CN105200015B

CN105200015B - A kind of porcine pseudorabies strain

Info

Publication number: CN105200015B
Application number: CN201510569963.8A
Authority: CN
Inventors: 陈红英; 杨明凡; 高晓云; 崔保安; 杨兴武; 张宇; 徐朋丽
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2015-09-09
Filing date: 2015-09-09
Publication date: 2019-04-12
Anticipated expiration: 2035-09-09
Also published as: CN105200015A

Abstract

The invention discloses a kind of porcine pseudorabies strains, it is characterised in that: porcine pseudorabies virus (Herpesviridae) strain, CCTCC NO:V201314.The present invention reached for the 7th generation for PRV/HN2012 plants, and had carried out virus titer TCID to the 7th generation virus₅₀Measurement, as a result TCID₅₀It is 10^9.0.And by PRV/HN2012 plants of Mice Inoculateds, after attacking 24 h of poison, there are nervous symptoms in PRV/HN2012 plants of infected group mouse, and after attacking 48 h of poison, mouse is all dead, show that the strain has very strong toxicity.Based on this, by PRV/HN2012 plants of Study on Physico-chemical, the results showed that pure chloroform is sensitive, and belonging to has togavirus to analyzing for the strain；There is stronger resistance to acid, alkali, the hydrochloric acid of pH3.0,1 h of NaOH processing of pH 11.0 make its inactivation；There is stronger resistance to heat, 56 DEG C of 1 h of water bath processing could be inactivated；Sensitive to trypsase, 30 min of UV treatment influences its infectivity unobvious.

Description

A kind of porcine pseudorabies strain

Technical field

The present invention relates to a kind of porcine pseudorabies poison strains.

Background technique

Pseudoabies (Pseudorabies, PR) is the domestic animal as caused by Pseudorabies virus (PRV) and a variety of wild dynamic Object one kind to generate heat, surprise is itched (in addition to pig) and encephalomyelitis is a kind of acute infectious disease of cardinal symptom.Disease head in 1813 The U.S. is seen, since its clinical manifestation is similar with rabies, therefore referred to as pseudoabies.1902, Hungary scholar Aujeszky It first reported the disease, therefore the disease is also named as AujeszkyShi disease.Pig is the natural reservoir (of bird flu viruses) and major source of infection of the disease, PRV can infect the pig of different age group, but the most serious with pregnant sow and suckling pig infection: pregnant sow being caused to be flowed Production, stillborn foetus and the mummification of fetus；There is apparent nervous symptoms, paralysis, failure death in newborn piglet, and mostly acute fatal passes through It crosses, the death rate is almost up to 100%.The disease is distributed widely in countries in the world.China Liu Yongchun nineteen forty-seven divides first out of cat body From arrive the virus.The disease China's prevalence quite extensively, this disease occurred for many pig farms, was that sow breeding difficulty, piglet are dead Die one of the main reason for rate is high.Especially in recent years, as China's pig breeding industry is intensive, the development of scale, pseudorabies Disease in many regional outbreak of epidemic has the tendency that sprawling is gradually expanded, causes grave danger to pig breeding industry, seriously hinder me The sustainable development of state's pig breeding industry.Since this disease of self-discovery, extremely extensive research has been carried out to it, and achieve very greatly into Exhibition, many countries successful eradications or have controlled the disease at present, and the research and prevention and control to the disease also have become that study molecule viral Learn and successfully control the model of virosis.

In production practice, it relies primarily on vaccine especially engineered deletion vaccine and anti-system is carried out to PR, to make this Disease is effectively controlled.It is immune using PRV marker vaccines in more areas such as Henan, Jilin, Heilungkiang since spring in 2012 Pig farm there is doubtful PR case, sow produces weak young, stillborn foetus or nervous symptoms, death occur in the mummification of fetus, piglet, and the death rate reaches 10%~50%, huge economic losses are caused to China's pig breeding industry.Therefore from the clinical sample that pig farm inspection is immunized in Henan, It separates and identifies PRV henan strains, illustrate the hereditary variation situation of PRV prevalence strain in Henan Province swinery, and carry out correlation Biological characteristic research is just particularly important.

