1320 strain positive bacterias in 8220 bacteriological detection, wherein 202 strain CNS, account for 15.3% (202/1320) of overall recall rate, in CNS take staphylococcus haemolyticus, staphylococcus epidermidis, slowly staphylococcus, Staphylococcus sciuri, imitation staphylococcus be as main front 5 pathogenic bacterium.In these positive strains, percentage of methicillin-resistant CNS accounts for 86.63%, and all bacterial strains keep responsive to vancomycin.Conclusion: CNS infects has become one of major issue in Pediatric Clinic curative activity, and wherein percentage of methicillin-resistant CNS bacterial strain is multidrug resistant; Infect for CNS, need to be on the basis of bacteriology checking Using adapted Antibios, vancomycin is still last treatment means.
Methicillin-resistant Staphylococcus (methicillin-resistant
staphylococci, MRS) become the main pathogenic fungi of ward infection at present, and expand to gradually community.Current, methicillin-resistant Staphylococcus (MRS) produces very serious Medical Crisis spreading of medical institutions and community, and the mankind's health in serious threat.
Staphylococcus sciuri was found by Kloos and his colleague for the first time in 1976.Staphylococcus sciuri belongs to coagulase negative staphylococcus (coagulase negative
staphylococci, CNS), be extensively present in occurring in nature, be important cause of diseases of the mankind simultaneously, mainly cause wound infection in the mankind's endocarditis, peritonitis, septicemia, urinary tract infection, pelvic inflammation and operation etc.
The nineteen ninety-fives such as domestic Zhao Qing are separated to Staphylococcus sciuri in the cerebrospinal fluid of 1 routine Children with Meningitis and blood simultaneously.Retrospective analysis .105 example septicemia of newborn blood culture with positive bacteria rate 100% is carried out in the aspect such as clinical characters and diagnosis and treatment of the 105 routine septicemia of newborn that Li Li accepts for medical treatment Shunyi hospital of Beijing of Chinese Medical Sciences University for 2012, respectively staphylococcus epidermidis 58 examples (55.24%), staphylococcus haemolyticus 28 examples (26.67%), Staphylococcus sciuri 6 examples (5.7%), streptococcus aureus 5 examples (4.76%), each 2 examples of staphylococcus hominis people subspecies and Hua Nashi staphylococcus (each 1.9%), B group streptococcus agalactiae, Staphylococcus auricularis, enterococcus faecalis, each 1 example of hen faecalis (each 0.9%).In 105 examples, take staphylococcus as main, totally 102 examples, account for 97.1%.Li Tingjun, Dong Yanjin, Qiu Tingting have reported that Staphylococcus sciuri causes septicemia 1 example for 2013.Tang Ying has reported Staphylococcus sciuri type scalded skin syndrome 1 example for 2008.
Chen Shixi etc. are separated to the pathogenic Staphylococcus sciuri of a plant height from suffer from the pig pericardial fluid of exudative dermatitis.Zhang Xi, Yang Zengqi, Song Changxu separate by sick pig the 4 strain Staphylococcus sciuris that obtain and carry out animal Orthogonal Rotational Regressive Tests, the pig that this bacterium has been inoculated in discovery shows as spiritual depressed, appetite declines, there is sheet exudate thin, taupe brown in whole body, dead successively subsequently, illustrate that Staphylococcus sciuri has larger harm to humans and animals.
Along with a large amount of uses of antibacterials in animal and human group, staphylococcic resistance is also more and more serious, the appearance of resistance Staphylococcus sciuri bacterial strain is also more and more, the generation of drug tolerant bacteria declines the curative effect of some common drugs and even loses curative effect, as the medicines such as penicillins, tetracyclines, aminoglycosides, sulfamido develop immunity to drugs in a large number in livestock and poultry, clinical effectiveness worse and worse.Along with the expanding day of veterinary antibacterial range of application and kind, the generation of bacterial drug resistance has presented surge trend, probably occurs " superbacteria " to main antibiotics resistance in the future.
Summary of the invention
Primary and foremost purpose of the present invention be to provide a strain Staphylococcus sciuri (
staphylococcus sciuri) NJ1306, this bacterial strain has pathogenic to BALB/c mouse and piglet.
Another object of the present invention is to provide Staphylococcus sciuri inactivated vaccine, can provide good protection to immune animal, avoided antibiotic abuse, in case " superbacteria " occurs.
