CN105695421A - Long-acting recombinant rabies virus vaccine strain and preparation method thereof - Google Patents
Long-acting recombinant rabies virus vaccine strain and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a long-acting recombinant rabies virus vaccine strain. SAD-B19 serves as a female parent strain, 194-bit aspartic acid on G protein of the female parent strain is mutated into serine, 333-bit arginine is mutated into glutamic acid, and a mouse IL-7 gene is inserted between a G gene and an L gene of a genome of the female parent strain. The invention further discloses a preparation method of the virus vaccine strain. A disclosed recombinant virus has good reproducibility and safety, immunogenicity is enhanced by inserting the mouse IL-7 gene, and long-time humoral immunity reaction of an organism can be maintained. Besides, when co-immunization is conducted again, the neutralizing antibody level of the recombinant rabies virus can be quickly increased, and the long-acting recombinant rabies virus vaccine strain is a good rabies vaccine candidate strain.
Description
Technical field
The invention belongs to animal vaccine technical field, be specifically related to a kind of long-acting recombinant rabies virus vaccine strain and preparation method thereof。
Background technology
Rabies are one of the most ancient infectious disease, are a kind of infectious diseases common to human beings and animalss caused by rabies virus (Rabiesvirus, RABV)。Rabies are the acute infectious disease that mankind's case fatality rate is the highest up to now, once morbidity, case fatality rate almost reaches 100%。In recent years, China there are about dying from rabies about 3000 people every year on average, occupies Death of Infectious Diseases number front three, is mostly due to by animal bite or scratches and infected。And vaccination in time can have good protective effect before exposure and after exposure; current wide variety of rabies vaccine is mainly the inactivated vaccine such as primitive cell culture vaccine and passage cell purified vaccine; although these vaccines have the advantage such as safely, effectively, but rabies exposure crowd typically requires and carries out repeatedly inoculation (at least 4 times) within the long period (21 days)。Additionally, this kind of vaccine price costliness, prevent it in the use of developing country further。So the immunity inoculation for some vagrant animals and wild animal is then difficulty with。In China, the animal such as the vagrant dog in street corner, cat is the currently main source of infection, and the animal of wandering carries out the key that effective immunity is rabies prevention and control。Therefore, one of main target of current rabies virus research is development low toxicity, can simplify immune programme for children and permanently effective rabies vaccine。
Recombinant rabies virus vaccine is that one utilizes Reverse Genetics that rabies virus is transformed, and purposively changes the genome structure of virus, thus changing the biological characteristics of virus, to increase safety and immunogenicity。Recombinant rabies virus vaccine has been applied to the rabies prevention and control of wild animal by American-European many countries。The first time such as Schnell in 1994 uses Reverse Genetics successfully to transform and save out rabies virus SADB19 strain (SchnellMJ1, MebatsionT, ConzelmannKK.InfectiousrabiesvirusesfromclonedcDNA.EMBOJ 1994, 13 (18): 4195-203.), the recombinant rabies virus vaccine of application includes SADB19 strain at present, RC-HL strain (ItoH, MinamotoN, WatanabeT, GotoH, RongLT, SugiyamaM, KinjoT, MannenK, MifuneK, KonobeT, etal. (AuniquemutationofglycoproteingeneoftheattenuatedRC-HLstr ainofrabiesvirus, aseedvirususedforproductionofanimalvaccineinJapan.Microb iolImmunol.1994, 38:479 482.), HEP-Flury strain (InoueKi, ShojiY, KuraneI, IijimaT, SakaiT, MorimotoK.Animprovedmethodforrecoveringrabiesvirusfromcl onedcDNA.JVirolMethods.2003Feb；107 (2): 229-36.) etc.。