CN1940063A - Pseudo-rabies gE/gI-gene loss poison strain, killed vaccine containing it and use - Google Patents

Abstract

A recombinant Pseudorabies virus PrV gene engineering strain WKQ-001, inactivated vaccine containing the poisonous strain and its use are disclosed. It can be used to discriminate and diagnose artificial immunity pig or natural infectious pig. It's safe and doesn't contain exogenous gene.

Description

A kind of pseudoabies gE -/ gI -Genetically deficient strain, the inactivated vaccine that contains this strain and application

Technical field

The invention belongs to the animal virology technical field, relevant with genetically engineered.The present invention is specifically related to a kind of Pseudorabies virus (Pseudorabies virus of reorganization, PrV) mutant strain (strain numbering: structure WKQ-001) and utilize the strain of this reorganization to prepare pseudoabies genetically deficient sign inactivated vaccine, strain of the present invention has lacked glycoprotein gene gI, the gE of PrV, cause the PrV virulence to descend, thereby can be used to prepare the pseudoabies safe vaccine.

The invention still further relates to the application of described recombinant pseudorabies virus strain and genetically deficient inactivated vaccine.

Background technology

Pseudorabies virus (PrV) belongs to herpetoviridae a simplexvirus subfamily, has growth fast on cell culture, characteristic (Roizmorn B such as neural preferendum of intensive and latent infection, Desrosiers R, Flecbenstein B, Lopez C, Minson Ad and studdertMJ.The family Herpesviridae; An update.Arch Virol.1992,123:425-449).Animals such as this virus under field conditions (factors) can infected pigs, ox, sheep, dog, cat, rabbit, mouse, wild boar, ermine, bear, fox.Except that pig, behind other zoogenetic infection Pseudorabies virus, have heating, very itch and classical symptom such as acute encephalomyelitis, be lethal infection.This disease is eruption and prevalence pig.Pig is the storage person and the contagium of this virus, mainly shows as breeding difficulty and the piglet mass mortality of sow, wherein 15 ages in days with interior piglet mortality ratio up to 100%.Pseudoabies causes enormous economic loss to pig industry.

In pseudoabies vaccine research and application facet, mainly comprise two kinds of attenuated vaccine and inactivated vaccines at present.Attenuated vaccine is because therefore this viral latent infection characteristic exists dangerous and diffusing malicious problem.It is generally acknowledged that the security of inactivated vaccine will be higher than attenuated vaccine, but its effectiveness is less better.Development along with genetic engineering technique, people have developed some gene-deleted vaccines at the anti-system of pseudoabies, promptly utilize gene engineering method to insert or the genomic one section sequence of deletion PrV, some gene is not expressed, reach and cause weak PrV and don't influence its immunogenic purpose.In addition, gene-deleted vaccine can also reduce the toxin expelling ability after immune swine is attacked poison, and the latent infection ability of himself also descends greatly.These engineered gene-deleted vaccines are not only more reliable at secure context, but also help setting up the differential diagnosis method.The two combines, and not only can prevent pseudoabies, and can distinguish natural infection swinery and immune swinery, by eliminating the natural infection pig gradually, sets up healthy swinery, so that finally reaches the purpose of eradicating pseudoabies.

The difficult point that porcine pseudorabies is eradicated is the immune evasion of the long-term latent of Pseudorabies virus and virus and the immuning failure that causes.Studies show that pseudoabies glycoprotein gene gE causes Pseudorabies virus to hide and the principal element of immune evasion.Glycoprotein gE is the equal energy of all PRV strains (except some vaccine strain) expressed proteins of finding so far, has group specificity, and is significant in the differential diagnosis of PRV.Therefore, prevent and control pseudoabies, become and generally accepted in the world and the popular method by inoculation Pseudorabies virus gE genetically deficient inactivated vaccine.

Aspect the development of Pseudorabies virus gE gene-deleted vaccine, Hua Zhong Agriculture University developed a kind of Pseudorabies virus TK in 2003 ^-/ gE ^-/ gI ^-Genetically deficient sign living vaccine, this vaccine has immune effect preferably, and described pseudoabies strain has lacked TK ^-/ gE ^-/ gI ^-Gene does not contain foreign gene, belongs to a kind of living vaccine (referring to the Chinese invention patent prospectus, number of patent application 03156706.1) that makes up with genetically engineered; Sichuan Agricultural University has reported a kind of pseudoabies TK ^-/ gE ^-/ gI ^-/ LacZ ⁺The genetically deficient sign causes weak living vaccine, this strain disappearance TK ^-/ gE ^-/ gI ^-Gene, but comprise the expression cassette (referring to, Guo Wanzhu etc., some biological characteristic research of pseudoabies gene-deleted vaccine strain (SA215), Chinese Preventive Veterinary Medicine newspaper, 2000 9 phases) of foreign gene LacZ; Harbin Veterinary Medicine Inst., China Academy of Agriculture makes up and has finished gE/TK genetically deficient mutant strain, this mutant strain contains the expression cassette of foreign gene LacZ (referring to Tian Zhijun etc., the structure of pseudorabies virus gE/TK genetically deficient mutant strain, Chinese Preventive Veterinary Medicine newspaper, 2004 5 phases); Agricultural University Of Nanjing has reported a kind of pseudorabies virus Shanghai strain (PRV-SH)/gE ^-/ gI ^-/ GFP ⁺The disappearance strain (referring to Jiang Yan etc., pseudorabies virus Shanghai strain (PRV-SH)/gE ^-/ gI ^-/ GFP ⁺The structure of disappearance strain) microorganism journal, 2003 (2): 15-20), this disappearance strain contains the expression cassette of foreign gene green fluorescent protein GFP.As a rule, foreign gene is introduced mutant strain, for this screening mutant strains, be undoubtedly a shortcut, because can improve the efficient of screening greatly like this.There is the expert to point out emphatically, LacZ, reporter genes such as LUC belong to alien gene, might influence its biological characteristics in vivo, and organism is produced some negative influences; Moreover, the biotechnological formulation that is used for organism is what not allow at will with people's alien gene, thereby these gene-deleted vaccines that insert reporter gene generally can only be used as the latent infection of research pseudoabies and dynamic (dynamical) a kind of means of proliferation in vivo thereof, can not be as the commodity (Jin Shengzao etc. that come into the market, pseudoabies gene-deleted vaccine progress, Scientia Agricultura Sinica, 2002,35 (1): 89-93).Therefore above-mentioned three kinds of gene-deleted vaccines are owing to depositing defective above-mentioned, in case come into the market to have the Biosafety problem.The applicant once developed a kind of pseudoabies gene vaccine of TK/gE/gI gene that lacked (referring to the Chinese invention patent ublic specification of application, number of patent application 03156706.1) has preferably immune effect and do not contain foreign gene, be a contribution greatly, but still may exist and return strong danger as causing weak living vaccine to existing vaccine.Therefore develop a kind of good immune effect, An Quan Pseudorabies virus genetically deficient inactivated vaccine is a urgent problem in the current production again.

