CN1452984A - Prepn of influenza-preventing antiserum - Google Patents
Prepn of influenza-preventing antiserum Download PDFInfo
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- CN1452984A CN1452984A CN03128871A CN03128871A CN1452984A CN 1452984 A CN1452984 A CN 1452984A CN 03128871 A CN03128871 A CN 03128871A CN 03128871 A CN03128871 A CN 03128871A CN 1452984 A CN1452984 A CN 1452984A
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Abstract
The present invention relates to biological products, and is especially preparation process of influenza-preventing antiserum. Influenza DNA vaccine is used to immunize animal to produce antiserum, and the antiserum is then used in passively immunize other animal to reach the preventing and protective effect. Without need of preparing antigen protein, the preparation process is simple, low in cost, convenient in use and high in immunological effect.
Description
Technical field
The invention belongs to field of biological product, be specifically related to a kind of sero-fast method for preparing flu-prevention.
Background technology
Existing influenza biological preventing goods and preparation method have following kind, and (one), vaccinum influenzae inactivatum comprise totivirus killed vaccine and lytic virus subunit vaccine.Wherein (1) totivirus inactivated vaccine (the human influenza vaccines of present unique registration) is on the basis of intact virus, the method for cause a disease virion Applied Physics or chemistry is made it complete inactivation and the viral vaccine made.Its preparation method is a techniques well known, has production method fairly simple than other vaccines, advantages such as the easy control of production process, but have significant disadvantages.At first; though vaccinum influenzae inactivatum can reach 70～90% at the effective percentage in influenza pandemic season; but precondition be in the vaccine antigenic component will with present popular influenza antigen height homology, illustrate that it can only infect effectively homologous virus, and do not have the effect of cross protection basically.Secondly, vaccinum influenzae inactivatum is fit to be inoculated in health adult, and old man and infant are had higher toxicity, if the immune renal failure and the immunosuppressant patient that use then can't bring out the antibody response with protectiveness level.Once more, because the influenza virus that the preparation vaccine is used needs incubation growth in the allantoic cavity of Embryo Gallus domesticus, its immunogenicity can be lowered or change.Four, tool is not long-lasting for the inactivated vaccine of influenza.By its inductive immunne response even also very of short duration, therefore need annual or twice inoculation in a year to the effect of congenerous disease strain.(2) influenza subunit vaccine wherein refers to the effective viral surface antigen composition that obtained by wild strains of influenza viruses behind cultivation, cracking, purification, mainly be HA and NA vaccine.Its traditional preparation process method production technology is loaded down with trivial details relatively, is difficult to obtain in a large number NA, therefore causes the NA subunit vaccine to cost an arm and a leg, and is difficult to apply.(2), Gripovax, verified in mice, Gripovax has more broad-spectrum immunne response.With the cold adaptation type Gripovax by gene resortment method preparation is example, and the cold adaptation strain of its gene resortment must have the coding HA that derives from wild strain and 2 RNA sections of NA, and segment then must derive from the strain of cold adaptation donor in addition.Before the inoculation crowd, also vaccine strain must be cultivated in special aseptic egg allantoic cavity, further detect its toxicity.Still there is the probability of back mutation in its virulence.Especially the cold adaptation vaccine strain need repeatedly go down to posterity, and is chronic, when viral prevalence, epidemic isolates carried out cold adaptation go down to posterity unrealistic.(3), the influenza synthetic peptide vaccine, need the identical peptide section of aminoacid sequence of synthetic and influenza virus protective antigen (HA, NA etc.) determinant, after being prepared into immunogen, animal or human's body is inoculated, impel body generation protection antibody.There is theoretical and actual difficulty: the polypeptide of seeking and make up best antigenic determinant; The less immunogenic of synthetic peptide, necessary adapted can be at the adjuvant of human body use during use.(4), influenza genetic engineering subunit vaccine; being about to coding induces influenza antigens determinant (HA, the NA etc.) gene of protective immune response to be inserted in the expression vector dna; then carrier is imported yeast, insecticide or mammalian cell, make it to express the virus antigen egg, get product behind the purification.Its weak point is that the workload of positive expression colony screening is big.Expressed proteins can not correctly fold or modify sometimes, thereby influences immunogenicity.The technology relative complex of recombiant protein separation and purification.(5), the influenza dna vaccine, for containing the expression plasmid of influenza antigen protein coding gene.Utilize recombinant DNA technology that protein coding genes such as HA, NA are cloned into expression vector, and in antibacterial, increase.To import in the body by methods such as intramuscular injection, particle gun injection, live body electric shocks behind the plasmid purification, thus the induction of immunity system influenza antigens albumen generation specific immune response expressed to coded sequence.1993, Ulmer JB etc. developed and has carried out the method that intramuscular transports with dna immunization is former, has released the nucleic acid vaccine of influenza.1997, will the encode nucleic acid vaccine intramuscular injection mice of HA gene such as Deck, the HA antibody titer that immune mouse produces is than the convalescent mice height of natural infection, and can keep more than 1.5 years.