A further object of the present invention is to provide the preparation method of Staphylococcus sciuri inactivated vaccine, and the method is simple, and cost is low.
Object of the present invention adopts following technical proposals to realize.
The invention provides Staphylococcus sciuri (
staphylococcus sciuri) NJ1306, preserving number is CCTCC NO:M 2013702.
The present invention also provides Staphylococcus sciuri inactivated vaccine, the described Staphylococcus sciuri that contains deactivation (
staphylococcus sciuri) NJ1306.
In the present invention, described Staphylococcus sciuri inactivated vaccine is oil-emulsion.
In the present invention, the adjuvant of described Staphylococcus sciuri inactivated vaccine is MONTANIDE ISA 760 VG.
The present invention also provides the preparation method of Staphylococcus sciuri inactivated vaccine, comprises following steps:
(1) by described Staphylococcus sciuri (
staphylococcus sciuri) nutrient solution of NJ1306, adopt formalin-inactivated, obtain the Staphylococcus sciuri nutrient solution of deactivation.
(2) Staphylococcus sciuri nutrient solution and the adjuvant of deactivation are mixed, obtain the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri.
In preferred technical scheme, the add-on of formaldehyde described in step (1) be Staphylococcus sciuri (
staphylococcus sciuri) 0.4-0.6% of NJ1306 nutrient solution volume.
In the present invention, Staphylococcus sciuri described in step (1) (
staphylococcus sciuri) preparation method of NJ1306 nutrient solution is as follows: by Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 is inoculated in THB liquid nutrient medium, cultivate 20-28h, obtain described nutrient solution for 36-38 ℃.
In preferred technical scheme, the condition that adopts formalin-inactivated described in step (1) is deactivation 45-50h under 36-38 ℃ of condition.In preferred technical scheme, the adjuvant described in step (2) is MONTANIDE ISA 760 VG.
The present invention confirms that through polymerase chain reaction, biochemical test, gramstaining, morphological observation the staphylococcus separating is Staphylococcus sciuri, name this bacterium be Staphylococcus sciuri (
staphylococcus sciuri) NJ1306.
Experimentation on animals through BALB/c mouse, new zealand rabbit, Chang-Bai piglet: 6 of BALB/c mouse, abdominal injection 2 × 10
9cFU/, interior dead 5 of 24h, interior dead 1 of 48h; 3 of 2 kilograms of new zealand rabbits, ear vein injection 5 × 10
9cFU/ only, is survived; 52 of age in days Chang-Bai piglets, intramuscular injection 1 × 10
10cFU/ head, 2 all fall ill (skin forms not whole erythema and the incrustation of beige shape), skin is all turned out identical bacterium with blood.Confirm that this bacterium has pathogenic to BALB/c mouse and piglet.
This bacterium is prepared into the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri, 5 of immunity BALB/c mouse in 7 weeks age, only, immunity is after 14 days for 0.2mL/, attacks poison, 2 × 10 with the viable bacteria of this bacterium 24h cultivation
9cFU/, 4 survivals, 1 death.5 of immunity contrasts are not attacked all death of poison with same dose.The revision test of BALB/c mouse immunity contrast: get 12 7 week age BALB/c mouse, choose at random 6 as oil emulsion vaccine group, remaining mouse as a control group.The oil-emulsion inactivated vaccinating agent of oil emulsion vaccine group mouse immune Staphylococcus sciuri, 0.2mL/ is only; Control group is not immune.After immunity 14 days, two groups of mouse are attacked to poison, 2 × 10 with the viable bacteria of this bacterium 24h cultivation
9cFU/ only.6 BALB/c mouse survivals of oil emulsion vaccine group, 6 mouse in control group are all dead.The oil-emulsion inactivated vaccinating agent of the above results explanation Staphylococcus sciuri can provide good immunizing power to BALB/c mouse, by immune simultaneous test, proves that this bacterium has good immunogenicity.
beneficial effect
1) Staphylococcus sciuri of the present invention (
staphylococcus sciuri) NJ1306 has pathogenic to BALB/c mouse and piglet.
2) Staphylococcus sciuri of the present invention (
staphylococcus sciuri) NJ1306 adopt PCR be accredited as methicillin-resistant Staphylococcus (methicillin-resistant
staphylococci, MRS), containing mecA gene, this genes encoding penicillin-binding protein 2a (PBP2a) is lower, therefore insensitive to this class microbiotic with β-lactam antibitics avidity,, make it show resistance.