It is subsequently based on these virus for female parent, by reverse genetics system basis constructs the various cytokines of expression, such as MIP-1alpha (ZhaoL, ToriumiH, WangH, KuangY, GuoX, etal.ExpressionofMIP-1alpha (CCL3) byarecombinantrabiesvirusenhancesitsimmunogenicitybyindu cinginnateimmunityandrecruitingdendriticcellsandBcells.J Virol.2010, 84:9642 9648.), GM-CSF (MingZhou, LeiWang, LingZhao, ZhenF.Fu, etal.RecombinantrabiesvirusexpressingdogGM-CSFisaneffica ciousoralrabiesvaccinefordogs.Oncotarget.2015Nov17；6 (36): 38504-16.), disappearance dependency structure albumen, as lacked the recombinant viral vaccine (McGettiganJP of M gene, P gene or G gene, DavidF, SchnellMJ, etal.Safetyandserologicalresponsetoamatrixgene-deletedra biesvirus-basedvaccinevectorindogs.Vaccine.2014Mar26；32 (15): 1716-9.CennaJ1, HunterM,, McGettiganJP, etal.Replication-deficientrabiesvirus-basedvaccinesaresa feandimmunogenicinmiceandnonhumanprimates.JInfectDis.200 9Oct15；200 (8): 1251-60.SatoS, OharaS, TsutsuiK, IijimaT.EffectsofG-geneDeletionandReplacementonRabiesVir usVectorGeneExpression.PLoSOne.2015May29；10 (5): e0128020.), process LAN dependency structure albumen (Hosokawa-MutoJ, ItoN, YamadaK, etal.Characterizationofrecombinantrabiesviruscarryingdou bleglycoproteingenes.MicrobiolImmunol, 2006, 50 (3): 187-196.TaoL1, GeJ, BuZ, etal.GenerationofrecombinantrabiesFluryLEPviruscarryinga nadditionalGgenecreatesanimprovedseedvirusforinactivated vaccineproduction.VirolJ.2011Sep25；The recombinant viral vaccine strain such as 8:454.)。But all there is many deficiencies in these vaccines, wherein it is important that NAT can not maintain in permanently effective level。Such as, HEP-dG NAT when the 21st day reaches to peak just decline (XiaohuiLiu, YoutianYang, XiaofengGuo, etal.ARecombinantRabiesVirusEncodingTwoCopiesoftheGlycop roteinGeneConfersProtectioninDogsagainstaVirulentChallen ge.PLoSOne.2014；9 (2): e87105.)。Therefore, one of main target of current rabies vaccine research is the rabies vaccine that development is permanently effective。
Summary of the invention
It is an object of the invention to provide a plant weight group rabies virus vaccine strain, this viral vaccine can promote rabies virus-immune Memorability, maintains the permanent humoral immune reaction of body。
The present invention is with the rabies vaccine strain LBNSE of report before us for maternal (WenY, WangH, WuH, YangF, TrippRA, etal. (2011) Rabiesvirusexpressingdendriticcell-activatingmoleculesen hancestheinnateandadaptiveimmuneresponsetovaccination.JV irol85:1634-1.), Mus source IL-7 gene is inserted in LBNSE strain genome, the recombinant rabies poison rLBNSE-IL7 that can express Mus IL-7 gene is saved out by reverse genetics manipulation technology, this Strain has higher virus titer, pathogenic low, and the permanent humoral immune reaction of body can be maintained, long-term induction produces notable high-caliber neutralizing antibody, especially again after immunity, neutralizing antibody level significantly quickly raises。
The above-mentioned purpose of the present invention is achieved through the following technical solutions:
LBNSE strain used derives from the vaccine strain SAD-B19 for Europe wild animal immunity, but on SAD-B19 viral G protein, the aspartic acid (AAT) of 194 is sported serine (TCC), the arginine of 333 (AGA) are sported glutamic acid (GAA), be equivalent to another strain vaccine strain SAG2 (being widely used in the vaccine strain of wild animal oral immunity in Europe), significantly reduce the pathogenic (WenY of virus, WangH, WuH, YangF, TrippRA, etal. (2011) Rabiesvirusexpressingdendriticcell-activatingmoleculesen hancestheinnateandadaptiveimmuneresponsetovaccination.JV irol85:1634-1.RasalingamP, RossiterJP, MebatsionT, JacksonAC.ComparativepathogenesisoftheSAD-L16strainofrab iesvirusandamutantmodifyingthedyneinlightchainbindingsit eoftherabiesvirusphosphoproteininyoungmice.VirusRes.2005, 111:55-60.ConzelmannKK, CoxJH, SchneiderLG, ThielHJ.Molecularcloningandcompletenucleotidesequenceoft heattenuatedrabiesvirusSADB19.Virology.1990, 175:485-499.)。It addition, also knocked out by pseudogene between G and L gene in SAD-B19 strain genome, between G gene and L gene, add two restriction enzyme site BsiWI and NheI as the site inserting exogenous gene。