Summary of the invention

Task of the present invention is to overcome the defective that prior art exists, obtain the aujeszkys disease geneticly engineered strain that a strain is recombinated by gene recombination method, utilize this strain to prepare the good He safe pseudoabies gE of a kind of effect of control, gI genetically deficient sign inactivated vaccine.

Based on the thought of foregoing invention, the present invention also comprise a kind of gE of disappearance, gI gene the Pseudorabies virus gene engineered strain, contain the inactivated vaccine and the application thereof of the preparation of this strain.

The present invention is achieved through the following technical solutions:

Key of the invention process, it is the applicant obtains strain reorganization by gene recombination technology Pseudorabies virus Pseudorabies virus strain, this strain be numbered WKQ-001, this strain is on September 2nd, 2005, be deposited in Chinese typical culture collection center (CCTCC), its deposit number is CCTCC-V200511.

The pseudoabies poison strain of the present invention's reorganization, this strain has following notable feature:

1) this recombinant strain has lacked gE, gI gene;

2) this recombinant strain does not contain foreign gene.

The applicant utilizes the Pseudorabies virus Pseudorabies virus strain WKQ-001 of this preservation, and CCTCC-V200511 prepares a kind of Pseudorabies virus deletion of vaccine.

Described gene-deleted vaccine is an inactivated vaccine, and it prepares according to following proportioning:

By umber:

3 parts of A, waters

Each part water adds the preparation of 4 parts of sterilization tween 80s by 96 parts of pseudoabies venom with the deactivation of claim 1 or 2 described pseudoabies strain preparations;

6 parts of B, oil phases

Each part oil phase is by 94 parts of No. 10 white oils for animals in milliliter, add in 2 parts of aluminum stearates of gram and in 6 parts of Si Ben-80 ratios preparations of milliliter and

C, the above-mentioned A of mixing and described water of B and oil phase promptly obtain described inactivated vaccine.

Technical scheme is as shown below in more detail:

The source strain that is used for gene recombination of the present invention is that pseudoabies poison strain Ea strain is (referring to Chen Huanchun etc., the isolation identification of porcine pseudorabies virus Hubei Province A strain, journal of animal science and veterinary medicine, 1998,29 (2), 972104) as parent plant, glycoprotein gene gI, gE coding region excalation with PrV, thereby obtain the deletion mutantion strain, this mutant strain is deposited in CCTCC, and preserving number is: CCTCC-V200511.

The concrete steps of preparation Pseudorabies virus genetically deficient sign inactivated vaccine are:

1) preparation of the strain of recombinant rabies poison: (this plasmid was deposited in Chinese typical culture collection center (CCTCC) on September 2nd, 2005 with intermediate transfer plasmid pIESE, preserving number is CCTCC NO:M205101), (be called for short PrV Ea with Pseudorabies virus Ea strain, genomic dna cotransfection pig kidney passage cell IBRS-2 down together), after pathology occurring, receive poison.Acquire PrV gE through PCR screening and plaque select ^-/ gI ^-Mutant strain, this strain be numbered WKQ-001, belong to a kind of Pseudorabies virus (Pseudorabies virus) strain, on September 2nd, 2005, be deposited in Chinese typical culture collection center (CCTCC), deposit number is CCTCC-V200511.

2) preparation of Pseudorabies virus genetically deficient sign inactivated vaccine: after the viral liquid that the pseudoabies poison strain (deposit number CCTCC-V200511) that obtains with step 1) is produced adds the abundant mixing of formaldehyde solution, put 37 ℃ of effects 36 hours, put then room temperature (about 25 ℃) continuation effect 12 hours antigen for vaccine.Umber meter by volume: (add the preparation of 4 parts of sterilization tween 80s in the pseudoabies venom of each part water by the reorganization of 94 parts of deactivations: (94 parts of No. 10 white oils for animals producing with Chinese Hangzhou, Zhejiang province city refinery of each part oil phase, adding aluminum stearate 2 parts (are unit in the gram) and 6 parts of Si Ben-80 are formulated for 6 parts of oil phases to get 3 parts of waters.

The invention has the beneficial effects as follows:

(1) recombinant pseudorabies virus strain of the present invention derives from the applicant at first from domestic autonomous separation and clone's pseudoabies strain, compare with external similar strain and products thereof, its effect is fit to the popular swinery of China's infection and preventing infection pseudoabies more, thereby specific aim of its anti-system is stronger;

(2) recombinant pseudorabies virus strain of the present invention derives from the pig of infection; compare with the pseudoabies strain that is located away from other animals of prior art report; the security of the pseudoabies genetically deficient sign inactivated vaccine of the present invention's preparation and protectiveness are more suitable for the prevention in porcine pseudorabies, help the earning processing of China's pig farm pseudoabies.

(3) recombinant pseudorabies virus strain of the present invention does not contain any foreign gene, compares with the pseudoabies gene vaccine of the marker gene such as the contained LacZ of similar Pseudorabies virus gene vaccine of prior art report, has higher biological safety.

Description of drawings

Fig. 1 has shown pIESE plasmid construction schema, and plasmid pIESE is the transferring plasmid that contains PRV Ea strain part gE gene and gI gene.