Chen Ze etc. are cloned in the β actin expression vector (pCAGGSP7) of chicken the immune protection of more different expression plasmids in BALB/c mouse respectively with the encoding gene of HA, NA, M1, NP and the NS1 of Strain A/PR/8/34.The result shows that the mice of inoculation HA or NA nucleic acid vaccine is highly resistant to viral infection.Use the same method, with expression plasmid inoculation BALB/c (H-2d), the B10 (H-2b) of HA, NA and NP and the mice of three strains of C3H (H-2k).The result shows that the NA expression plasmid all shows very high protection level in all three kinds of mices, and the HA expression plasmid is only effective in BALB/c mouse, and the NP expression plasmid only causes low titer antibody reaction then completely without effect.Prove that thus the NA gene is the important component part of influenza nucleic acid vaccine.Afterwards, use the expression plasmid combined immunization mice of HA and NA again, its immune effect will be significantly better than with HA or the independent immune mouse of NA plasmid.On this basis, the cross protection effect of NA expression plasmid is studied.Obtain 3 kinds of NA expression plasmids from A/Guizhou/54/89 (H3N2), A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1), BALB/c mouse is inoculated respectively, the A/Guizhou/54/89 with lethal dose behind the booster immunization attacks.The result shows that the mice of inoculation A/Guizhou NA plasmid can resist the attack of homology virus fully; the mice of inoculation A/AichiNA plasmid also has very high cross protection effect to the attack of variant (A/Guizhou), and the mice of inoculation PR8 NA plasmid then can't be resisted the attack of different subtype Strain.NA is proved again to the cross protection effect of influenza variant.
Based on above relative analysis, the common deficiency that exists in the prior art of described as can be seen influenza biological preventing goods preparation is that preparation technology is loaded down with trivial details relatively, causes producing manufacturing cycle and increases, and production cost increases.Secondly; because these influenza biological preventing goods are vaccine product substantially; its immunoprotection mechanism is to produce initiatively to reply in inoculator's body, and therefore for old man and infant and immune renal failure or other immunosuppressant patient, described vaccine can't bring out the antibody response with protectiveness level.For dna vaccination, be directly used in human body and also exist safety issue.
Summary of the invention
The purpose of this invention is to provide the sero-fast method of a kind of preparation flu-prevention.
This method adopts influenza dna vaccine immunity animal, produces antiserum, with this antiserum passive immunity animal, plays prevention protection effect.The experiment proved that the antiserum that is produced by the influenza dna vaccine has the protection effect.The inventive method need not prepare antigen protein, uses the dna vaccination immune animal, obtains antiserum. and the prevention of this influenza has overcome the deficiency that known influenza is prevented goods with antiserum, and a kind of preparation method of safer, effective influenza prevention goods is provided.
The inventive method realizes with step by following technical solution,
1) extracts influenza antigen HA, NA gene; 2) make up HA, NA dna vaccination; 3) identify HA, NA dna vaccination; 4) select laboratory animal; 5) HA, NADNA vaccine immunity animal; 6) measure anti-HA, NA antiserum titre; 7) gather antiserum; 8) antiserum passive immunity; 9) influenza viruse attack experiment.
Described viral DNA extracts from breeding in the PR8 virus that obtains the instar chicken embryo on 10th by known method.RNA gets strand cDNA through reverse transcription reaction.With cDNA is template, and HA and NA gene are carried out pcr amplification, and primer separately is well known in the art, all contains Xho I and Sma I restriction enzyme site.The PCR product is cloned in the expression vector pCAGGSP7 that same enzyme action is handled with Xho I-Sma I digestion.PCAGGSP7 contains the ori site (Ori) of chicken β-actin promoter composition and SV40.
(PR8, expression vector pCAGGSP7 is gone in HA H1N1) and NA gene clone, obtains recombiant plasmid pCAGGSP7/HA, pCAGGSP7/NA with strains of influenza viruses A/PR/8/34.
The plasmid of coding HA and NA increases in Escherichia coli XL1-blue, with purification kit (commercial) purification.Order-checking confirms HA and NA DNA nucleotide sequence.Concentration and purity with ultraviolet scene spectrphotometric method for measuring plasmid.