The gene of rabies virus vaccine strain SAD-B19, its nucleotide sequence is such as shown in SEQIDNO:1。
The clone of the Mus IL-7 gene in the present invention is by rabies virus infection mice, extracts cerebral tissue RNA, then obtains Mus source IL-7 gene with reverse transcriptional PCR amplification。The Mus IL-7 gene of amplification is inserted between the genomic G gene of LBNSE strain and L gene after sequence verification is correct, constructs the infection clones LBNSE-IL7 expressing Mus IL-7 gene。
The nucleotide sequence of Mus IL-7 gene is such as shown in SEQIDNO:2, and the gene of recombinant rabies poison rLBNSE-IL7, its nucleotide sequence is such as shown in SEQIDNO:3。
Infection clones LBNSE-IL7 and the helper plasmid expressing tetra-albumen of rabies virus N, P, G and L respectively prove to have saved the recombinant rabies virus that can express Mus IL-7 gene through immunofluorescence after jointly transfecting bsr cell。In order to verify whether that Mus IL-7 gene has been inserted in rabies virus genome, the viral rLBNSE-IL7 of rescue is infected bsr cell, then extracts viral RNA, be verified with reverse transcriptional PCR, it was demonstrated that Mus IL-7 gene has been inserted in rabies virus genome。
The recombinant virus rLBNSE-IL7 in present invention growth curve on bsr cell and NA cell is similar to LBNSE virus, it was shown that exogenous gene Mus IL-7 inserts the growth and propagation that do not have influence on virus。It addition, the titre that recombinant virus rLBNSE-IL7 is on NA and bsr cell all can reach 108.25FFU/ml, reaches the requirement of panimmunity approach titre。
By approach Mice Inoculated in recombinant virus rLBNSE-IL7 brain, in 21 days observed, mice displayed no goes out any disease symptom, and body weight does not have significant change, it was shown that this recombinant virus safety is high。
Recombinant virus rLBNSE-IL7 intramuscular immunisation once just can induce the greater number of memory B cell of generation, it is possible to keeps high-caliber neutralizing antibody in a long time。Maternal strain LBNSE after immunity when the 4th week neutralizing antibody rise to peak, and progressively decline；And recombinant virus rLBNSE-IL7 7 weeks antibody horizontals after immunity reach peak, and in 3 months, still maintain significantly high neutralization levels antibody。Co-immunization LBNSE again after initial immunity 6 months, the neutralizing antibody level of recombinant rabies poison rLBNSE-IL7 can raise rapidly, and is significantly higher than parent poison LBNSE。
In a word, in the present invention, recombinant virus rLBNSE-IL7 has good replicability, safety, and enhance immunogenicity by the insertion of Mus IL-7 gene, maintain the permanent humoral immune reaction of body, after intramuscular immunisation can the neutralizing antibody of induced high levels up to 3 months, it is significantly better than LBNSE virus (Europe is for the vaccine strain of wild animal immunity), and again during co-immunization, recombinant rabies poison neutralizing antibody level can raise rapidly, therefore, recombinant virus rLBNSE-IL7 can as the Candidate Strain of rabies vaccine。
Accompanying drawing explanation
Fig. 1 is the operation chart inserting Mus source IL7 gene in hydrophobia strain LBNSE genome。
Fig. 2 is the immunofluorescence experiment testing result of the recombinant rabies virus rLBNSE-IL7 saved out。
Fig. 3 is the genome identification electrophoretogram that Mus IL-7 gene is inserted into rabies virus。
Fig. 4 is recombinant virus rLBNSE-IL7 growth curve chart in different cell lines。
Fig. 5 is that recombinant virus rLBNSE-IL7 is to the body weight change curve chart after counteracting toxic substances in mouse brain。
Fig. 6 is the recombinant virus rLBNSE-IL7 result of the test that inducing memory B cell produces in Mice Body。
Fig. 7 is the NAT testing result after recombinant virus initial immunity。
Fig. 8 be ICR mice initial immunity after 21 days with the protective rate testing result of counteracting toxic substances in rabies virus virulent strain CVS24 brain。
The NAT testing result of Fig. 9 recombinant virus immunity one week after again。
