CN101979504A - Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine - Google Patents
Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine Download PDFInfo
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Abstract
The invention provides a Yersinia pestis ghost, a preparation method thereof, and application of the Yersinia pestis ghost serving as a vaccine. The ghost has a complete outer membrane structure, can induce an organism to generate a protective antibody by inoculating a mouse without an adjuvant, generate strong immune memory and protect the mouse from being attacked by strong dose of plague live strains, and has higher safety compared with a live vaccine.
Description
Technical field
The present invention relates to a kind of Yersinia pestis ghost, its preparation method with and as the application of molecule delivery system, belong to molecular biology and immunological technique field.
Background technology
(Bacterial Ghost is the E genetic expression of inducible phage PhiX174 in Gram-negative bacteria BG) to the bacterium ghost, the complete bacterium ghost that the host cell cracking is formed.Hydrophobic protein of being made up of 91 amino acid of PhiX174E genes encoding, this albumen itself be without any enzymic activity, but can play the effect that connects inside and outside film by oligomerization.Show that through electron photomicrograph interior adventitia is realized at the passage place merging, and forms the periplasmic space of a sealing.The diameter range of cracking passage is between 40nm and 200nm.The size of cracking passage is subjected to the influence of cell individual difference, relates generally to the degree that aqtocytolysis takes place, the variation that cytolemma took place in the size cases of sieve aperture and the cracking process on the peptidoglycan layer.In a single day passage forms, and under the ordering about of high osmotic pressure, intracellular most contents thing comprises nucleic acid in cell, and rrna and other components are excluded out, have formed a complete ghost.
The preparation of bacterium ghost is to realize by the strict expression regulation to lysis genes E.The lysis genes E of PhiX174 can be controlled by a plurality of expression systems, as the control of transcribing of thermally sensitized λ pL/pR-cI857 promotor, or some chemical induced promoter L ac PO-LacI
q, or expression system such as tol.The temperature control expression system that the present invention utilized is that using at present maximum is λ pL/pR-cI857 Controlling System.
Experiment confirm E gene also can effectively cracking pleuritis pneumonia actinobacillus except that the cleavable intestinal bacteria, Klebsiella pneumonia, haemolysis mannheim bacterium, p pestic, helicobacter pylori, Salmonella typhimurium, vibrio cholerae, Edwardsiella, Vibrio anguillarum etc.Infer that thus E cracking system can mediate all Gram-negative bacteria generation cracking.
Because preparation process is gentle and need not Denaturation, so cell walls is got off by complete preservation to a great extent.The bacterium ghost itself can be used as a kind of good vaccine because it has kept coating structure and associated antigen protein under the viable bacteria state, as lipopolysaccharides, CPG and pili etc., thus can be effectively by antigen presenting cell (APCs) engulf, processing and submission.Present known ghost can and have been obtained good immune effect by animal models such as approach immune mouse, rabbit, pigs such as subcutaneous, vein, collunarium.The pleuropneumonia actinomycetes (A.pleuropneumoniae, APP) ghost has been induced the provide protection at APP through the intramuscular injection immune swine; While is in APP aerosol infection model, but APP ghost inducing producing specificity mucosal immune response is more effective than whole-cell vaccines.(V.cholerae Ghost VCG) through mode immune rabbits such as subcutaneous or intramuscular injection, can induce body fluid and the cellullar immunologic response of generation at the cell envelope composition to the vibrio cholerae ghost.