Summary of the invention

The object of the present invention is to provide a kind of porcine pseudorabies strain, classification namings: herpetoviridae herpesvirus suis I type Porcine pseudorabies virus PRV/HN2012, Latin name: Herpesviridae.Depositary institution: in China typical culture collection The heart, depositary institution's abbreviation: CCTCC, depositary institution address: the Wuhan University of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16 is protected Hide the date: on May 28th, 2013, deposit number CCTCC NO:V201314.

The technical scheme is that a kind of porcine pseudorabies strain, porcine pseudorabies virus (Herpesviridae) poison Strain, CCTCC NO:V201314.

The beneficial effects of the present invention are: the present invention reached for the 7th generation for PRV/HN2012 plants, and carried out to the 7th generation virus The measurement of virus titer TCID50, as a result TCID50 is 109.0.And by PRV/HN2012 plants of Mice Inoculateds, after attacking poison for 24 hours, There are nervous symptoms in PRV/HN2012 plants of infected group mouse, and after attacking malicious 48h, mouse is all dead, show that the strain has very Strong toxicity.Based on this, by PRV/HN2012 plants of Study on Physico-chemical, the results showed that the strain is quick to pure chloroform is analyzed Sense, belonging to has togavirus；There is stronger resistance to acid, alkali, the hydrochloric acid of pH3.0, the NaOH processing 1h of pH 11.0 make its mistake It is living；There is stronger resistance to heat, 56 DEG C of water bath processing 1h could be inactivated；Sensitive to trypsase, UV treatment 30min is right Its infectivity influences unobvious (table 1).These physicochemical properties meet the physicochemical property of porcine pseudorabies virus.It is observed through Electronic Speculum, It can be seen that the virion that size is uniform, approximate circle, size about 110~150nm, there is the fibre being arranged radially on cyst membrane surface It is prominent, it coincide substantially with reported PRV form.Therefore, the present invention is successfully separated out PRV/HN2012 plants, and to PRV/HN2012 Strain has carried out physicochemical property test and morphological observation, and the prevalence for control porcine pseudorabies in Henan provides theoretical foundation.

Detailed description of the invention

Fig. 1 is the PCR amplification result of PRV in tissue pathological material of disease, M.DNA Marker DL2000；PRV in 1-9. pathological material of disease PCR product；10. negative control.

Fig. 2 is the ST cell (100 ×) of infection separation poison PRV, the normal ST cell controls of A.；B. separation poison PRV infection ST cell.

Fig. 3 is PRV gE gene magnification result, M.DNA Marker DL2000；1-3.PCR product；4. negative control.

Fig. 4 is PRV PRV/HN2012 plants of electromicroscopic photograph.

Fig. 5 is PRV gE full genome amplification, M.DNA Marker DL2000；1-4.PCR product；5. negative control.

Fig. 6 is recombination plasmid PCR identification, M.DNA Marker DL2000；1-9.PCR product；N. negative control.

Fig. 7 is the digestion qualification figure of recombinant plasmid, M.DNA Marker DL5000；1-3. recombinant plasmid is through I He of EcoR I double enzyme digestion product of Pst；4. the digestion products (negative control) of empty plasmid.

Fig. 8 is that the nucleotide sequence homology of PRV difference separation strains gE full genome is analyzed.

Fig. 9 is the comparison for the amino acid sequence that PRV gE gene derives.

Figure 10 is the systematic evolution tree of PRV separation strains gE gene nucleotide series.

Figure 11 is the PCR amplification of PRV gC gene, M.DNA marker DL2000；1.PCR product；2.PCR product；3. Negative control.

Figure 12 is recombination plasmid PCR identification, M.DNA Marker DL2000；1-4.PCR product；5. negative control.

Figure 13 is the digestion qualification figure of recombinant plasmid, M.DNA Marker DL5000；The digestion products of 1-2. empty plasmid (negative control)；3-4；Recombinant plasmid is through I double enzyme digestion product of EcoR I and Pst.

Figure 14 is that the nucleotide sequence homology of PRV difference separation strains gC full genome is analyzed.

Figure 15 is the amino acid sequence homology analysis that PRV gC gene derives.