Fig. 2 has shown the electrophoretogram that the dual-gene disappearance transfer vector of pseudorabies virus gE, gI pIESE identifies.1:15000Marker among the figure; 2:Stu I and BstEII double digestion plasmid pIESE.Through StuI+BstE II double digestion, produce the fragment of a 6200bp size, rather than 1247bp and two electrophoretic bands of 6200bp.Fig. 2 has shown that these two restriction enzyme sites eliminate.

Fig. 3 has shown recombinant virus PrV gE ^-/ gI ^-Make up flow process.

Fig. 4 has shown recombinant virus PrV gE ^-/ gI ^-The electrophoretogram that PCR identifies, among the figure, 1-18 is gE ^-/ gI ^-Recombinant virus; 19 are the water contrast; 20 are the cell contrast; 21 are the contrast of pIESE positive plasmid; 22 is the contrast of PRV EA pnca gene group.

As shown in Figure 4, recombinant pseudorabies virus of the present invention can amplify the gE of the about 1400bp of size ^-/ gI ^-Fragment, and traditional isolating pseudoabies Ea strain (PRV EA strain) and negative control all can not expand and this fragment, proves that gE, gI gene lack in this PrV genomic dna.

Fig. 5 has shown recombinant virus PrV gE ^-/ gI ^-The genetic stability electrophoretogram, among the figure, 1-5 be the 5th generation recombinant virus pcr amplification product; 6-10 be the tenth generation recombinant virus pcr amplification product; 11-15 be the 15 generation recombinant virus pcr amplification product; 16 are the water contrast; 17 are the cell contrast; 18 are the contrast of pIESE positive plasmid.

Fig. 6 has shown that the indirect immunofluorescence of recombinant virus is identified the picture result, and 1 is gE among the figure ^-/ gI ^-Mutant strain; 2 are the cell contrast; 3 is traditional PRV Ea strain.By the indirect immunofluorescence result as can be known: owing to lacked the gE-gene, pseudoabies mutant strain of the present invention (being the pseudoabies recombinant strain) can't with gE ^-The monoclonal antibody combination, therefore, 1,2 is shown as feminine gender among the figure.

Fig. 7 has shown recombinant pseudorabies virus PrV gE ^-/ gI ^-The plaque of protein dot blot identify collection of illustrative plates, among the figure 1: Pseudorabies virus Ea strain parental virus strain contrast; The contrast of 2:PK-15 cell; 3: recombinant pseudorabies virus PrV gE ^-/ gI ^-Results of hybridization.By the plaque of protein dot blotting; After the gE gene elmination, PrV Ea gE ^-/ gI ^-Can not express gE glycoprotein.

The present invention is further illustrated below in conjunction with chart and embodiment.

Embodiment

One, design of primers

The Prv Ea strain gI (the gene database accession number is AF306511) that has delivered according to GeneBank; The synthetic a pair of Auele Specific Primer of gE (the gene database accession number is AF171937) gene order design abbreviates P1, P2 as, and the dna fragmentation size after the dual-gene disappearance of amplification gI, gE is 1400bp.

Primer sequence is as follows:

P1：5’-TAGACGGGACGCTGCTGTTTCTGGAGG-3’，

P2：5’-CGGTCACGCCATAGTTGGGTCCATTCG-3’

The pcr amplification condition is as shown in table 1:

Table 1 PCR condition of the present invention and primer

Primer	The PCR reaction conditions	PCR reaction conditions system
			gE -/gI -	94℃4min，94℃1min 51.8℃1min，72℃1.5min 35cycles，72℃10min	10×buffer(without Mg 2+)5.0μL， Template 5.0μL 25mmol/L MgCl 2 8.0μL 2mmol/LdNTP 3.0μL 10mmol/L P1 2.0μL 10mmol/L P2 2.0μL Taq 2.0μL DMSO 5.0μL H 2O 11μL 500mmol/L kcl 6.0μL

Two, the structure of general transfer vector pIESE

1, plasmid pIECMV (containing PRV Ea strain gD, gI, gE, 28k Gene Partial encoding sequence, the multiple clone site district of complete encoder block of 11k and plasmid pcDNA3.1) is made up by animal virus laboratory doctor Fang Liurong of Hua Zhong Agriculture University.Its detailed preparation process is referring to the Chinese invention patent application, application number: 200510012147.3) with restriction enzyme STUI, and BSTEII double digestion plasmid pIECMV, remove its multiple clone site district, reclaim rest segment, connect with T4 DNA Ligase, 16 ℃ of water-baths are spent the night, transformed into escherichia coli DH _{5 α}, cultivated 16-20 hour for 37 ℃, picking mono-clonal bacterium colony was cultivated 12 hours in the LB of routine substratum then, extracted plasmid, and is after enzyme is cut evaluation, stand-by in-20 ℃ of preservations.This plasmid is deposited in CCTCC, and preserving number is CCTCC NO:M205101

2, the mensuration of transfer vector sequence adopts the terminal cessation method of two deoxidations (referring to molecular cloning handbook, Chinese science press) order-checking and DNAclub commonly used, DNAsis2.5 software analysis.It the results are shown in shown in accompanying drawing 1, the accompanying drawing 2.

Three, the structure of Pseudorabies virus Ea strain gE genetically deficient mutant strain

1, the extraction of Pseudorabies virus Ea pnca gene group: the PK-15 cell transfer of the pathology of will detoxifying to the 500mL centrifuge tube (5000rpm, 5min).Discard nutrient solution, it is resuspended to add PBS (PH7.4), change in the 50mL centrifuge tube (5000rpm 5min), abandons supernatant, and is resuspended with 10mL LCM, ice bath 15min, and (2500rpm 8min), gets supernatant to add 1mL freonll-11.With 24000rpm, 2 hours ultracentrifugations are abandoned supernatant, and it is resuspended to add 10mLTEN.Add 10%SDS 500UL, effect 3min, the imitative extracting of phenol 3 times, each 10000rpm, 10min. dehydrated alcohol precipitation 30min, 8000rpm, 15min, resuspended ,-20 ℃ of preservations are stand-by.