The plague is to suffer from deadly infectious disease altogether by the people beast that Yersinia pestis (Yersiniapestis) causes, the several hundred million people of historical records die from the plague.It does not rely on the people and long-term existence in the specific geographical environment of nature.The plague comprises pneumonic plague, glandular plague and 3 kinds of forms of septicemic plague.Traditional prevention and therapeutic vaccine at the plague comprise killed vaccine and living vaccine, so far in the world widespread use USP bacterin and plague EV live vaccine arranged.The USP bacterin in nineteen forty-six first Application in the mankind, be the full vaccine that obtains through formalin or thermal treatment.Its shortcoming comprises: production security is poor; Protection efficient is low, and validity period is short, need repeat immunity after 6 months; Side reaction rate height, side effect is serious; Cost an arm and a leg, and to pneumonic plague protection poor effect.Attenuation plague living vaccine has EV strain and EV
BlueStrain.The used EV of China
BlueVaccine is the Pgm mutant strain, and its attenuation is brought into play by changing iron metabolism mechanism.Animal experiment shows that this vaccine all has quite high protection to mouse and cavy, but not parallel at animal body and human body result of use.The vaccine strain immunogenicity that laboratory, various countries different condition is preserved is difference to some extent also; Produce with 28 ℃ of cultivations, can not fully produce protective antigen (F1), evaluation EV in 1974
BlueF1 antigen fail to detect; Subcutaneous injection reactivity height, side effect is obvious; The cut reaction of inoculation is slight, but can not guarantee to enter intravital bacterium number, influences vaccine protection effect.In the research of plague new generation vaccine, Sub-unit Vaccine of Plague is the most popular direction at present, mainly carries out around two kinds of antigenic components (F1 antigen and V antigen).F1 antigen is the protein clostridium antigen of yersinia pestis, has the anti-cell phagolysis in vivo.Many studies show that, the F1 subunit vaccine all is a kind of main protective antigens to animal and human's class; V antigen is a kind of adjusting albumen, also is a kind of virulence factor, is unique albumen with protectiveness in the yersinia pestis secretory protein.Other has test to confirm that V antigen and F1 antigen have very strong coordinating protection effect.In addition, effectively new immunogen such as Yop secretory protein (YscF) day by day becomes the research focus.Subunit vaccine (tropina of recombinant protein or purifying) is though can effectively induce body to produce protective immune response; but still have some limitation, as complicated process of preparation, need adjuvant; cost is higher, and composition is single can not to provide effective protection etc.Except that above-mentioned protein vaccine, people also are devoted to develop other new generation vaccine, as dna vaccination etc.
Based on some shortcomings of existing plague vaccine, so this area needs more efficient, stable, safe plague vaccine.
Summary of the invention
The invention provides a kind of Yersinia pestis ghost, described ghost is to make the host cell cracking by the abduction delivering product of lysis genes E in Yersinia pestis, and the intact bacterial ghost that forms.
In an optimized technical scheme, described Yersinia pestis is EV
BlueStrain.
In an optimized technical scheme, described gene E is arranged in plasmid pMBE.
The present invention also provides a kind of method for preparing the Yersinia pestis ghost, said method comprising the steps of:
(1) cultivates described Yersinia pestis;
(2) carrier that will contain lysis genes E is transformed into described Yersinia pestis;
(3) 42 ℃ of expression of inducing described gene E;
(4) collect the thalline that step (3) obtains.
In an optimized technical scheme, described Yersinia pestis is EV
BlueStrain.
In an optimized technical scheme, described carrier is pMBE.
In an optimized technical scheme, the employed method for transformation of step (2) is that electricity transforms.
The present invention also provides a kind of freeze-dried preparation that contains above-mentioned ghost.
In an optimized technical scheme, described preparation is an injection formulations, and described preparation also contains phosphate buffered saline buffer.
In an optimized technical scheme, the administering mode of described preparation is subcutaneous injection.
Plague EV disclosed in this invention
BlueGhost by morphological observation, has typical ghost feature, has kept its outer membrane structure effectively, and materials such as the interior nucleic acid of most born of the same parents are released, and have reduced the possibility of reverse mutation.And preparation is simple for described ghost, cost is low, the time is short, compares with traditional live bacterial vaccines preparation, has certain economic.Confirm that through the BALB/c mouse immunization experiment side effect of plague EV ghost is little, safe, but inducing mouse produces protection antibody, compares with living vaccine, can produce stronger immunological memory, can protect mouse to avoid strong dosage plague EV
BlueThe attack of living vaccine strain.
Description of drawings
Fig. 1 expresses the reorganization plague EV bacterium PCR qualification result of E gene;
The lysis efficiency of Fig. 2 plague EV ghost;
The scanning electron microscope and the transmission electron microscope results of Fig. 3 plague EV ghost;
BALB/c body weight change situation behind Fig. 4 initial immunity;
The 6th all BALB/c mouse serum IgG antibodies are tired behind Fig. 5 initial immunity;
Fig. 6 attacks poison back BALB/c mouse body weight change situation;
Fig. 7 attacks poison back BALB/c mouse serum IgG antibody and tires.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment and replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Test materials
1 bacterial strain and plasmid
(Yesinia Pestis, YP) the EV strain is the plague bacillus less-virulent strain to plague bacillus, available from Lanzhou Institute of Biological Products.