Figure 10 is the protective rate testing result of recombinant virus rLBNSE-IL7 offer in 1 week after the immunity again of ICR mice。
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail。It should be noted that embodiments of the invention are only limitted to, the present invention will be described, without restriction effect。Relevant test method involved in embodiment and other various experimental implementation, it is the ordinary skill in the art, the part being not particularly illustrated in literary composition, those of ordinary skill in the art is referred to the various common tool books before the present patent application day, scientific and technical literature or relevant description, handbook etc. and is practiced。
The design of embodiment 1 recombinant virus rLBNSE-IL7, rescue, qualification and immune efficacy preliminary assessment
1 material and method
1.1 materials
1.1.1 virus
LBNSE strain is provided by agricultural microorganism National Key Laboratory of Hua Zhong Agriculture University, the specifying information of this virus and construction method are referred to relevant document (WenY, WangH, WuH, YangF, TrippRA, etal. (2011) Rabiesvirusexpressingdendriticcell-activatingmoleculesen hancestheinnateandadaptiveimmuneresponsetovaccination.JV irol85:1634-1.)。
1.1.2 plasmid
Construction strategy and concrete grammar for saving the infection clones pLBNSE of LBNSE strain are referred to relevant document (WenY, WangH, WuH, YangF, TrippRA, etal. (2011) Rabiesvirusexpressingdendriticcell-activatingmoleculesen hancestheinnateandadaptiveimmuneresponsetovaccination.JV irol85:1634-1.)。
1.1.3 cell and monoclonal antibody
Bsr cell (cloned cell line of BHK-21 cell), it is grown on containing 10% hyclone (Fetalbovineserum, FBS) (Gibco, GrandIsland, DMEM NY) (Dulbecco ' smodifiedEagle ' smedium, DMEM) (Mediatech, Herndon, VA) culture fluid。Mus neuroma cell (Mouseneuroblastomacells, NA), is grown on the RMPI1640 culture fluid (Mediatech) containing 10%FBS。The Rables virus glycoprotein gene monoclonal antibody of Fluorescein isothiocyanate (Fluoresceinisothiocyanate, FITC) labelling is purchased from Fujirebio (Melvin, PA) company。
1.1.4 laboratory animal
The ICR mice of 6 week old, the Balb/c mice of 6 week old, it is purchased from Disease Prevention Control Center, Hubei Prov。
1.1.5 main agents
Glue reclaims that test kit, plasmid are little to be carried and big extraction reagent kit, Superfect transfection reagent box are all purchased from QIAGEN company, various restricted enzyme are purchased from NewEnglandBiolabs, Beverly, MA company, AMVReverseTranscriptaseXL and PrimerSTARHSPCR is purchased from TAKARA company。
The design of 1.2 primers and synthesis
Detect the primer of Mus IL-7 gene in Table 1 for expanding Mus IL-7 gene and RT-PCR, Wuhan Qing Ke company synthesize。
Table 1
Note: underscore part is restricted endonuclease site sequences: in Mus IL-7 gene amplification forward primer, underlined sequences is BsiWI, and in downstream primer, underlined sequences is NheI。
1.3 rabies virus infection mices, extract the amplification of cerebral tissue RNA and Mus IL-7 gene
1. Balb/c mice is infected with rabies virus B2c intramuscular routes；
2. after 7 days, with in shears, the femur of tweezers separating mouse and tibia to clean plate, once proceed to afterwards in another plate filling aseptic PBS with 75% alcohol rinse。
3. carefully cut the muscle around femur and tibia with shears, note cutting off bone, reject after totally until muscle and bone is transferred in another plate filling aseptic 1640 culture medium。
4. carefully cut off bone two ends epiphysis with shears, repeatedly rinse the medullary cavity of femur and tibia, the cell in medullary cavity is developed。Medullary cell in piping and druming flushing liquor, makes medullary cell be separated from each other as far as possible, becomes single cell suspension repeatedly。
5. single cell suspension is collected in 50ml centrifuge tube, 4 DEG C of centrifugal 5min sedimentation cells of 300g/min, abandon supernatant, with complete medium washed cell once, count with cell counting count board, be taped against in cell plates after cell is adjusted suitable concentration。