2 Pseudorabies virus screening mutant strains purifying

1) cotransfection IBRS-2 cell: utilize liposome mediated-method (referring to the Lipofectamine of invitrogen company ^TM2000 process specificationss), with transfer vector pIESE and Pseudorabies virus Ea pnca gene group cotransfection IBRS-2 cell, pathology appears and after, collect enchylema, be connected in the 24 porocyte culture plates of the IBRS-2 cell that grows up to individual layer with 100 μ L, pathology appears and after, receive poison.Make viral template with 200 μ L, PCR screens positive mutant strain.

2) the plaque purifying of Pseudorabies virus mutant strain mutant strain: with the cotransfection product multigelation of test positive 3 times, 10 ^-2～10 ^-7Dilution is got 400 μ L respectively and is seeded to the PK-15 cell monolayer culture that grows in 6 porocyte culture plates, and in 37 ℃ of absorption 1 hour, the nutrient solution that inclines was washed 3 times with the PBS of no calcium magnesium, and every hole covering low melting-point agarose 2.5mL puts 37 ℃ of CO ₂Incubator is cultivated, treat that plaque forms after, with 1/10000 neutral red solution dyeing, 37 ℃ of senses are done 1 hour, with its sucking-off, then 6 porocyte culture plates are done 2 hours 37 ℃ of senses, and residual neutral red solution is fully volatilized.With glass capillary picking plaque, be connected in 24 orifice plates that cover with monolayer cell, treat that pathology fully after, receive poison ,-20 ℃ of preservations.Take out 200 μ L then and make viral template, the pcr screening, the plaque purifying, so repeatedly, till obtaining 100% positive mutant strain.See shown in accompanying drawing 3, the accompanying drawing 4.

Four, the biological characteristics of recombinant pseudorabies virus strain WKQ-001

This sudden change strain belongs to herpetoviridae a simplexvirus subfamily herpesvirus suis I type, the rounded or oval outward appearance of virus particle, and about 9.5 * 106 dalton of molecular weight, its nuclear stamen diameter is 75nm, the about 12nm of the length of capsid capsomere, wide 9nm; Be positioned at the about 110-150nm of virus particle diameter of the no cyst membrane of nucleus, be positioned at the about 180nm of diameter of the mature virion of endochylema band cyst membrane.The process that Pseudorabies virus enters host cell can be divided into three phases, and promptly absorption, film fusion, nucleocapsid release etc. are gone down to posterity in the propagation mode.Pseudorabies virus can breed on pig kidney passage cell, and substratum commonly used is DMEM (pH7.0) growth media that contains new-born calf serum.This virus can be preserved 4 ℃ of short-terms, can be-70 ℃ of following prolonged preservation after the lyophilize and non-loss of activity.

Table 2 recombinant pseudorabies virus strain WKQ-001 is to ether, chloroform, trypsinase sensitivity test

Group	TCID 50(0.1mL)	Group	TCID 50(0.1mL)	Group	TCID 50(0.1mL)
						The ether contrast	No CPE produces 10 -8.0	The trypsinase contrast	10 -5.36 10 -7.96	The chloroform contrast	10 -1.36 10 -7.76

The data sheet of table 2 understands that recombinant pseudorabies virus strain WKQ-001 of the present invention is to ether, chloroform and trypsinase sensitivity.

Table 3 recombinant pseudorabies virus strain WKQ-001 is to the resistibility of soda acid

The pH value	Experimental group TCID 50(0.1mL)	Control group TCID 50(0.1mL)
			3.0 4.0 5.0 6.0 9.0 10.0 11.0	No CPE produces 10 -2.1 10 -7.9 10 -7.6 10 -7.6 10 -4.0No CPE produces	10 -5.3 10 -7.7 10 -7.6 10 -7.6 10 -7.6 10 -7.6 10 -7.6

Table 3 has shown that recombinant pseudorabies virus strain WKQ-001 of the present invention keeps stable between pH5.0-9.0.

Table 4 recombinant pseudorabies virus strain WKQ-001 is to 56 ℃ of tolerances

Heat-up time (min)	15	20	30	45	Control group
						TCID50(0.1Ml)	10 -5.1	10 -4.0	10 -7.8	No CPE produces	No CPE produces

Table 4 shows recombinant pseudorabies virus strain WKQ-001 of the present invention 56 ℃ of heating, and 30min is by complete inactivation.

4, the plaque test shows, recombinant pseudorabies virus strain WKQ-001 is after gE and gI disappearance, and formed plaque is less than traditional formed plaque of the Pseudorabies virus Ea of street strain strain.Recombinant pseudorabies virus strain WKQ-001 inoculation pig kidney passage cell PK-15 during 15 generations, still can expand the gE-/gI-fragment that the about 1400bp of size in vaccinization, shows this recombinant pseudorabies virus strain WKQ-001 inheritance stability, can not return strong.See accompanying drawing 5

5, pseudorabies virus Ea strain gE ^-/ gI ^-The detection of mutant strain (being WKQ-001)

1) indirect immunofluorescence detects deletion mutantion strain WKQ-001: wash the 6 porocyte culture plates 3 times that cover with the PK-15 cell with PBS solution, and natural air drying, the formaldehyde fixed 5min with 100% (0.5mL/ hole), PBS solution is washed 3 times, blots.Every hole adds hyper-immune serum 200uL, 37 ℃, CO ₂Incubator is cultivated 30min.PBS washes 1 time, ddH ₂O washes 5 times.Add two and resist the 200uL/ hole.37 ℃, CO ₂Incubator is cultivated 30min.。PBS washes 3 times, adds an amount of PBS, fluorescence microscope.See accompanying drawing 6.

2) the plaque of protein dot blot detects deletion mutantion strain WKQ-001 results inoculation PrV Ea gE ^-/ gI ^-The PK-15 cell of CPE occurs with PrVEa ,-20 ℃ of freeze thawing 3 times are got freeze thawing liquid 3 ц l and are dripped on the nitrocellulose membrane film, repeat to drip secondary after air-dry.With mouse-anti gE monoclonal antibody is one anti-, and sheep anti mouse HRP-IgG is two anti-, develops the color through diaminobenzidine.After getting ready, PrV Ea shows Vandyke brown, and positive; And PrV Ea gE ^-/ gI ^-And the contrast of PK-15 cell is negative.After this showed gE gene elmination, WKQ-001 can not express gE glycoprotein.See accompanying drawing 7.