Plasmid pMBE has lysis genes E, is made up voluntarily by this laboratory.Construction process: the method (forward primer 5 '-GAATTCGGCATGGTACGCTG-3 ' of using PCR, reverse primer 5 '-GGATCCGGCTCACTCCTTCC-3 '), obtain lysis genes E from phage PhiX174 (available from ATCC13706-B 1 U.S. typical case culture), then lysis genes E orientation is connected among the temperature control expression plasmid pMB, obtains cracking plasmid pMBE.The temperature control expression system of described pMB is a λ pL/pR-cI857 Controlling System.Described pMB plasmid is on the basis of commercialization plasmid pBV220 (brilliant bio tech ltd is dodged in Shanghai), and directed RepA gene (the GenBank accession number Z49272.1) structure that inserts the helicobacter pylori of synthetic forms at distance initiating terminal 1300bp place.
2 main agents and instrument
Key instrument has: PCR instrument (Thermocyler, Biometra), horizontal strip electrophoresis instrument (Liuyi Instruments Plant, Beijing), enzyme connection instrument (Thermo, Multiskan MK3), supercentrifuge (HITACHI, CR22GIII), electronic balance (YUEPING), electric shock instrument (BIO-RAD Gene Xcell), flow cytometer (BD Bioscience), scanning electronic microscope (HITACHI, S-2400) and transmission electron microscope (PHILIPS, Megaview II), flow cytometer (BD Bioscience).Main agents has: Tryptones, yeast extract are available from Oxoid company, and plasmid extraction kit is available from TIANGEN company, and primer, restriction enzyme, PCR test kit are available from TAKARA company.
Preparation, the evaluation of embodiment 1 plague EV ghost
1.1 plasmid pMBE electricity transforms plague EV bacterium
The making method of EV competent cell is as follows: with the single colony inoculation of plague EV in 5ml LB liquid nutrient medium, 37 ℃ of incubated overnight enlarge and are transferred in the 100ml LB substratum, and 37 ℃ are cultured to logarithmic growth mid-term, centrifugal collection thalline, change substratum (0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, 10% glucose with PBS (PH7.4) and electricity, the distilled water constant volume) respectively washs 2 times, the electricity that adds the 1ml precooling at last changes substratum, and the 100ul packing is stored in-70 ℃.
Electricity conversion operation method is as follows: after will identifying correct plasmid pMBE and 100 μ l competent cells mixing, inject the 0.1cm electric shock cup (BIO-RAD) of precooling, set on the electric shock instrument: voltage 1500v, and electric capacity 25uF, resistance 200 Ω carry out electricity and transform.After conversion is finished, its sucking-off is added in the blank substratum of 3ml about 2h that recovers, coating paraxin (GC+) flat board, 28 ℃ of cultivations.Transformant is identified (see figure 1) by bacterium colony PCR.
Primer:
P1：5’-GAATTCGGCATGGTACGCTG-3’
P2：5’-GGATCCGGCTCACTCCTTCC-3’。
Test-results: the reorganization plague EV bacterium (pMBE) after will transforming is through pcr amplification, 1% agarose electrophoresis, and stripe size is correct, illustrates that plasmid pMBE successfully changes in the plague EV bacterium.Among Fig. 1, the 1st swimming lane is molecular weight marker DL2000, and second swimming lane is a recombinant clone, the negative contrast of the 3rd swimming lane, the positive contrast of the 4th swimming lane.
1.2 the preparation of plague EV ghost and lysis efficiency are measured
To identify that correct reorganization plague EV bacterium (pMBE) is cultured to OD in 28 ℃
600Be about 0.4.Culture is divided into 2 groups, and one group is elevated to 42 ℃ rapidly with culture temperature and induces E genetic expression, and another is organized in 28 ℃ and continues to cultivate, and enumeration is carried out in the different time points sampling, infers its lysis efficiency (see figure 2).
With identify correct reorganization plague EV bacterium (pMBE) through 28 ℃ of enlarged culturing to OD
600Be about 0.4, induce E genetic expression for 42 ℃, induce certain hour after, centrifugal collection thalline.PBS washing back freeze-drying is preserved standby.
