CN101264322A - Listeria monocytogenes inactivated vaccine and preparation thereof - Google Patents
Listeria monocytogenes inactivated vaccine and preparation thereof Download PDFInfo
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- CN101264322A CN101264322A CNA2008100253353A CN200810025335A CN101264322A CN 101264322 A CN101264322 A CN 101264322A CN A2008100253353 A CNA2008100253353 A CN A2008100253353A CN 200810025335 A CN200810025335 A CN 200810025335A CN 101264322 A CN101264322 A CN 101264322A
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- listeria monocytogenes
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Abstract
The invention relates to a Listeria monocytogenes inactivated vaccine and the preparation method, belonging to biotechnical field. The preparation method of Listeria monocytogenes inactivated vaccine is characterized in that Listeria monocytogenes is processed by <SUP>60</SUP>Co irradiation and inactivation to prepare the inactivated vaccine. The protection antigen component in the pathogen surface is remained, which can excite the protection T cell immune response and body fluid immune response. The advantages are significantly important when the inactivated vaccine aiming at intracellular pathogen is researched.
Description
Technical field
The present invention relates to biological technical field, specifically is a kind of Listeria monocytogenes inactivated vaccine and preparation method thereof.
Technical background
Listeria monocytogenes (Listeria monocytogenes, LM, be called listeria monocytogenes again) be growth, amphimicrobian Gram-positive bacillus in a kind of born of the same parents, be a kind of important foodborne pathogens, can cause the infecting both domestic animals and human listeriosis.LM causes a disease to multiple poultry, and higher fatality rate arranged, susceptible colony is anemia of pregnant woman, baby, old people and immunocompromised in the crowd, mainly cause meningitis, miscarriage and immunodeficiency patient and neonatal central nervous system's infection, though sickness rate is low, but mortality rate is up to 30%～70%, and therefore the research to LM has important public health meaning.Given this, people are developing the prevention and control that vaccine safely and effectively is used for listeriosis.
At present, widely used vaccine mainly comprises types such as inactivated vaccine, attenuated live vaccine and recombinant vaccine.Attenuated live vaccine is used the microorganism of attenuation or avirulent work and is made.Behind the animal inoculation, the immunity that is obtained is lasting and strong, but has the danger of reversion on remaining virulence and the virulence.Recombinant vaccine is divided into multiple, wherein gene-deleted vaccine is the disappearance gene relevant with the pathogen virulence on DNA or cDNA level, but not obvious its replication capacity that influences, do not destroy its immunity as vaccine strain, a little less than this kind vaccine virulence, can reversion, safety is good, but generally the lead time long, safety waits further checking.The inactivated vaccine method of production is simple, quick, and most of vaccine of developing at present all belongs to this type.Traditional inactivated vaccine generally passes through formalin-inactivated or hot inactivation treatment, and the immunne response that body produces is based on humoral immunization, and the cellular immunization that causes is very little.For class born of the same parents endophytes such as Listeria monocytogenes, cellular immunization seems even more important in the effect of removing and produce in the long-acting immunoprotection.Therefore, it is significant to seek the new technology of preparing of inactivated vaccine.
Antibacterial loses the ability that causes disease through the effect of irradiation complete deactivation, and irradiated antibacterial has kept complete architectural feature simultaneously, can the excitating organism immune system start comprehensively defence.The most important thing is that irradiation deactivation antibacterial can bring out body and produce cellullar immunologic response, cytotoxic T cell kills and wounds parasitic the target cell of LM, and it is removed completely.Inactivated vaccine is simple and safe, cost is low, fabrication cycle is short so produce by irradiance method, but does not also have the report of being correlated with so far.
Summary of the invention
An object of the present invention is to overcome the defective of above-mentioned inactivated vaccine through the traditional method preparation, the preparation method that can excitating organism produces the inactivated vaccine of humoral immunization and cellular immunization is provided, promptly pass through
60Co irradiation prepares Listeria monocytogenes inactivated vaccine.
Purpose of the present invention is achieved by the following technical programs:
A kind of method for preparing Listeria monocytogenes inactivated vaccine is characterized in that, with the Listeria monocytogenes warp
60The deactivation of Co irradiation makes inactivated vaccine.
The condition of above-mentioned said irradiation, specifically: bacteria samples be in freezing state with
60The irradiation of Co irradiation bomb, irradiation dose is not less than 15KGy.Radiation mode is static irradiation, close rate 60Gy/min.
Can realize that the object of the invention antibacterial comprises all Listeria monocytogenes, specifically can be Listeria monocytogenes yzu LM 1-2, or Listeria monocytogenes LM-4.
Another object of the present invention is to provide by above-mentioned
60The Listeria monocytogenes inactivated vaccine that Co irradiation makes.