Five, the preparation of recombinant pseudorabies virus inactivated vaccine

1, Bing Du inoculum size and cultivation

Pseudorabies virus recombinant strain WKQ-001 is inoculated in the cell monolayer that grew up in 37 ℃ of rotating and culturing 24-36 hours, connects the poison amount and be 1/10 of growth media.37 ℃ are rotated absorption 1 hour, add an amount of DMED substratum (containing each 100u/mL of 3% calf serum and mycillin), put 37 ℃ and hatch.Inoculation back 24-48 hour when cytopathy (CPE) reaches 80% when above, stops cultivation.

2, Bing Du deactivation

Behind the viral liquid multigelation of pathology three times, collect viral liquid.Through steriling test, and to the TCID of PK-15 cell ₅₀〉=10 ^-6The viral liquid of/0.1mL mixes, and makes inactivator with formaldehyde, and behind the abundant mixing of adding formaldehyde solution, the ultimate density of formaldehyde solution is 8/1000ths.Put 37 ℃ of effects 36 hours, put then room temperature (about 25 ℃) continuation effect 12 hours antigen for vaccine.Get the viral liquid after the deactivation, be inoculated in 3 bottles of PK-15 cell monolayers, cultivate for 37 ℃ and observe 7 days two generations of blind passage again, should not have CPE.

Table 5 formaldehyde is to the check result of PRV inactivating efficacy

	PK-15 cell infection titre
						Virus liquid lot number	Log 10TCID 50
Before the deactivation	After the deactivation	1 generation of blind passage	2 generations of blind passage	Not deactivation contrast
					20050501 20050510 20050520 20050530		6.8 6.5 7.0 6.5	0 0 0 0	0 0 0 0	0 0 0 0	6.8 6.5 7.0 6.5

In the table 5 after listed 4 batches of viral suspension deactivations, in inoculating cell and two generations of blind passage,, CPE does not appear in cell, TCID ₅₀Be 0, prove that inactivation of virus loses replication completely.

3, emulsifying process research

(1) preparation of oil phase: get No. 10 white oils for animals (production of Chinese Hangzhou, Zhejiang province refinery) 94 parts (is unit with the milliliter), add 2 parts of aluminum stearates (is unit with the gram), the limit edged stirs, till transparent fully.Add 6 parts of Si Ben-80 (is unit with the milliliter) again, fully behind the mixing, the autoclaving of (for example sterilizing 30 minutes down for 121 ℃) according to a conventional method is standby.

(2) water preparation: by volume, in the recombinant pseudorabies virus liquid of 96 parts of deactivations, add 4 parts of sterilization tween-80s, fully shake and thoroughly dissolve up to tween-80.

(3) emulsification: get 6 parts of oil phases and put into colloidal mill, machine and stir at a slow speed, add 3 parts of waters simultaneously slowly, add the back with 8000-10000rpm, emulsification 2.5 minutes adds 1 part of water then, continue to stir 30 seconds, be emulsified into emulsion-type Pseudorabies virus inactivated vaccine.

Six, the check of Pseudorabies virus inactivated vaccine technical indicator

(1) physical behavior

Outward appearance this product is the even emulsion of oyster white.

Formulation is got a cleaning suction pipe for the oil-emulsion type and is drawn a small amount of vaccine and drip in cold water, should be the oil droplet shape, indiffusion.

Viscosity: draw vaccine 1mL with the outlet internal diameter under 25 ℃ of left and right sides room temperatures for the 1.2mm suction pipe, make its vertical outflow, 8 second the planted agent flow out be judged to more than the 0.4mL qualified.

(2) steriling test is undertaken by " Chinese veterinary drug allusion quotation " appendix 169-171 page or leaf, answers asepsis growth.

(3) small white mouse about safety verification inoculation 16-18 gram is 5, and every subcutaneous injection 0.3mL observes 14 days mouse and should be good for work.2 of inoculation 1.5-2kg healthy rabbits, every buttocks subcutaneous injection 5mL observes should be good in 14 days and lives and have no adverse reaction.

(4) Prv negative antibody, the healthy weanling pig about efficacy test inoculation body weight 10-20kg is 4, each musculi colli vaccinate 3mL.Blood sampling in 28 days after annotating seedling, separation of serum is measured the antibody neutralization index for 315 pages by " People's Republic of China's veterinary biologics quality standard " appendix, and immune swine serum neutralization index answers 〉=316.

5 formaldehyde content are measured by " Chinese veterinary drug allusion quotation " appendix and are carried out for 177 pages, should be no more than specified amount.

The every index test of table 6 Pseudorabies virus inactivated vaccine of the present invention

Interventions Requested		Survey date	Assay	Judge (S-is up to specification, and U-is against regulation, and I-does not have the result, and NI-does not check)
					1	Outward appearance	2005.05.02	Even oyster white emulsion	S
2	Formulation	2005.05.02	Be the indiffusion of oil droplet shape	S
					3	Viscosity	2005.05.02	Near 8 seconds	S
4	Stability	2005.05.02	Not stratified, breakdown of emulsion not	S
					5	Steriling test	2005.05.02-2005.05.04	No bacterial growth	S
6	Formaldehyde content is measured	2005.05.03	0.185％	S
					7	Safety verification	2005.05.04-2005.05.18	Rabbit 2/2 health	S
2005.05.04-2005.05.18	Small white mouse 5/5 strong living	S
				8			Efficacy test (neutralizing antibody index)	2005.05.04-2005.05.31	316 398 398 512	S

Illustrate: the unification of (1) genetically deficient inactivated vaccine of the present invention is prepared into oil-emulsion (down together)

(2) detect foundation: " People's Republic of China's veterinary biologics quality standard ", Chinese agriculture science and technology press, in 2001 version

Seven, the security of vaccine, immune efficacy, protection, duration of immunity and preservation period test

(1) safety evaluation of gene-deleted vaccine of the present invention and biological test

1, small white mouse proof test

With 4 batches of vaccines of prepared in laboratory (20 bottles/batches, 100 milliliters/bottle) at random every batch get 3 bottles, after mixing, 10 of the small white mouses about inoculation 18 grams, 0.3 milliliter of every subcutaneous injection was observed 14 days.