The lead-in mode of dna vaccination of the present invention has intramuscular injection, intradermal injection, intranasal instillation or nasal spray, liposome method and particle gun immunity and live body electric shock immunity etc.The Antiserum Preparation that the inventive method relates to can be according to animal species difference, and the needs of antiserum amount and type and antigen requirement select 1) animal, comprise horse, mule, or rabbit, Cavia porcellus, or goat and rabbit; 2) immunizing dose, time and approach, wherein the about 0.5～1mg/ of large animal antigen dose (being as the criterion with proteantigen), the about 0.1～0.6mg/ of toy only.The approach of inoculation generally adopts multi-point injection, always counts to be about 8～10 points, comprises around vola and the fossa cubitalis lymph node, locates Intradermal or subcutaneous under both sides, back, the jaw, behind the ear etc.Immunity blanking time with at interval 10～20 days for well.What secondary was later on each is spaced apart 7～10 days; 3) animal blood sampling is after the last immunity 5～7 days, uses the carotid artery blood-letting, and heart is taken a blood sample and vein blood collection method repeatedly; 4) antiserum separates the room temperature natural coagulations of adopting more, places 37 ℃ or 4 ℃ then and treats clot retracts; 5) double immunodiffusion is adopted in antiserum titre evaluation or ELISA tests and hemagglutination inhibition test and radioimmunoassay technique.
The immune programme for children that the inventive method relates to can adopt a dna vaccination immunity, twice dna vaccination immunity, three dna vaccination immunity or the panimmunity program of strengthening with dna vaccination immunity reuse protein vaccine earlier.
The specific embodiment
Embodiment 1
HA, NA dna vaccination immune animal;
Choose 6～8 the week age BALB/c mouse.
Behind the lumbar injection general anesthesia mice, injection is dissolved in the dna solution of TE and goes into the right rear leg quadriceps femoris, inserts the electrode of two electric shock instrument in the both sides, injection site, at a distance of 0.5cm, (voltage 100v, electric shock time 50ms shock by electricity, shock by electricity positive and negative each three times, at interval 1s), finish once immunity.This method adopts 3 kinds of different immune programme for children that mice is carried out immunity: 1) each immunity of the 0th, 3 weeks is 1 time, totally 2 times.Each every mice 50 μ gDNA (1 μ g/ μ l).If do not insert the negative contrast of carrier DNA immune mouse of gene.The 4th week was used the chloroformization mice, took a blood sample from heart.2) each immunity of the 0th, 3 weeks is 1 time, totally 2 times.Each every mice 50 μ gDNA (1 μ g/ μ l).If do not insert the negative contrast of carrier DNA immune mouse of gene.The 6th week was used the chloroformization mice, took a blood sample from heart.3) each immunity of the 0th, 3,6 weeks is 1 time, totally 3 times.Each every mice 50 μ gDNA (1 μ g/ μ l).With the carrier DNA immune mouse that does not insert gene as negative control.The 9th week was used the chloroformization mice, took a blood sample from heart.
Get the mice serum sample and make ELISA, survey IgG antibody.By 37 ℃ of 96 hole ELISA Plate 2 hours, the PBS-T time back of giving a baby a bath on the third day after its birth added confining liquid with the A/PR/8/34 inactivated vaccine bag of 10 μ g/mL, and 4 ℃ are spent the night.With confining liquid with 2 multiple dilute serum after, add ELISA Plate, 37 ℃ of incubations 1 hour.After PBS-T gives a baby a bath on the third day after its birth time, add with biotin labeled mountain sheep anti-mouse igg two and resist, 37 ℃ of incubations 1 hour.After PBS-T gives a baby a bath on the third day after its birth time, add the chain mycoprotein of alkali phosphatase enzyme mark, 37 ℃ of incubations 1 hour.At last, after PBS-T gives a baby a bath on the third day after its birth time, add the p-NPP colour developing.In 30 minutes, utilize dual wavelength (414nm-405nm) to measure the OD value with microplate reader, finally determine the high dilution of IgG antibody, determine the height of antibody amount with this.
Use the chloroformization mice, take a blood sample from heart.The blood of collecting placed room temperature 1 hour.Separate out serum after solidifying.4000 rev/mins centrifugal 10 minutes.Sucking-off serum under aseptic condition, by every pipe 50～200 μ l packing ,-20 degree refrigerators are preserved behind the mixing.
Embodiment 2
Antiserum passive immunity animal
With HA, the NA antiserum passive immunity mice in 6～8 ages in week.Syringe is drawn antiserum and aerofluxus, and warm water soaking Mus tail distends the blood vessels, with 70% ethanol wiping fixing Mus tail also.The antiserum tail vein injection is after 24 hours, with A/PR/8/34 (H1N1) virus (20LD
50) attack mice by nasal drip (17 μ l viral suspension).Observe mice body weight change and survival rate and judge HA and the sero-fast protection effect of NA.After the virus attack the 3rd day, HA and NA experimental mice average body weight average descended about 10%, and the control group mice average weight descends about 20%.After the virus attack the 6th day, experimental mice average body weight average descended about 20%, and the control group mice average weight descends about 35%.Experiment shows, is 90% with the mice survival rate of 100 μ l HA and NA antiserum passive immunity, is 100% with the mice survival rate of 300 μ l HA and NA antiserum passive immunity.The result confirms that the antiserum passive immunity adult mice with HA, NA dna vaccination mice immunized produce can make it to resist the lethal dose influenza viruse attack, plays the protection effect.