3) adopt Staphylococcus sciuri of the present invention (
staphylococcus sciuri) oil-emulsion inactivated vaccinating agent that NJ1306 and MONTANIDE ISA 760 VG make, after immunity, attack poison, can provide good protection to BALB/c mouse, illustrate Staphylococcus sciuri of the present invention (
staphylococcus sciuri) NJ1306 has good immunogenicity, can be used as vaccine bacterial strain.Adopt the vaccine of the present invention can preventing infection Staphylococcus sciuri, avoided antibiotic abuse, in case there is " superbacteria ".
4) the present invention provides the good Staphylococcus sciuri of a kind of immune effect oil-emulsion inactivated vaccinating agent first.The preparation method of the oil-emulsion inactivated vaccinating agent of this Staphylococcus sciuri is simple, and cost is low.This vaccine of evidence has immanoprotection action, can be for zooprophylazis.For Staphylococcus sciuri disease, there is no desirable applicable animal and human group's vaccine at present, so prevention Staphylococcus sciuri disease is not only the protection to animal, the guarantee to people especially.Therefore, the market outlook of the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri of the present invention are wide.
Accompanying drawing explanation
Fig. 1 is that PCR detects Staphylococcus specificity 16S rRNA(Staph 16S rRNA), leueocidin gene (PVL) and drug resistant gene (
meca) electrophorogram, wherein swimming lane M is Marker; Swimming lane 1 be the special 16S rRNA of Staphylococcus, PVL and
mecthe multiplex PCR amplified production of A gene, swimming lane 2 is the pcr amplification product of Staphylococcus specificity 16S rRNA, swimming lane 3 is the pcr amplification product of leueocidin gene (PVL), swimming lane 4 be drug resistant gene (
meca) pcr amplification product.
Fig. 2 PCR detects 23S rDNA, Exh-A, Exh-B, Exh-C, the electrophorogram of Exh-D gene, wherein swimming lane M:Marker; Swimming lane 1 is 23S rDNA amplified production; Swimming lane 2-5 is respectively the amplified production of decortication malicious A, B, C, D, and the product of 23S rDNA is the positive control of PCR for this reason, and the successful amplification of this gene confirms that the genome quality and the content that extract are all no problem.
Fig. 3 is the microphotograph after the gramstaining of bacterial strain.
Fig. 4 is the picture of bacterial strain of the present invention to Chang-Bai piglet Virulence detection.
Fig. 5 shown infection various dose Staphylococcus sciuri (
staphylococcus sciuri) the mouse survival situation of NJ1306.
Fig. 6 has shown that in BALB/c mouse immunity contrast experiment, each group mouse is attacked the rear survival rate of poison over time.
Fig. 7 has shown that in the immune revision test contrasting of BALB/c mouse, each group mouse is attacked the rear survival rate of poison over time.
Embodiment
Material:
PCR Mix: Dongsheng bio tech ltd, Guangzhou;
DNA glue reclaims test kit: Axygen company;
Staphylococcus TH-16S bacteria bio coding assessor: sky, Hangzhou and microorganism reagent company limited;
Bacto
tMtodd Hewitt Broth(THB) substratum: BD company;
THB liquid nutrient medium: take 30g THB
substratum, with being settled to 1000mL after deionized water dissolving, 121 ℃ of autoclavings 20 minutes;
THB solid medium: add 15g agar powder in every 1000mL THB liquid nutrient medium, 121 ℃ of autoclavings 20 minutes, solidify in appropriate impouring plate;
Commodity oil emulsion adjuvant MONTANIDE ISA 760 VG: French SEPPIC company.
Gather morbidity piglet skin incrustation sample and pericardial effusion from pig farm, Nanjing, be separated to Staphylococcus sciuri (
staphylococcus sciuri) NJ1306.
(1) increasing bacterium cultivates:
The piglet skin of falling ill incrustation sample and pericardial effusion, put into 5mL THB liquid nutrient medium, and 37 ℃ leave standstill cultivation 16～18h, and results culture is for subsequent use.