2, newborn piglet proof test

With (20 bottles/batches of 4 batches of vaccines of prepared in laboratory, 100 milliliters/bottle) at random every batch get 3 bottles, after mixing, select healthy 1 age in days newborn piglet, 10 pigs of every batch of vaccine inoculation, each intramuscular injection 2 multiple dose 4mL pseudo-rabies oil emulsion vaccine, other establishes 4 and stays and do blank, all piglets are a week before vaccinate and behind the notes seedling, and upgrowth situation, spirit, the appetite measuring 2 body temperature every day and observe pig were observed 14 days altogether.

3, weanling pig proof test

With 4 batches of vaccines of prepared in laboratory (20 bottles/batches, 100 milliliters/bottle) at random every batch get 3 bottles, after mixing, inoculate about 30 ages in days 4 of healthy weanling pigs, inoculate 6 milliliters for every, by 2 times of immunizing doses inoculations, other establishes 4 pigs of the same age and does blank observation 14 days.

4, pregnant sow proof test

With 4 batches of vaccines of prepared in laboratory (20 bottles/batches, 100 milliliters/bottle) at random every batch get 3 bottles, after mixing, by 2 of about 80 days sows of 2 times of immunizing doses inoculation gestation, 10 milliliters of every intramuscular injection.Establish 2 of blanks simultaneously, the same terms is raised to farrowing down.Observe influence and newborn piglet development condition that vaccine produces reproduction performance of gestation sow.

Its result is as shown in table 7

The safety verification result of table 7 genetically deficient inactivated vaccine of the present invention

Vaccine batch	The injection animal
														18 gram small white mouses			Weanling pig			Newborn piglet			80 days left and right sides sows of gestation
	Dosage (mL)	Number of elements	Observations	Dosage (mL)	Number	Observations	Dosage (mL)	Number	Observations	Dosage (mL)	Number	Observations
														Sow	Fetus
												0501 0510 0520 0530	0.3 0.3 0.3 0.3			10 10 10 10	Reactionless	6 6 6 6	4 4 4 4	Healthy	4 4 4 4	10 10 10 10	Healthy	10 10 10 10	2 2 2 2	Healthy	It is normal to grow normal development normal development normal development

Untoward reaction does not all appear in small white mouse after inoculation.1 age in days newborn piglet, the inoculation of weanling pig doubling dose, the mental status is good, and appetite is normal, and inoculation position does not have untoward reactions such as swelling heating.Have no adverse reaction with the negative control group under the condition, about 80 days sow of doubling dose injection gestation is not all miscarried yet, and normal labor does not on schedule influence sow gestation, and sow reproductive performance is not exerted an influence.The pig that farrows grow normal.Inoculation position does not have untoward reactions such as heating swelling, with 2 no abnormal clinical responses of contrast pregnant sow of raising under the condition.

(2) efficacy test of vaccine

1, replacement gilt immuning effect test

With 4 batches of vaccines at random every batch get 3 bottles, after mixing, without 4 of replacement gilts of breeding, 5 milliliters of musculi colli injected articles, other establishes 4 as non-immunity contrast,, immunity back 14,21,28 day blood sampling preceding respectively at immunity, separation of serum is done microneutralization test, detects Prv neutralizing antibody index.

2, pregnant sow immuning effect test

With 4 batches of vaccines at random every batch get 3 bottles, after mixing, select 4 of multiparity sows, every intramuscular injection is 2 milliliters before breeding, other establishes 4 and contrasts for non-immunity.To gestation 70 days in kind with the dosage booster immunization once, gather immunity respectively before, immunity back 28 days, 56 days, 80 days, 14 days serum is used for detecting antibody behind the booster immunization.Attack poison in booster immunization after 2 weeks, every pig is through ear vein injection 10 ^-7TCID ₅₀The strong malicious 2mL of/0.1mL Prv Hubei Province A strain, collunarium 2mL.Promptly attack back 14 days of poison before the expected date of childbirth and immune swine and contrast pig are cutd open killed, check every nest fetation situation.

3, weanling pig immuning effect test

With 4 batches of vaccines at random every batch get 3 bottles, after mixing, vaccine is injected each 4 of the healthy weanling pigs of 25 ages in days respectively with 3 milliliters of dosage, establish 4 of blanks.Attack poison after 14 days, every pig is through ear vein injection 10 ^-7TCID ₅₀The strong malicious 2mL of/0.1mL Prv, collunarium 1mL attacks, and observes 21 days, calculates protection ratio.

4, newborn piglet immuning effect test

With 4 batches of vaccines at random every batch get 3 bottles, after mixing, select healthy 1 age in days newborn piglet, 4 nests for use, 10 pigs of every nest, the 4 batches of vaccines are injected 8 piglets in 1 nest respectively, each injects 2mL, stays 2 as immunity contrast.Inject 10 together with the contrast pig through ear vein in back 14 days each immune swines of immunity ^-6TCID ₅₀The strong malicious 1mL of/0.1mL Prv Hubei Province A strain, collunarium 1mL attacks poison, observes and calculates protection ratio in 21 days.The result is shown in table 8-

4.1 replacement gilt

Table 8 inactivated vaccine of the present invention is to replacement gilt immuning effect test result

Vaccine batch	Immunizing dose (mL)	Test pig number (head)	The antibody neutralization index
							Before the immunity (my god)	The immunity back (my god)
			0	14	21	28
							0,501 0,510 0,520 0530 contrast	2 2 2 2	4 4 4 4 4	1 1 1 1 1	218-316 251-398 198-380 258-398 1	398-1000 398-1258 398-1258 398-1258 1	398-1000 398-1258 308-1412 398-1258 1

Illustrate: the lot number of the vaccine of this table is 20050501-20050530,4 totally batches

Lot number is 4 batches of vaccines of 20050501-20050530 immune replacement gilts respectively, and immunity is after 14 days, and the neutralizing antibody index rises to 218-398.In the time of 21-28 days, the neutralizing antibody index is up to 1412.