6. add stimulating factor GM-CSF and IL-4, put into 5%CO2, 37 DEG C of cell culture incubators, partly change liquid every other day and add GM-CSF and IL-4。
7. within the 9th day, collect cell, extract RNA, after carrying out reverse transcription with AMVReverseTranscriptaseXL, expand Mus source IL-7 with TAKARAPrimerSTARHSPCR。
The rescue of 1.4 recombinant virus rLBNSE-IL7
Referring to document (WenY, WangH, WuH, YangF, TrippRA, etal. (2011) Rabiesvirusexpressingdendriticcell-activatingmoleculesen hancestheinnateandadaptiveimmuneresponsetovaccination.JV irol85:1634-1.)。Specific as follows: bsr cell is inoculated in six orifice plates by day before transfection, cell is made to reach the coverage of 80% used time in the second angel, say according to the description of product of transfection reagent Superfect (QIAGEN company) and be operated, by the full-length infectious clone of 2ug, 0.5ugN helper plasmid, 0.25ugP helper plasmid, 0.15ugG helper plasmid and 0.1ugL helper plasmid transfect bsr cell jointly, cell after transfection puts into 37 DEG C of incubators, CO2Concentration is 5%, hatches 4 hours, is then outwelled by supernatant, washes once by the DMEM culture medium containing 10%FBS, adds fresh culture and cultivates 4 days, supernatant is transferred on BSR cell monolayer, at 34 DEG C containing 5%CO2Incubator cultivate 3 days, collect supernatant ,-80 DEG C of subpackages preserve。
The qualification of 1.5 Revive virus
1.5.1 Immunofluorescence test
By the virus of rescue and bsr cell after 96 porocyte plates co-culture 48 hours, supernatant is discarded, 30 minutes are fixed in 4 DEG C with 80% acetone of pre-cooling, then 2 times are washed with PBS, by Fluorescein isothiocyanate (Fluoresceinisothiocyanate, FITC) the Rables virus glycoprotein gene monoclonal antibody (being purchased from Fujirebio company) of labelling carries out 1:70 dilution, every hole adds 50ul, put into 37 DEG C hatch 1 hour after wash three times with PBS, observe at fluorescence microscope (LeicaDMIRES)。
1.5.2 reverse transcription (RT) PCR detects Mus IL-7
After the virus of rescue and bsr cell are co-cultured 48 hours, cell scraping extraction total serum IgE, RT-PCR detects。
1.6 titration of virus
Take 96 porocyte culture plates, it is classified as a sample area with 4 row × 9, two subregions of every plate, will add the RPMI1640 cell culture fluid containing 10%FBS of 90 μ l in each hole, each sample sets 4 repetitions, the virus stock solution used of 10 μ l is joined the first row of 96 porocyte plates, draw 10 μ l mixed liquors after being sufficiently mixed to secondary series, fully mix, then sucking-off 10ul mixed liquor is to next column, continuously perform the doubling dilution of 10 times successively, after arranging hole to the 9th, discard the mixed liquor of 10 μ l。In 37 DEG C after the NA cell of every hole addition 5 × 104,5%CO248h is made in sense, then abandons cell conditioned medium liquid, with the fixing infection cell of 80% cold acetone (80%Ice-coldacetone)。After dyeing with rabies poison N protein fluorescent antibody (FITC-conjugatedanti-RVNantibodies), by fluorescence microscope and record antigen positive region (Antigen-positivefoci)。Carry out the calculating of virus virulence according to Reed-muench method and be recorded as FFU/ml (Fluorescentfocusunitspermilliliter)。
1.7 recombinant virus rLBNSE-IL7 growth characteristics on BSR and NA cell
Virus is infected respectively with infection multiplicity for 0.01 (MOI=0.01) BSR and NA cell, distinguishes the 1st day after infection, the 2nd day, 3rd day, the 4th day and within the 5th day, take the supernatant of 50 μ l, carry out titration of virus, record virus is in the titre of different time points, and draws out growth curve。
1.8 zooperies
1.8.1 the pathogenic experiment of recombinant virus rLBNSE-IL7
By the ICR mice of 6-8 week old, often group 12, respectively with the DMEM or 10 of 50 μ l7The recombinant virus of FFU and LBNSE, infect in the way of intracerebral injection (i.c.), observes and observes twice every day, records clinical onset and the death condition of mice。