Beneficial effect of the present invention is embodied in: by the method for irradiation deactivation, kept the protective antigen composition on pathogen surface, can excite protectiveness T cellullar immunologic response and humoral immunoresponse(HI).These advantages seem particularly important in development during at the inactivated vaccine of intra-cellular pathogens.
Figure of description
Fig. 1 is the electron microscopic observation figure as a result of irradiation deactivation LM
Fig. 2 is antibacterial separating resulting figure in organizing behind the immune counteracting toxic substances
Fig. 3 is antibacterial separating resulting figure in organizing behind the counteracting toxic substances in the experiment of T cell transfer
The specific embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with concrete preparation embodiment and effect embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person indicate when occurring first that all used thereafter identical reagent if no special instructions, and is all identical with the content of indicating first.
Bacterial strain: Listeria monocytogenes yzu LM 1-2, preserving number CCTCC NO.M206107.
Listeria monocytogenes LM-4 is separated and preservation by the applicant.
Embodiment 1: irradiation deactivation antibacterial
Antibacterial is prepared: yzu LM 1-2 rules on the BHI flat board, behind 37 ℃ of cultivation 24h, and picking list colony inoculation liquid B HI culture medium, 37 ℃ of joltings are cultured to exponential phase, centrifugal results thalline with phosphate buffered solution washing, suspension, is adjusted bacterial concentration to 1 * 10
10CFU/ml.
The irradiation deactivation: the bacteria samples of prepared fresh be in freezing state with
60The irradiation of Co irradiation bomb, irradiation dose is 15KGy, and radiation mode is static irradiation, and close rate is 60Gy/min.
The scanning electron microscopic observation ne ar: the yzu LM 1-2 of irradiation deactivation is through scanning electron microscopic observation, and the result does not see that bacterium surface has tangible structural damage as shown in Figure 1.
Embodiment 2: irradiation inactivated vaccine immune effect is estimated
Animal grouping and immunity inoculation: with 6～8 ages in week female BALB/c mouse be divided into 5 groups at random, be respectively viable bacteria group, irradiation deactivation group, hot deactivation group, formalin-inactivated group and negative control group, every group of 24 mices.The yzu LM1-2 and the sterilization PBS of lumbar injection viable bacteria yzu LM 1-2, the yzu LM 1-2 of irradiation deactivation, heat-inactivated yzu LM 1-2, formalin-inactivated respectively in the 0th, during 7d.Immunizing dose is respectively 1 * 10
3CFU/, 1 * 10
9CFU/, 1 * 10
9CFU/, 1 * 10
9CFU/ and 0.1ml/.
The mensuration of antibody titer in the mice serum of immunity back: each is organized mice and exempts from back 7d one respectively, and two exempt from back 14d, 21d eye socket venous blood collection, centrifugal preparation serum.Best antigen coated concentration is determined in the square formation experiment, and indirect ELISA is measured antibody titer in the immune mouse serum.Concrete grammar is as follows: every hole adds 100 μ l, 5% glutaraldehyde solution in (1) elisa plate, 37 ℃ of effect 2h, distilled water wash 3 times; (2) the yzu LM 1-2 of fresh cultured suspends with the PBS washing, adjusts bacterial concentration to 5 * 10
8CFU/ml adds above-mentioned bacterial suspension 50 μ l/ holes, 37 ℃ of oven dry; (3) every hole adds 200 μ l confining liquids (the PBS solution that contains 10% calf serum), and 4 ℃ are spent the night, and PBST (the PBS solution that contains 0.05%Tween-20) washes 5min/ time 3 times; (4) test serum doubling dilution, 50 μ, 1/ hole adds in the entering plate, hatches 1.5h for 37 ℃, and PBST washes 3 times, 5min/ time; (5) the horseradish peroxidase-labeled sheep anti-mouse igg and the IgM of adding working concentration, 37 ℃ of effect 1.5h, PBST washes 3 times, 5min/ time; (6) the every hole 50 μ l of OPD colour developing liquid of the fresh configuration of adding, 37 ℃ of water-bath lucifuges colour developing 10～15min; (7) 2M H
2SO
4Cessation reaction, OD is measured in the every hole of 50 μ l
490Value.Be judged to the positive with P/N 〉=2.1, with inverse the tiring of the antibody maximum dilution multiple that can satisfy P/N 〉=2.1 as antibody.The result is as shown in table 1, can produce higher antibody horizontal after the irradiation inactivated vaccine immunity BALB/c mouse, reaches 1: 1280, is higher than the antibody horizontal that other two kinds of inactivated vaccines produce, and also is higher than the antibody titer that produces after the viable bacteria immunity simultaneously.Antibacterial had still kept better immunogenicity after the irradiation deactivation was described thus, equally with wild-type bacterium can excite high-level antibody.