Test-results: the enumeration result shows that reorganization EV bacterium (pMBE) is inducing back 1h to begin bacteriolyze, cracking fullest when 4h, and lysis efficiency reaches 99%.
1.3 the electron microscopic observation of plague EV ghost
Get plague EV ghost cell through PBS washing 3 times, 3% glutaraldehyde is resuspended, and 4 ℃ are fixedly spent the night, and ethanol dewaters step by step, acetate penta 2 ester interchanges, and dry back metal spraying is observed under the scanning electron microscope, is taken a picture as shown in Figure 3A.
Get plague EV ghost cell through PBS washing 3 times, 3% glutaraldehyde, 4 ℃ are fixedly spent the night, and ethanol dewaters step by step, acetate penta 2 ester interchanges, dry back metal spraying is observed under the transmission electron microscope, is taken a picture (Fig. 3 B).
Test-results: referring to Fig. 3, most bacteriums are cavity shape structure, and can see the bacteriolyze duct inducing the back to form ghost, and diameter is between 200-400nm.Ghost has kept complete bacterial outer membrane, but because intracellular organic matter is released out, obvious shrinkage takes place bacterium.
Experimental example 2 plague EV ghost immune effects are analyzed
Plague EV ghost by subcutaneous injection mode immunity BALB/c mouse, and is analyzed ghost inductive immunne response and immune protective.
2.1 experimental animal and grouping
Healthy BALB/c female mice, 6-8 age in week is about body weight 18g, available from Military Medical Science Institute's Experimental Animal Center.Be divided into 3 groups at random, every group of 5-6 only.Experimental group (EV ghost group) directly is dissolved in freeze dried ghost among the PBS, does not add any adjuvant, and every mouse is through subcutaneous route inoculation 10
7CFU/100 μ L, twice (the 21st day booster immunization) of immunity altogether; Set up positive controls (be EV living vaccine group, Lanzhou Institute of Biological Products produces human plague EV living vaccine) simultaneously, immunity once, every mouse inoculation dosage is 10
7CFU/100 μ L; Negative control group (be PBS group), every mouse subcutaneous route are given the PBS with equal volume.Collected serum respectively at the 0th, 14,21,28,42 day.
2.2BALB/c general condition detection such as mouse body weight
Just exempt from initial two weeks of back, every morning, 9:30-10:00 measured the mouse body weight, and was X-coordinate with time, was ordinate zou drafting growth curve (see figure 4) with body weight change situation (BWLP, the preceding body weight of body weight/immunity before immunity back body weight-immunity).Observe simultaneously whether mouse alarms hair, general situations such as feed situation and mental status.
Test-results: just exempt from two weeks of back, EV living vaccine group mouse occurs seriously alarmming hair, symptom such as One's spirits are drooping, and EV ghost group and PBS control group non-evident sympton; From the body weight situation, EV living vaccine group mouse weight loss is maximum, is the ghost group secondly.Sum up general situations such as immunity back mouse body weight, after the EV ghost immunity BALB/c mouse, its side effect obviously alleviates than the EV living vaccine.
2.3 immunity back BALB/c serum IgG antibody detects
Respectively at the 0th, 14,21,28,42 day collection serum ,-70 ℃ of preservations were standby.Bag is extracted F1 antigen, reorganization V antigen (final concentration 0.5ug/ml) with plague bacillus; The full bacterium antigen of plague EV (final concentration is 5ug/ml), the coating buffer dilution.4 ℃ of bags are spent the night.The TSTA confining liquid, 37 ℃ of sealing 1h.Mice serum TSTA doubling dilution is provided with negative control and blank simultaneously, hatches 1h for 37 ℃.PBST washes 5 times.Goat anti-mouse IgG/horseradish the enzyme labelling two that adds final concentration then and be 1ug/ml is anti-, hatches 1h for 37 ℃.PBST washes 5 times.Colour developing 15min, stop buffer termination reaction (see figure 5).
Test-results: the ELISA detected result shows, the equal nonreactive V antigen-antibody of immunity back the 6th all 3 groups of mouse; EV living vaccine group has stronger anti-F1 antigen-antibody than EV ghost group; EV ghost group has strong anti-full bacterium antigen-antibody than EV living vaccine group, illustrates that the EV ghost induced the specific antibody at plague bacillus membrane-associated protein and polysaccharide antigen.Therefore, the EV ghost can effectively induce BALB/c mouse to produce protection antibody, induces intensity and EV living vaccine not to have significant difference.
