CN102114241A - Attenuated live vaccine and application thereof - Google Patents
Attenuated live vaccine and application thereof Download PDFInfo
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- CN102114241A CN102114241A CN 201010292145 CN201010292145A CN102114241A CN 102114241 A CN102114241 A CN 102114241A CN 201010292145 CN201010292145 CN 201010292145 CN 201010292145 A CN201010292145 A CN 201010292145A CN 102114241 A CN102114241 A CN 102114241A
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Abstract
The invention provides an attenuated live vaccine and an application thereof. The collection number of the attenuated live vaccine is CCTCC M2010185. The attenuated live vaccine is a nontoxic enterohemorrhagic escherichia coli O157: H7 strain, which contains enterohemorrhagic escherichia coli O157: H7 antigens. The attenuated live vaccine can be used for preparing preparations for preventing enterohemorrhagic escherichia coli O157: H7 infection. The strain of the attenuated live vaccine is convenient to prepare, has all the antigens of the enterohemorrhagic escherichia coli O157: H7, can stimulate the systemic anti-enterohemorrhagic escherichia coli O157: H7 immunoreaction and further has a good protective effect.
Description
Technical field
The present invention relates to a kind of vaccine, relate in particular to a kind of attenuated live vaccine and application thereof.
Background technology
Enterohemorrhagic Escherichia coli (enterohermorrhangic E.coli, EHEC) O 157:H7 (EHECO157) is a kind of important infectious diseases common to human beings and animals cause of disease, it infects has become global public health problem.Since nineteen eighty-two, this bacterium was confirmed to be serious pathogenic bacterium first, a lot of countries and regions, the whole world comprised all eruption and prevalences repeatedly of China.EHEC O157 infection host can cause disease and severe complications such as diarrhoea, hemorrhagic enteritis, haemolysis uraemic syndrome.Because antibiotic therapy can aggravate to infect the danger of the state of an illness, still there are not effectively prevention and control measure at present.EHEC O157 bacterium is cultivated easily, appeal is strong, the route of transmission is various, makes it very likely as the bacteriological weapon and the bio-terrorism war agent of following military struggle, and the strong virulence factor of EHEC O157 bacterium also might be used for the structure of genetic weapon.The Center for Disease Control has been classified it as category-B bio-terrorism pathogen and has strictly been taken precautions against.Therefore, still be the needs of biological anti-terrorism no matter from public health, development EHEC O157 vaccine is strengthened its prevention and control research and is had great strategic importance.
EHEC O157 causes what the serious infectious disease of host was mainly realized by three aspects.1) shiga toxin (shiga toxin, Stx): shiga toxin enters cell at cell surface receptor under in conjunction with mediation, suppresses the synthetic of cell protein, finally causes organizing serious pathology damage, causes infectious disease; 2) EHECO157 induces the violent inflammatory reaction in intestinal infection position by the effect of the surface antigen and the inflammatory stimulus factor (OmpA, intimin etc.), causes the electrolyte balance disorder; 3) adhere to and wiping damage (Attaching andeffacing lesions, A/E lesions): the antibacterial tight adhesion is in the enterocyte surface, interaction (intimin-Tir by a series of signal transducin, Tir-Cortactin, TCCP-N-WASP etc.), cause the gathering of bacterial adhesion position actin, cause enterocyte characteristic cases such as microvillus destruction, brush border damage to change, the barrier cell function is destroyed, and causes infectious disease.
OmpA, Intimin and Stx2B are the important virulence factor of enterorrhagia Bacillus coil 0157: H7, also be the protective antigen of antibacterial simultaneously, intravital anti-OmpA (Torres AG, Kaper JB et al.Infect Immun 2003 Sep have been evidence suggests at present; 71 (9): 4985-95.), anti-intimin (Gu; J.; Y.Liu; etal._Microbes Infect 2,009 11 (10-11): 835-841) with anti-Stx2B (Ma; Y.; X.Mao, et al.Immunol Lett 2008121 (2): antibody 110-115.) can be protected the infection of the enterorrhagia Bacillus coil 0157 of fatal dose: H7.
Summary of the invention
The object of the present invention is to provide a kind of attenuated live vaccine, its preserving number is: CCTCC M 2010185.
Described attenuated live vaccine comprises enterorrhagia Bacillus coil 0157: the antigen of H7.