1.8.2 recombinant virus rLBNSE-IL7 neutralizing antibody level experiment
By the ICR mice of 6-8 week old, often group 12, respectively will containing 1 × 10 with 1ml asepsis injector6The LBNSE strain of FFU and 1 × 106The recombinant virus LBNSE-IL7 of FFU carries out intramuscular routes immunity, and carries out weekly blood sampling mensuration neutralizing antibody level after immunity, and after the tenth week, blood sampling measures neutralizing antibody level every other month。Co-immunization 1 × 10 again after initial immunity 6 months6The LBNSE strain of FFU, after immunity, blood sampling in 1 week measures neutralizing antibody level。
1.8.3 recombinant virus rLBNSE-IL7 protective rate experiment
With 50LD when initial immunity 21 days50Approach counteracting toxic substances ICR mice in CVS-24 brain, observes 21 days, the protective rate of detection rLBNSE-IL7。And after immunity again 1 week with 50LD50In CVS-24 brain, situation is effectively protected in the detection of approach counteracting toxic substances mice。
2. experimental result
The structure of 2.1 infection clones LBNSE-IL7 and Revive virus detection
LBNSE strain is to suddenly change G gene 194 and 333 on the basis of SAD-B19, and is knocked out by the pseudogene between G and L gene, adds two restriction enzyme site BsiWI and NheI。Recombinant virus rLBNSE-IL7 is on the basis of LBNSE virus, it is inserted between G gene and L gene (as shown in Figure 1) after correct to Mus IL-7 gene amplification order-checking, utilize Reverse Genetics Revive virus, the virus immunofluorescence experiment of rescue detects, as shown in Figure 2, it can be seen that the fluorescence stove of green, being the recombinant rabies virus rLBNSE-IL7 saved out, cell negative control does not then observe fluorescence stove。Additionally, in order to detect whether Mus IL-7 gene has been inserted in the virus of rescue, after the viral infection bsr cell of rescue, RNA carries out reverse transcriptional PCR detection in advance, the BsiWI upstream, site that forward primer design is inserted at Mus IL-7 gene, downstream primer designs in the downstream of another insertion point NheI, and concrete primer sequence is in Table 1。Testing result is as shown in Figure 3,1st swimming lane is the purpose band that LBNSE strain can expand 325bp, and the LBNSE-IL7 of the 2nd swimming lane is it can be seen that the purpose band of 790bp, the 465bp differed between the two is the Mus IL-7 gene order of insertion, identifies through order-checking and shows that Mus IL-7 gene has been inserted into the genome of rabies virus。
The growth characteristics of cultured of 2.2 viruses
Whether the insertion in order to study Mus IL-7 gene affects duplication and the propagation of rabies virus self, and LBNSE strain and LBNSE-IL7 strain infect BSR and NA cell simultaneously, and measures the titre of its virus in different time points, draws out the growth curve of virus。Such as Fig. 4, it can be observed that recombinant virus rLBNSE-IL7 has similar growth curve with virus LBNSE, this shows that the insertion of exogenous gene Mus IL-7 gene does not affect duplication and the propagation of virus, and the highest titre is also more or less the same with female parent virus。The highest titre that recombinant virus rLBNSE-IL7 reaches on bsr cell and NA cell respectively 1 × 108.25FFU/ml and 1 × 108.5FFU/ml, reaches titre peak in the 3rd day and the 4th day respectively after infection。
The 2.3 pathogenic situation detections of recombinant virus
In the ICR mice of recombinant virus rLBNSE-IL7 intracerebral injection 6-8 week old, observing 3 weeks, as it is shown in figure 5, any disease symptom does not occur in period, there is not significant change in body weight, illustrates that recombinant virus is to mice no pathogenicity, and safety is high。
The generation of 2.4 recombinant virus inducing memory B cell