(2) pure culture of bacterium:
Adopt method of scoring to be seeded to THB solid medium culture described in step (1), 37 ℃ leave standstill cultivation 24h, picking list colony inoculation THB liquid nutrient medium, and 37 ℃ leave standstill cultivation 24h, obtain the propagation bacterium liquid of single bacterium.
(3) gene identification of bacterium:
The described propagation of step (2) bacterium liquid is carried out centrifugal, adopt ordinary method to extract total DNA of thalline as template, take StaphF and StaphR as primer amplification l6S rRNA gene fragment, then order-checking, sequence is as shown in SEQ ID NO:1.
The sequence of StaphF is: AACTCTGTTATTAGGGAAGAACA; The sequence of StaphR is: CCACCTTCCTCCGGTTTGTCACC.
Known array on sequencing result and GenBank is carried out to homology comparison, finds that the nucleic acid sequence homology of this bacterium and Staphylococcus sciuri DSM20345 reaches 99.6%, determine the bacterial strain that is separated to be Staphylococcus sciuri (
staphylococcus sciuri), called after Staphylococcus sciuri (
staphylococcus sciuri) NJ1306.
With Staphylococcus sciuri (
staphylococcus sciuri) total DNA of NJ1306 is template, adopts PCR to identify that whether it is containing killing white corpuscle gene (Panton-Valentine leukocidin, PVL), resistance mecA gene, result is as shown in Figure 1.Take Luk-PVF and Luk-PVR as primer, amplification is not to PVL gene, illustrate Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 is containing killing white corpuscle gene.Take MecAF and MecAR as primer, amplification is to the MecA gene of 310 bp, illustrate Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 is containing mecA gene, the penicillin-binding protein 2a (PBP2a) of this genes encoding is lower, therefore insensitive to this class microbiotic with β-lactam antibitics avidity, therefore, Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 is methicillin-resistant Staphylococcus (methicillin-resistant
staphylococci, MRS).
From Fig. 2, can find out with Staphylococcus sciuri (
staphylococcus sciuri) total DNA of NJ1306 is template, the 23S rDNA(positive control of 662 bp that can increase), but can not increase decortication toxin gene (Exh-A, Exh-B, Exh-C, Exh-D), illustrate Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 containing decortication toxin gene (Exh-A, Exh-B, Exh-C, Exh-D).
In above-mentioned PCR, increase the primer of each gene and object product as shown in Tables 1 and 2.
Table 1 PCR detects the primer sequence of Staphylococcus specificity 16S rRNA, leueocidin gene and drug resistant gene
Table 2 PCR detects the primer sequence of 23S rDNA and decortication toxin gene
(4) ne ar feature:
Adopt method of scoring to be seeded to THB solid medium the propagation bacterium liquid in step (2), 37 ℃ leave standstill cultivation 24h, observe colonial morphology.Result is as follows: bacterium colony size 1-2mm, be that meat white, rule are circular, opaque, smooth surface, neat in edge.Single colony inoculation is cultivated after 24 h in blood agar culture-medium, do not occurred zone of hemolysis.
Using gram staining method to dye to bacterium colony to be checked, can be observed hepatic coccus (Fig. 3) under microscope, be thyrsiform and arrange, is circular or ovate gram-positive cocci, but when its aging, dead negative.Tentatively be defined as staphylococcus.
(5) biochemical identification:
Single colony inoculation is carried out to biochemical identification in Staphylococcus TH-16S bacteria bio coding assessor (project: urea, sucrose, arginine hydrolysis, wood sugar, lactose, seminose, maltose, Xylitol, N.F,USP MANNITOL, gill fungus sugar, acetylglucosamine, nitrate reduction, melibiose, fructose, V-P, sorbyl alcohol), and concrete outcome is as shown in table 3.Contrast Staphylococcus TH-16S bacteria bio coding testimonial, determines the bacterial strain coding and the identification rate that are separated to after analyzing, result shows that this bacterium is encoded to 233721, coincidence rate %ID>=99.9, expected value T=1, be accredited as Staphylococcus sciuri (
staphylococcus sciuri), conform to gene identification result.
The biochemical identification result of table 3 isolated strains
Note: "+" represents the biochemical test positive: variable color."-" represents biochemical test feminine gender: nondiscoloration.
In addition, this bacterium catalase is positive, and plasma-coagulase is negative, and oxydase reaction is positive, to Vulkamycin. PA-93 resistance.
Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 preservation information is as follows:
Classification And Nomenclature: Staphylococcus sciuri NJ1306
Staphylococcus sciuri NJ1306；
Depositary institution: Chinese Typical Representative culture collection center;
Address: China. Wuhan. Wuhan University; Preservation date: on December 24th, 2013;
Deposit number: CCTCC NO:M 2013702.
(1) BALB/c mouse Virulence detection:
7 week age 6 of BALB/c mouse, abdominal injection Staphylococcus sciuri (
staphylococcus sciuri) NJ1306, dosage is 2 × 10
9cFU/, interior dead 5 of 24h, interior dead 1 of 48h.It is pathogenic that the above results illustrates that this bacterium has BALB/c mouse.
(2) new zealand rabbit Virulence detection:
3 of 2 kilograms of new zealand rabbits, ear vein injection Staphylococcus sciuri (
staphylococcus sciuri) NJ1306, dosage is 5 × 10
9cFU/ only, is survived.The above results illustrates that this bacterium is to new zealand rabbit no pathogenicity.
(3) Chang-Bai piglet Virulence detection:
52 of age in days Chang-Bai piglets, intramuscular injection Staphylococcus sciuri (
staphylococcus sciuri) NJ1306, dosage is 1 × 10
10cFU/ head.All fall ill for 2: be short of breath, skin forms not whole erythema and the incrustation (Fig. 4) of beige shape, and skin is all turned out identical bacterium with blood, confirms that this bacterium has piglet pathogenic.Attacking malicious the 14th day to the sick pigs of 2 hairs and 2 contrast pigs blood sampling cultivations, be all separated to identical bacterium, illustrate that Staphylococcus sciuri can be communicated by contact.
(4) test of different infective dose mouse survival situations:
By the Staphylococcus sciuri of various dose (
staphylococcus sciuri) NJ1306 infects BALB/c mouse in 7 week age by abdominal injection, respectively organizes 5, result is as shown in table 4 and Fig. 5, and abdominal injection dosage is greater than every 2.5 × 10
8two groups of CFU, mouse is all dead.Therefore, can be with being more than or equal to 5 × 10
8cFU/ the toxic agent amount of attacking as BALB/c mouse.
The toxicity test of table 4 Staphylococcus sciuri to BALB/c mouse
| |
1×10 9CFU/ only | 5×10 8CFU/ only | 2.5×10 8CFU/ only |
| Survival number (only) | 0 | 0 | 5 |
| Death toll (only) | 5 | 5 | 0 |
(5) BALB/c mouse immunity simultaneous test:
The preparation of the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri: by Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 adopt method of scoring be seeded to THB solid medium (
bacto tM todd Hewitt Broth, THB), 37 ℃ of incubated overnight, the single colony inoculation obtaining, in THB liquid nutrient medium, is cultivated 24h for 37 ℃, obtains viable bacteria nutrient solution, adjusts concentration to 5 × 10
9cFU/mL.Be 5 × 10 by concentration
9the viable bacteria nutrient solution of CFU/mL is that 1:0.005 mixes with formaldehyde according to volume ratio, and deactivation 48h under 37 ℃ of conditions obtains the Staphylococcus sciuri nutrient solution of deactivation.Be that 2:3 mix with adjuvant MONTANIDE ISA 760 VG according to volume ratio by the Staphylococcus sciuri nutrient solution of deactivation, obtain the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri.
Get 10 7 week age BALB/c mouse, be divided at random 2 groups, 5 every group; Wherein first group as oil emulsion vaccine group, the oil-emulsion inactivated vaccinating agent of immune Staphylococcus sciuri, and 0.2mL/ is only; Second group as a control group, not immune.Immunity 14 days after, with 24h cultivate Staphylococcus sciuri (
staphylococcus sciuri) NJ1306 viable bacteria attacks poison, attacking toxic agent amount is 2 × 10
9cFU/ only.Attack after poison 4 mouse survivals in oil emulsion vaccine group, 1 dead mouse; The all mouse of control group are all dead, and result is as shown in table 5 and Fig. 6.The above results explanation, the oil-emulsion inactivated vaccinating agent of this Staphylococcus sciuri can provide good protective capability to BALB/c mouse, by immune simultaneous test, proves that this bacterium can be used for the development of oil-emulsion inactivated vaccinating agent.