Table 1. is respectively organized the test result of inactivated vaccine to antibody in the BALB/c mouse serum
The protectiveness of vaccine experiment: exempt from back 21d in two and respectively organize the yzu LM 1-2 of mouse peritoneal injection fresh cultured (BALB/c mouse LD50 is 1.47 * 10
4CFU, counteracting toxic substances dosage are 2.5 * 10
6CFU/ only observes 14d continuously, respectively organizes the dead mouse situation behind the record counteracting toxic substances, calculates immune protective rate.The result is as shown in table 2, warp
60The protective rate that produces after the inactivated vaccine immunity of Co irradiation deactivation antibacterial preparation is up to 100%, and is suitable with the viable bacteria immunity, the attack that can resist the high dose wild-type bacterium fully; And the protective rate that the inactivated vaccine of the preparation of the method by hot deactivation and formalin-inactivated causes only is 35%, 30%.
Table 2. is respectively organized the immunoprotection result of inactivated vaccine to mice
Infect dynamic experiment: behind counteracting toxic substances the 1st, 3,5,7,9,13d slaughters 2 mices for every group, the antibacterial amount of carrying in separating spleen and the liver.The negative control group number that carries disease germs is maximum, and occurs obviously reducing in time.In spleen, the viable bacteria group is carried disease germs to count in time with irradiation deactivation group and is descended rapidly, separates less than antibacterial to the 5d spleen.Hot deactivation group and formalin-inactivated group discharge of bacteria speed are slower, reach aseptic condition respectively at 9d, 13d.In liver, the viable bacteria group is carried disease germs to count in time and is descended rapidly, organizes to 5d to reach aseptic.Irradiation deactivation group discharge of bacteria is slow slightly, organizes to 7d to reach aseptic.And hot deactivation group and formalin-inactivated group reach aseptic condition to 9d, 13d respectively, and the result as shown in Figure 2.
The experiment of T cell transfer: exempt from back two all cervical vertebra dislocations in two and put to death mice, aseptic separating immune Mus spleen is made single cell suspension, the gentle washing of pre-cooling PBS three times, and each 10min suspends the microscopically cell counting with 500 μ l PBS; Add the fluorescently-labeled CD3 monoclonal antibody of 10 μ l in the cell suspension, in 4 ℃ of black out effect 30min, the gentle washing of pre-cooling PBS 3 times, each 10min; Selected by flow cytometry apoptosis is the T cell wherein, the microscopically cell counting.T cell after the sorting suspends with 200 μ l PBS, the tail vein injection BALB/c mouse.This mice is lumbar injection 2.5 * 10 simultaneously
6The yzu LM 1-2 of CFU fresh cultured.Slaughter this mice after three days, sterile working's isolating cardiac, liver, spleen, colony counting.The result as shown in Figure 3, the spleen of negative control group mice, liver, heart carry disease germs the number be respectively 2 * 10
7CFU, 7.8 * 10
6CFU, 5.7 * 10
3CFU, and the spleen of positive mice, liver, the heart number that carries disease germs is respectively 5.5 * 10
5CFU, 6.6 * 10
4CFU, 2 * 10
1CFU obviously is less than negative control group, illustrates that protective immune response comprises the protective effect that is produced by the T cell.
Embodiment 3: substantially the same manner as Example 1, difference is that used strain is Listeria monocytogenes LM-4.The irradiation inactivated vaccine immune effect evaluation effect that it obtains is substantially the same manner as Example 2.
The invention is not restricted to these disclosed embodiments, the present invention will cover the scope described in the patent book, and the various modification of claim scope and equivalence variation.
Claims (5)
1, a kind of Listeria monocytogenes inactivated vaccine preparation method is characterized in that, with the Listeria monocytogenes warp
60Co radiation deactivation.
According to the said Listeria monocytogenes inactivated vaccine preparation method of claim 1, it is characterized in that 2, said radiation specifically is, make bacteria samples place ice with
60The Co radiation source irradiates, radiation dose is not less than 15KGy.
According to the said Listeria monocytogenes inactivated vaccine preparation method of claim 2, it is characterized in that 3, radiation mode is static irradiation, close rate 60Gy/min.
According to the said Listeria monocytogenes inactivated vaccine preparation method of claim 3, it is characterized in that 4, radiation dose is 15KGy.
5, a kind of Listeria monocytogenes inactivated vaccine that makes by the method for claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
| CN104189898B (en) * | 2014-06-27 | 2017-09-08 | 四川大学 | P. aeruginosa bacteria vaccine and preparation method thereof |
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Open date: 20080917 |