4.2 after the pregnant sow immunity 28 days, test pig neutralizing antibody index Gao Zheke reaches 1412, back 80 days of immunity, and serum neutralizing antibody index still can reach 1000, and with behind the same dose booster immunization 14 days, serum neutralizing antibody index Gao Zheke reached 3548.Attack poison and cutd open that the low person of serum neutralizing antibody index can be 1258 when killing in back 14 days, Gao Zheke reaches 3548.16 sows of 4 batches of vaccine immunities are cutd open and add up the fetus number extremely, get 151 on tire altogether, and the strong tire of living is 145, totally 6 on stillborn foetus and weak tire.4 non-immunity contrast pigs are total to pregnant 38, only 2 on strong tire alive, totally 36 on stillborn foetus and weak tire.

Table 9 inactivated vaccine of the present invention is to pregnant sow immuning effect test result

Vaccine batch	Immunizing dose	The test pig number	Attack the toxic agent amount	The neutralizing antibody index						The fetus number
														Before the immunity (my god)	Head exempts from			Two exempt from		Amount to	Strong tire	Stillborn foetus	Weak tire
				Back 28 days of immunity	Back 55 days of immunity	Back 80 days of immunity	Back 14 days of immunity	Attacked poison back 14 days	Number	Number	Number	Number
													20050501 20050510 20050520		5 5 5	4 4 4	4 4 4	0 0 0	389-1412 380-1258 316-1122	389-1122 398-1000 389-1000	389-1000 316-1000 316-639	1000-3548 1000-3548 398-3548	1258-3548 1258-3548 1258-3548	38 36 37	36 35 36	1 1 0	1 0 1

20050530 contrasts

5

4 4

4 4

0

316-1258

389-1000

316-1000

1000-3548

1258-3548

40 38

38 2

1 31

1 5

4.3 poison was attacked in the weanling pig immunity in back 14 days, piglet protection number average is 4/4, and protection ratio is 100%.At viewing duration, immune swine does not have any untoward reaction, and immunity contrast piglet had 3 temperature of pig body to raise in back 2 days to be respectively 40 ℃, 41 ℃, 41.8 ℃ attacking poison, and spirit is depressed, shakes vomiting, diarrhoea.Wherein 1 contrast pig has nervous symptoms, shows as ataxia, in attacking poison death in back 7 days; 3 contrast pigs are at viewing duration, and symptom alleviates gradually, recover normal at last.

Table 10 inactivated vaccine of the present invention is to weanling pig immuning effect test result

Vaccine batch	Immunizing dose (mL)	Test pig number (head)	Attack toxic agent amount (mL)	The immunity time (my god)	Attack poison protection number (head)	Protection ratio (%)
							20,050,501 20,050,510 20,050,520 20050530 contrasts	3 3 3 3	4 4 4 4 4	2 2 2 2 2	14 14 14 14	4/4 4/4 4/4 4/4 1/4	100 100 100 100 25

4.4 newborn piglet after immunity, is attacked poison after 14 days, is attacking poison back in the observation period, has 1 attacking the poison back 24-72 hour, fervescence to 40.5 ℃-41.8 ℃, and other immune swines all have no adverse reaction after attacking poison.Total protection ratio is 90.6%.8 not immune contrast pigs are in attacking poison back 24 hours, fervescence to 40.5 ℃-42 ℃, and lassitude is shaken, and vomiting and diarrhoea wherein have 6 attacking poison back death in 36-50 hour respectively.Dead pig is cutd open the inspection pathology and shows as upper respiratory tract mucosa and hemorrhage of tonsil, pulmonary edema, and kidney has the big blutpunkte of needle point, meninx hyperemia, hemorrhage, oedema.2 contrast pigs are in attacking the 10th day sx in poison back in addition, and body temperature descends to some extent, but still keeps the low-grade fever about 40 ℃ in the observation period.

Table 11 inactivated vaccine of the present invention is to newborn piglet immuning effect test result

Vaccine batch	Immunizing dose (mL)	Test pig number (head)	Attack toxic agent amount (mL)	The immunity time (my god)	Attack poison and protect a number (head)	Protection ratio (%)	Morbidity contrast (head)
								20050501 20050510 20050520 20050530	2 2 2 2	8 8 8 8	1 1 1 1	14 14 14 14	7/8 7/8 7/8 7/8	87.5 87.5 87.5 100	2/2 2/2 2/2 2/2

The detection of the piglet source of parents neutralizing antibody level after the 5 sow immunity

Vaccinate 2mL before breeding of picking produces previous month booster immunization once (2mL), and it is 1: 16 that blood sampling in antenatal 2 days is surveyed in it with antibody.And the antibody in the first day postpartum milk reaches 1: 64-128.Young 11 of common property is chosen wherein 7 piglets and is taken a blood sample in different days, has carried out the complete detection of antibody horizontal, and it the results are shown in Table

The detection of the piglet source of parents neutralizing antibody level of table 12 inactivated vaccine of the present invention after to the sow immunity

13	1∶11	1∶18.7	1∶11	1∶11	1∶18.7	1∶18.7	1∶22
								21	1∶9.4	1∶11	1∶11	1∶8	1∶8	1∶11	1∶8
30	1∶5.6	1∶9.4	1∶9.4	1∶9.4	1∶5.6	1∶5.6	1∶56
								40	1∶4	1∶4	1∶8	1∶2	1∶4	1∶5	1∶2
50	1∶2	-	1∶4	-	1∶2	-	-
								60	1∶2	-	-	-	-	-	-

Experimental result all exceed antibody horizontal in the sow serum through the colostrum milk antibody horizontal of vaccine immunity sow, thereby the antibody horizontal in the newborn piglet serum that records is also higher.But along with the increase of age in days, it is also very fast that antibody horizontal descends, lower to 30 ages in days, and therefore clinical, it is comparatively suitable carrying out when the immunity of piglet pseudoabies is fixed on wean that head exempts from.