Described antigen has antigen OmpA, Intimin and Stx2B.
The application of attenuated live vaccine in the preparation that preparation prevention enterorrhagia Bacillus coil 0157: H7 infects.
Attenuated live vaccine in the described application is induced anti-OmpA, the Intimin of generation protectiveness and the antibody of Stx2B.
EHEC O157 bacterium itself is cultivated convenient, economy.In the attenuated live vaccine mice immunized serum that through preserving number is CCTCC M 2010185, detect tangible anti-enterorrhagia Bacillus coil 0157: the antibody of H7 bacterial strain, and mice has certain protection during through the virulent strain counteracting toxic substances.
Attenuated live vaccine bacterial strain of the present invention is easy to prepare, and itself has enterorrhagia Bacillus coil 0157: whole antigens of H7, can the anti-enterorrhagia Bacillus coil 0157 of stimulating system: the H7 immunoreation has the better protect effect.
In preservation on July 23 in 2010, its preserving number was CCTCCNO:M2010185 to attenuated live vaccine of the present invention; Depositary institution: Chinese typical culture collection center; Address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University; Classification name: enterohemorrhagic Escherichia coli EHECO157:H700B015 (Enterohemorrhagic Escherichia coli EHEC O157:H700B015).
The specific embodiment
The embodiment that provides below is used for further illustrating the present invention, and does not constitute limitation of the scope of the invention.
Embodiment 1The immunoreation that enterorrhagia Bacillus coil 0157: H7 bacterial strain 00B015 (preserving number is CCTCC M 2010185) stimulates
1 experimental technique
1) enterorrhagia Bacillus coil 0157: the isolation identification of H7 bacterial strain 00B015:
Enterorrhagia Bacillus coil 0157: H7 bacterial strain 00B015 separates from the common diarrhoea patient of Jiangsu Disease Control and Prevention Center, the method of its separation and evaluation is carried out with reference to " national enterorrhagia Bacillus coil 0157: H7 district monitoring scheme (trying) ", culture presevation is in China typical culture collection center, and preserving number is CCTCC M 2010185.
2) with 20 6 age in week the Balb/c mice be equally divided into 2 groups, 10 every group, each organizes mice before oral vaccination, at one night of fasting, prohibits water 4h.Give 3% sodium bicarbonate solution 300ul earlier and irritate stomach, in and gastric acid, prevent the destruction of gastric acid to bacterial strain, behind the 20min, get the above-mentioned bacterium liquid feed for preparing of 300ul and respectively organize mice (about 1 * 10
9CFU/ is only), recover feed to mice behind the 30min.0,14, each feed of 28d once, totally 3 times.Respectively before feed, feed 2 times and feed 3 after this 7 days are taked mice serum from the tail vein, preserve-80 ℃ standby.
3) ELISA detects behind the feed antigenic specificity IgG level of anti-enterohemorrhagic Escherichia coli main immunogens OmpA, Intimin and Stx2B in the mice serum:
Immunogen OmpA, the Intimin and Stx2B (Torres AG, Kaper JB et al.Infect Immun 2003 Sep that prepare purification according to the method for document; 71 (9): 4985-95, Gu, J., Y.Liu, et al._MicrobesInfect 2,009 11 (10-11): 835-841).Gather venous blood from mouse tail vein, beginning was with normal saline doubling dilution serum to be checked in 1: 100, by the elisa plate of 100 μ l/ holes adding corresponding antigens albumen bag quilt, place 1h for 37 ℃, PBS-Tween20 washes 4 times, 100 μ l/ holes add the sheep anti-mouse igg (1: 20000) of HR labelling, place 1h for 37 ℃, PBS-Tween20 washes 4 times, adds OPD substrate colour developing liquid, room temperature lucifuge reaction 15min, 2mol/L H
2SO
4Cessation reaction is made blank with the PBS hole, and 492nm measures each hole OD value.OD
SampleWith OD
NegativeRatio 〉=2.1 positive.