The each group of table 5 mouse is attacked survival and the death toll after poison
| Project | Oil emulsion vaccine group | Control group |
| Survival number (only) | 4 | 0 |
| Death toll (only) | 1 | 5 |
(6) revision test of BALB/c mouse immunity contrast:
Get 12 7 week age BALB/c mouse, choose at random 6 as oil emulsion vaccine group, remaining mouse as a control group.The oil-emulsion inactivated vaccinating agent of oil emulsion vaccine group mouse immune Staphylococcus sciuri (the present embodiment title (5) preparation), 0.2mL/ is only; Control group is not immune.After immunity 14 days, use Staphylococcus sciuri (
staphylococcus sciuri) viable bacteria of NJ1306 attacks poison, 2 × 10 to two groups of mouse
9cFU/ only.Result, as shown in table 6 and Fig. 7, can be found out, attacks after poison, and 6 BALB/c mouse survivals of oil emulsion vaccine group, 6 mouse in control group are all dead.Revision test has been verified the reliability of vaccine immunity, and the oil-emulsion inactivated vaccinating agent immune effect of proving again Staphylococcus sciuri is good.
The each group of table 6 mouse is attacked survival and the death toll after poison
| Project | Oil emulsion vaccine group | Control group |
| Survival number (only) | 6 | 0 |
| Death toll (only) | 0 | 6 |
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> mono-strain Staphylococcus sciuri, vaccine and preparation method thereof
<130> 20140401
<160> 17
<170> PatentIn version 3.3
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agtggaattc catgtgtagc ggtgaaatgc gcagagatat ggaggaacac cagtggcgaa 300
ggcggctctc tggtctgtaa ctgacgctga tgtgcgaaag cgtggggatc aaacaggatt 360
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Claims (9)
- Staphylococcus sciuri ( staphylococcus sciuri) NJ1306, preserving number is CCTCC NO:M 2013702.
- 2. Staphylococcus sciuri inactivated vaccine, Staphylococcus sciuri described in the claim 1 that it is characterized in that containing deactivation ( staphylococcus sciuri) NJ1306.
- 3. Staphylococcus sciuri inactivated vaccine according to claim 2, is characterized in that described vaccine is oil-emulsion.
- 4. Staphylococcus sciuri inactivated vaccine according to claim 3, the adjuvant that it is characterized in that described oil emulsion vaccine is MONTANIDE ISA 760 VG.
- 5. the preparation method of Staphylococcus sciuri inactivated vaccine, is characterized in that comprising following steps:(1) by Staphylococcus sciuri described in claim 1 ( staphylococcus sciuri) nutrient solution of NJ1306, adopt formalin-inactivated, obtain the Staphylococcus sciuri nutrient solution of deactivation;(2) Staphylococcus sciuri nutrient solution and the adjuvant of deactivation are mixed, obtain the oil-emulsion inactivated vaccinating agent of Staphylococcus sciuri.
- 6. the preparation method of Staphylococcus sciuri inactivated vaccine according to claim 5, the add-on that it is characterized in that formaldehyde described in step (1) be Staphylococcus sciuri ( staphylococcus sciuri) 0.4-0.6% of NJ1306 nutrient solution volume.
- 7. the preparation method of Staphylococcus sciuri inactivated vaccine according to claim 6, is characterized in that: Staphylococcus sciuri described in step (1) ( staphylococcus sciuri) preparation method of NJ1306 nutrient solution is as follows: by Staphylococcus sciuri ( staphylococcus sciuri) NJ1306 is inoculated in THB liquid nutrient medium, cultivate 20-28h, obtain described nutrient solution for 36-38 ℃.
- 8. according to the preparation method of the described Staphylococcus sciuri inactivated vaccine of one of claim 5-7, it is characterized in that: the condition that adopts formalin-inactivated in step (1) is deactivation 45-50h under 36-38 ℃ of condition.
- 9. the preparation method of Staphylococcus sciuri inactivated vaccine according to claim 8, is characterized in that: the adjuvant described in step (2) is MONTANIDE ISA 760 VG.
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| CN108220463A (en) * | 2018-03-14 | 2018-06-29 | 江苏省农业科学院 | A kind of PCR primer, kit and detection method for detecting Staphylococcus sciuri |
| CN111733106A (en) * | 2020-07-06 | 2020-10-02 | 山东卓苒生物科技有限公司 | Culture method and application of staphylococcus squirrel F-E8-1 |
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