(3), the duration of immunity of vaccine

The immunity of 1 pig began to occur antibody in back 14 days, though the neutralizing antibody index does not reach positive criteria 316, only compared obviously rising with non-immunity contrast pig.Immunity is back 28 days the time, and 5 batches of vaccine neutralizing antibody indexes all reach more than 398, peak at immune 35-42 days, and later antibody horizontal descends gradually, and antibody horizontal equals or near threshold value during by 180 days.According to antibody horizontal growth and decline situation, the duration of immunity of this vaccine is as preventing with being decided to be six months.To pregnant sow, for first Ruzhong has high-caliber maternal antibody to protect newborn piglet, advise just before giving birth previous month booster immunization once, can protect piglet to arrive wean fully.When fattening the weaned piglet of usefulness injection once, up to delivering for sale.During to the weaned piglet of kind of usefulness injection once, every 4-6 week again booster immunization once, as fundamental immunity, later immunity every half a year once, concrete outcome sees the following form.

The detection of the neutralizing antibody behind the table 13 boar initial immunity

The vaccine lot number	Immune swine number (head)	Clear body of the anti-middle finger of blood (mean value) and number
												14 days	21 days	28 days	35 days	42 days	52 days	85 days	125 days	155 days	180 days
																						0,501 0,510 0,520 0530 contrast	4 4 4 4 2	199 223 251 223 1	354 316 316 398 1	457 355 501 355 1	954 1258 1122 819 1	1513 1412 1258 1096 1	1412 1122 1203 1096 1	758 891 794 891 1	630 398 316 398 1	407 398 316 316 1	199 199 112 199 1

Antibody horizontal measurement result behind the 2 boar second immunisatioies

Antibody horizontal peaks when immune back 42 days left and right sides once, thereby carries out the immunity second time in the time of 42 days, and back 14 days antibody horizontals of immunity obviously rise, during by 180 days, antibody horizontal is apparently higher than once immunity, and the immunity back antibody time length is also longer, and concrete outcome sees the following form.

Neutralization index behind the table 14 boar second immunisation

The vaccine lot number	Immune swine number (head)	Serum antibody neutralization index (mean value)
													14 days	21 days	28 days	35 days	42 days	52 days	62 days	85 days	125 days	155 days	180 days

0,501 0,510 0,520 0530 contrast

4 4 4 4 2

7162 7891 7019 7891 1

6818 6511 6818 6818 1

6511 6238 6511 6238 1

5995 5584 5584 5584 1

5258 5122 5412 5258 1

4912 4309 4912 4079 1

4141 4309 4309 4309 1

3162 2511 3162 3162 1

2511 2511 2818 2511 1

794 891 1029 891 1

620 630 891 630 1

(4), the preservation period of vaccine

Vaccine leaves 4-8 ℃ in to be preserved 12 months and 18 months; Preserved 1 month and 3 months for 20-25 ℃, with the 3mL using dosage to 1 the monthly age weanling pig carry out immunity, respectively in back 21 days of immunity and blood sampling in 28 days, detect in it and antibody horizontal, all between 316-398, the level that produces antibody is all in normal range, and use strong virus attack, pig is only all normal, confirms that fully vaccine quality does not change in above-mentioned preservation condition and preservation period.Thereby the standard of drafting is decided to be 4-8 ℃ and preserves December, preserves 1 month for 20-25 ℃

The preservation period test (is example with the weanling pig) of table 15 inactivated vaccine of the present invention

The vaccine lot number	Preservation condition ℃	Shelf time (moon)	Test pig number (head)	Neutralization index (mean value)
								Preceding 1 day of immunity	Back 21 days of immunity	Back 28 days of immunity	Attacked poison back 14 days
				0,501 0,510 0,520 0530 contrast	20-25 4-8 20-25 4-8 20-25 4-8 20-25 4-8 20-25	1 3 12 15 1 3 12 15 1 3 12 15 1 3 12 15 1 3	2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4					1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1	398 354 346 380 380 363 354 295 372 324 380 331 478 346 380 355 380 363 1	501 407 436 426 602 478 489 426 537 501 602 588 616 490 478 467 501 407 1	398 436 407 416 512 467 457 489 537 436 501 575 588 380 467 446 537 436 331

As shown in Table 15, inactivated vaccine of the present invention 4-8 ℃ preserved 12 months and 4-8 ℃ preserve 15 months after, difference immune animal (for example pig, small white mouse, rabbit etc.), the neutralizing antibody index differential that is produced in the time of 28 days is significantly (P＞0.05) not, illustrates that inactivated vaccine of the present invention can preserve 12-15 month at 4-8 ℃; 20-25 ℃ preserved 1 month and 20-25 ℃ preserve 3 months after, the neutralizing antibody index differential that is produced in the time of 28 days is (0.01＜P≤0.05) significantly.Vaccine of the present invention can be preserved 1 month at 20-25 ℃.

Claims (7)

1, a kind of Pseudorabies virus Pseudorabies virus strain WKQ-001 of reorganization is deposited in CCTCC, and deposit number is CCTCC-V200511.

2, the pseudoabies poison strain of the described reorganization of claim 1 is characterized in that:

1) said strain has lacked gE, gI gene;

2) said strain does not contain foreign gene.

3, the gene-deleted vaccine that comprises claim 1 or 2 described recombinant pseudorabies virus strains.

4, the described gene-deleted vaccine of claim 3 is characterized in that, described gene-deleted vaccine is an inactivated vaccine.

5, claim 3 or 4 described gene-deleted vaccines by the proportioning of umber are:

3 parts of A, waters

Each part water adds the preparation of 4 parts of sterilization tween 80s by 96 parts of pseudoabies venom with the deactivation of claim 1 or 2 described pseudoabies strain preparations;

6 parts of B, oil phases

Each part oil phase is by 94 parts of No. 10 white oils for animals in milliliter, add in 2 parts of aluminum stearates of gram and in 6 parts of Si Ben-80 ratios preparations of milliliter and

C, the above-mentioned A of mixing and described water of B and oil phase.

6, claim 1 or the 2 described recombinant pseudorabies virus strains application in preparation Pseudorabies virus gene-deleted vaccine.

7, the application of each described gene-deleted vaccine of claim 3-5 in anti-system pseudoabies.

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