Dna vaccination that the present invention adopts and tradition are used for the vaccine of the prevention of infectious disease such as inactivated vaccine, attenuated live vaccine, subunit vaccine etc. to be compared, and following characteristics are arranged:
(1) good immune effect, different with common protein vaccine, gene vaccine can produce extrinsic protein in self cell, this albumen more resembles natural molecule than the protein that produces in the prokaryotic expression system, its pass living process and natural infection quite similar.Therefore, such immunogen will contain epi-position relevant on conformation, induce the immunne response of generation corresponding to native antigen.In addition, nucleic acid vaccine still can work to the individuality inoculation of existing immunity.
(2) the same with the carrier live vaccine CTL of causing with attenuated live vaccine of nucleic acid vaccine replys, but the danger that does not exist both virulence of back to go up, there is not the poison that looses yet, the virulence that the sensitivity of the viral pollution and the individual source of infection is relevant changes, be difficult to cultivate or dangerous pathogen for conventional vaccine, nucleic acid vaccine then is easy to make up.
(3) immunne response is lasting, because can there be the long period in vivo in exogenous gene, and constantly expresses foreign protein, it can provide stimulation to immune system constantly, and therefore, very micro-antigen can stimulate body to produce strong and lasting immune response.
(4) method is easy, and is cheap.Nucleic acid vaccine only needs to produce in antibacterial, makes up efficient expression plasmid, compares with common vaccine, and loaded down with trivial details time-consuming procedure such as antigen extraction and purification have been saved in the making of nucleic acid vaccine, make manufacturing cycle shorten greatly.The nucleic acid vaccine consumption is few, and is more more economical than other vaccine.
(5) nucleic acid vaccine has identical physicochemical property, for combined immunization provides possibility.Genetic immunization does not only produce immunne response to carrier to the antigen of expressing, so can being used to transport different target genes, repeatedly uses same plasmid, also can be on a carrier the multi-functional vaccine of multiple antigenic being called as of construction expression " combination vaccine " or polyvalent vaccine, reach once immunity and can obtain repeatedly the identical immune effects of different immunity.
(6) nucleic acid vaccine can be processed into exsiccant granule be convenient to the storage and the transportation.Exsiccant DNA granule is at room temperature relatively stable, does not need refrigerating equipment, and is therefore also comparatively convenient to backwoodsman use.Nucleic acid vaccine is just being restored to the original state simply with the preceding water that adds at once, and is all favourable with the public health aspect economically.
Claims (6)
1, the sero-fast method of a kind of preparation flu-prevention is characterized in that adopting influenza dna vaccine immunity animal, produces antiserum, with this antiserum passive immunity animal.
2, the sero-fast method of preparation flu-prevention according to claim 1 is characterized in that described method comprises the steps 1) extract influenza antigen HA, NA gene; 2) make up HA, NA dna vaccination; 3) identify HA, NA dna vaccination; 4) select laboratory animal; 5) HA, NA dna vaccination immune animal; 6) measure anti-HA, NA antiserum titre; 7) gather antiserum; 8) antiserum passive immunity; 9) influenza viruse attack experiment.
3, the sero-fast method of preparation flu-prevention according to claim 1 is characterized in that described method wherein uses the dna vaccination immune animal, and immunization ways adopts the immunity of live body electric shock, particle gun immunity and intramuscular injection immunization ways.
4, the sero-fast method of preparation flu-prevention according to claim 1, it is characterized in that described method wherein uses the dna vaccination immune animal, immune programme for children adopts a dna vaccination immunity, twice dna vaccination immunity, three dna vaccination immunity or elder generation dna vaccination immunity reuse protein vaccine booster immunization.
5, according to claim 1 and the sero-fast method of 4 described preparation flu-preventions, it is characterized in that described method wherein uses the dna vaccination immune animal, immune programme for children adopts dna vaccination immunity reuse protein vaccine booster immunization.
6, preparation influenza according to claim 1 is prevented sero-fast method, it is characterized in that described method wherein makes up HA, the described expression vector of NA dna vaccination is pCAGGSP7, and recombiant plasmid is pCAGGSP7/HA, pCAGGSP7/NA.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393358C (en) * | 2004-02-23 | 2008-06-11 | 中国人民解放军军需大学军事兽医研究所 | Preparation of horse family animal anti human, pultry grippe immune globulin and its medicinal preparation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393358C (en) * | 2004-02-23 | 2008-06-11 | 中国人民解放军军需大学军事兽医研究所 | Preparation of horse family animal anti human, pultry grippe immune globulin and its medicinal preparation |
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