By recombinant virus rLBNSE-IL7 and LBNSE strain (Europe is for wild animal immune vaccine strain) intramuscular routes immunity Balb/c mice, after initial immunity 7 days and 14 days, and after initial immunity 6 months co-immunization 1 × 106After the LBNSE strain of FFU, the lymph node that gathers of 1 week carries out the activation situation of memory B cell in Flow Cytometry Assay mouse lymphocyte。As shown in Figure 6, it has been found that after initial immunity 7 days and 14 days, and immunity all can activate the generation of memory B cell for 7 days again, thus maintaining the permanent humoral immune reaction of body。
2.5 recombinant virus intramuscular immunisation effect assessments
By recombinant virus rLBNSE-IL7 and LBNSE strain (Europe is for wild animal immune vaccine strain) intramuscular routes immunity ICR mice, carrying out weekly blood sampling after immunity and measure neutralizing antibody level, after the tenth week, blood sampling measures neutralizing antibody level every other month。Inducing high neutralizing antibody level is antirabic effective means, therefore, and the method that the mensuration of neutralizing antibody is evaluated as Efficacy of Rabies Vaccine Prepared。As shown in Figure 7, the neutralizing antibody level of recombinant virus rLBNSE-IL7 induction to be significantly higher than LBNSE strain, due to the IL-7 impact that memory B cell is produced, rLBNSE-IL7 the 7th week neutralizing antibody level after immunity is the highest, 65.49IU/ml can be reached, and the neutralizing antibody level of LBNSE strain is the highest at the 4th week, also only has 12.90IU/ml。It is noted that after initial immunity when 3 months, most senior middle school and antibody horizontal that rLBNSE-IL7 then induced neutralizing antibody and LBNSE can induce are suitable。It addition, when initial immunity 21 days with 50LD50Approach counteracting toxic substances ICR mice in CVS-24 brain, rLBNSE-IL7 has good protective rate, as shown in Figure 8。Further, co-immunization 1 × 10 again after initial immunity 6 months6The LBNSE strain of FFU, after immunity, the neutralizing antibody level of 1 week is quickly raised to 22.86IU/ml, is significantly higher than the 10.98IU/ml of LBNSE strain, and has good protected effect, such as Fig. 9, shown in 10。
In a word, recombinant rabies virus rLBNSE-IL7 in the present invention to mice without obvious body weight change, no pathogenicity, safety is high, on immune effect, namely compared with the female parent strain LBNSE in testing for the vaccine strain that wild animal is immune with Europe, rLBNSE-IL7 can induce and produce significantly high neutralizing antibody level, the highest can reach 65.49IU/ml, and rose to peak at the 7th week, maintain notable high-caliber NAT up to 3 months, additionally, significantly high NAT can be produced rapidly to 22.86IU/ml after immunity again, again within 1 week, it is respectively provided with good protective rate after co-immunization LBNSE strain after initial immunity 21 days and 6 months。Therefore, recombinant virus rLBNSE-IL7 of the present invention has possessed the safety and low toxicity as vaccine strain, permanently effective feature, it is possible to as the Candidate Strain of rabies vaccine。
Claims (2)
1. recombinant rabies virus vaccine strain one kind long-acting, it is characterized in that: it is with SAD-B19 for maternal strain, it is that serine, the arginine of 333 sport glutamic acid, and insert the nucleotide sequence such as Mus IL-7 gene shown in SEQIDNO:2 between genome G gene and the L gene of described maternal strain by the Aspartic acid mutations of 194 in described maternal strain G-protein。
2. the preparation method of recombinant rabies virus vaccine strain one kind long-acting, it is characterised in that comprise the following steps:
1) with SAD-B19 for maternal strain, it is that serine, the arginine of 333 sport glutamic acid by the Aspartic acid mutations of 194 in described maternal strain G-protein；
2) pseudogene between G gene and L gene in maternal strain genome is pounded out, two restriction enzyme site BsiWI and NheI are added between G gene and L gene, by nucleotide sequence, such as the Mus IL-7 gene shown in SEQIDNO:2 is inserted between G gene and L gene, and is saved out the recombinant rabies virus of expression Mus IL-7 gene by reverse genetic operating system。
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