2.4 challenge trial
2.4.1 general growth indexes such as body weight detects
Attack poison back in two initial weeks, every morning, 9:30-10:00 measured the mouse body weight, and was X-coordinate with time, was ordinate zou drafting growth curve (see figure 6) with body weight change situation (BWLP, the preceding body weight of body weight/immunity before immunity back body weight-immunity).Observe mouse simultaneously and alarm a hair situation, ulcer level, feed situation, and general situation such as mental status.
Test-results: in two weeks after attacking poison, 3 groups of mouse all present in various degree towering hair, feature such as One's spirits are drooping, and wherein the PBS control group mice is the most serious, and EV ghost group and EV living vaccine group symptom are lighter.In addition, festering in various degree, tissue adhesion take place in right leg injection site, so that crippled, and wherein the crippled rate of PBS control group reaches 100%, and EV living vaccine group and EV ghost group only 20%.By the body weight situation, PBS group weight loss is the most obvious, is EV ghost group, EV living vaccine group secondly.
2.4.2 attack the BALB/c serum I G antibody test of poison front and back
Before attacking poison, 2 weeks collected serum respectively, the ELISA method mensuration serum IgG antibody (see figure 7) of tiring with attacking poison back.
Attack the 2nd week of poison back, before EV ghost group mouse anti F1 antigen and anti-full bacterium antigen I gG antibody are attacked poison remarkable increase (p＜0.05, p＜0.05) is arranged all, there were significant differences before attacking poison.And EV living vaccine group and PBS control group mice the two all do not have obvious increase, even the trend of reduction is arranged.So behind the EV ghost initial immunity mouse, but inducing mouse produces strong immunological memory, thus the protection mouse avoids the attack of high dosage EV viable bacteria, thus mouse is formed provide protection effectively.
Claims (10)
1. Yersinia pestis ghost, described ghost is to make the host cell cracking by the abduction delivering product of lysis genes E in Yersinia pestis, and the intact bacterial ghost that forms.
2. ghost according to claim 1 is characterized in that, described Yersinia pestis is EV
BlueStrain.
3. ghost according to claim 2 is characterized in that, described gene E is arranged in plasmid pMBE.
4. method for preparing the Yersinia pestis ghost said method comprising the steps of:
(1) cultivates described Yersinia pestis;
(2) carrier that will contain lysis genes E is transformed into described Yersinia pestis;
(3) 42 ℃ of expression of inducing described gene E;
(4) collect the thalline that step (3) obtains.
5. method according to claim 4 is characterized in that, described Yersinia pestis is EV
BlueStrain.
6. method according to claim 5 is characterized in that, described cracking plasmid is pMBE.
7. method according to claim 6 is characterized in that, the employed method for transformation of step (2) is that electricity transforms.
8. freeze-dried preparation that contains ghost described in the claim 1-3.
9. preparation according to claim 8 is characterized in that, described preparation is an injection formulations, and described preparation also contains phosphate buffered saline buffer.
10. preparation according to claim 9 is characterized in that, the administering mode of described preparation is subcutaneous injection.
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| CN102352338A (en) * | 2011-09-30 | 2012-02-15 | 中国人民解放军军事医学科学院军事兽医研究所 | Brucella shell as well as preparation method and application thereof |
| CN103275914A (en) * | 2013-06-03 | 2013-09-04 | 中国人民解放军军事医学科学院微生物流行病研究所 | Bacterial ghost presenting protective antigens and application thereof |
| CN104232502A (en) * | 2014-03-06 | 2014-12-24 | 北京市水产科学研究所 | Bacterial ghost of aeromonas veronii as well as preparation method and application thereof |
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
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| CN104232502A (en) * | 2014-03-06 | 2014-12-24 | 北京市水产科学研究所 | Bacterial ghost of aeromonas veronii as well as preparation method and application thereof |
| CN105784984A (en) * | 2016-03-01 | 2016-07-20 | 南京财经大学 | Preparation method and application of gram-positive bacterium ghost |
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Application publication date: 20110223 |