2 experimental results
The variation of three anti-OmpA antibody of oral back mice serum
*Compare with the PBS group, there were significant differences (P＜0.01)
The variation of three anti-Intimin antibody of oral back mice serum
*Compare with the PBS group, there were significant differences (P＜0.01)
The variation of three anti-Stx2B antibody of oral back mice serum
*Compare with the PBS group, there were significant differences (P＜0.01)
Embodiment 2Enterorrhagia Bacillus coil 0157: the immune protective of H7 bacterial strain 00B015 is identified
1 experimental technique
Mice is drunk 3d in the back aseptic aqueous solution that contained the 5g/L streptomycin in 10 days of 3 immunity, again with 1 * 10
9The O157-SM of CFU/ dosage
R2 (Cheng Jianping, Yi Yong, Mao Xuhu, the foundation [J] of the bright .O157:H7 coli-infection of Zou Quan animal model. JOURNAL OF MICROBIOLOGY, 2004, (5) .), full bacterium liquid at twice, 6h at interval, per os pours into mouse GI tract, attack the activity of close observation mice behind the bacterium, ingest and the change of the mental status, calculate the death toll of mice when 28 day observation period finished, get behind the mice counteracting toxic substances 3 simultaneously, 7,14,28d feces number, physiological saline solution is resuspended, vortex mixer shakes broken, coats SMAC selective medium flat board, analyzes the situation that stool in mice behind the bacterium is got rid of EHEC O157:H7 of attacking.Calculate infection rate and protective rate at last.
The calculating of infection rate and protective rate:
Infect number=dead animal number+feces and cultivated number positive in 14 days
2 experimental results
1) clinical manifestation:
PBS group mice is at O157-SM
RAll show lethargy, anorexia, the fluffy color jaundice of Mus hair behind 2 counteracting toxic substances behind 2～3d, psychological problem, tic.Recombinant immune group mice has symptoms such as slight Mus hair is fluffy, drowsiness in initial infection, and it is normal that 3～4d recovers.
2) mice O157 infection conditions behind the counteracting toxic substances
*Compare with the PBS group, there were significant differences (P＜0.05),
*Compare with the PBS group, there were significant differences (P＜0.01)
Can learn that by the data in the table immunoreation that causes behind the 00B015 feed can protect mice to be subjected to the attack of the EHEC O157:H7 of fatal dose, illustrates that 00B015 has significant protection in mice animal level.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
Claims (5)
1. attenuated live vaccine, its preserving number is: CCTCC M 2010185.
2. attenuated live vaccine according to claim 1 is characterized in that, comprises enterorrhagia Bacillus coil 0157: the antigen of H7.
3. attenuated live vaccine according to claim 2 is characterized in that described antigen has antigen OmpA, Intimin and Stx2B.
4. the application of the described attenuated live vaccine of claim 1 in the preparation that preparation prevention enterorrhagia Bacillus coil 0157: H7 infects.
5. application according to claim 4 is characterized in that, described attenuated live vaccine is induced anti-OmpA, the Intimin of generation protectiveness and the antibody of Stx2B.
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| CN2010102921455A CN102114241B (en) | 2010-09-26 | 2010-09-26 | Attenuated live vaccine and application thereof |
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| CN2010102921455A CN102114241B (en) | 2010-09-26 | 2010-09-26 | Attenuated live vaccine and application thereof |
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| CN102114241A true CN102114241A (en) | 2011-07-06 |
| CN102114241B CN102114241B (en) | 2012-07-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018228A (en) * | 2017-09-28 | 2018-05-11 | 江苏省农业科学院 | One plant of Escherichia coli O 157:H7 low virulent strains and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1631438A (en) * | 2004-03-27 | 2005-06-29 | 中国人民解放军第三军医大学 | 0157 bacterium gene engineering multivalence subunit vaccine of human and sensitive animals and its preparing method |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1631438A (en) * | 2004-03-27 | 2005-06-29 | 中国人民解放军第三军医大学 | 0157 bacterium gene engineering multivalence subunit vaccine of human and sensitive animals and its preparing method |
| CN101062410A (en) * | 2007-02-05 | 2007-10-31 | 中国人民解放军第三军医大学 | Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《免疫学杂志》 20060731 毛旭虎等 致泻性大肠杆菌疫苗研究进展 466-468 1-5 第22卷, 第4期 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018228A (en) * | 2017-09-28 | 2018-05-11 | 江苏省农业科学院 | One plant of Escherichia coli O 157:H7 low virulent strains and its application |
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| CN102114241B (en) | 2012-07-04 |
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