Figure 16 is the systematic evolution tree of PRV separation strains gC gene nucleotide series.

Specific embodiment

A kind of porcine pseudorabies poison strain, classification naming: herpetoviridae herpesvirus suis I type porcine pseudorabies virus PRV/ HN2012, Latin name: Herpesviridae.Depositary institution: China typical culture collection center, depositary institution's abbreviation: CCTCC, depositary institution address: the Wuhan University of Wuhan City, Hubei Province Wuchang District Luo Jia Shan road 16, preservation date: in May, 2013 28 days, deposit number CCTCC NO:V201314.

The present invention carries out PCR detection using pathological material of disease of the PCR method to the doubtful PR that Henan acquires, by being inoculated with pig testis (ST) cell is separated to 1 strain virus, is named as HN2012.Pass through observation cytopathy, biological characteristic research, Electronic Speculum observation disease The approach such as the form of poison confirm that isolated viral is porcine pseudorabies virus.

By PRV/HN2012 strain virus liquid, oil emu, inoculation health 6 weeks are mixed and made into Freund's adjuvant after formalin-inactivated Age female KM mouse, while Bartha-K61 vaccine strain commercial seedling, 98 vaccine strain commercial seedling immune group of Hubei and yin are set Property control group, blood sampling separation serum after 28d carries out serum neutralization test respectively, and detection neutralizing antibody is horizontal, and uses PRV/ HN2012 plants of progress protest tests, as a result PRV/HN2012 plants of oil emulsion adjuvant emulsion immune group mouse are protected completely, show PRV/HN2012 has good immunogenicity.

One, the separation identification of porcine pseudorabies virus PRV/HN2012 and biological characteristic research

1 materials and methods

1.1 material

1.1.1 pathological material of disease, cell strain and experimental animal are organized

The clinical onset sample of the doubtful PRV infection in pig farm is immunized from Henan PR for pathological material of disease, acquires in March, 2013, packet Include brain, liver, spleen and the lymph node of the piglet that dies of illness.Pig testis (ST) cell is purchased from China Veterinary Drugs Supervisory Inst., by room of the present invention It freezes.6 week old female KM mouse are purchased from experimental animal center of henan province.

1.1.2 main agents

Prime GXL enzyme, EX Taq enzyme, DNA Marker DL2000 etc. are purchased from Dalian treasured bioengineering Co., Ltd； RPM-1640 culture solution, fetal calf serum, trypsase etc. are purchased from Suo Laibao Science and Technology Ltd..Agarose is public purchased from Promega Department；Proteinase K is purchased from Huamei Bio-Engrg Co.,；Disodium ethylene diamine tetraacetate (EDTA), is satisfied at lauryl sodium sulfate (SDS) Shanghai bioengineering Co., Ltd is purchased from phenol, chloroform, isoamyl alcohol.

1.2 design of primers

Referring to the PRV delivered in GenBank gH(X61696),gE(AF171937) gene order, it is soft using design of primers Part devises 2 pairs of primers, and primer P1 and P2 are for expanding partgHGene, target fragment length are 352 bp, primer P3 and P4 Expanding fragment length is 425 bp, for expanding partgEGene, primer are synthesized by Shanghai bioengineering Co., Ltd.

P1:5'-GCGTGTGCGACTGCGTGTT-3',

P2:5'- CCTGGCGTTTATTAACCGAGA-3'；

P3:5'- TACACGTTCGACCTGATG-3'

P4:5'- CCTTGACCGTGACGTACT-3'.

The PCR detection of PRV DNA in 1.3 tissue pathological material of diseases

Take the tissues such as brain, lymph node, spleen, liver, kidney, the lungs of acquisition to shred, mix after be ground into homogenate, be added PBS liquid (penicillin and streptomycin containing 1000 units), is made 10% ~ 30% suspension, and multigelation 3 times.400 μ L of supernatant is taken, is adopted DNA is extracted with proteinase-K pathway.Using pathological material of disease tissue DNA as template, PCR amplification is carried out.In PCR reaction system, template DNA 3 Each 12 μ L of 0.5 μ L, Taq archaeal dna polymerase of μ L, upstream and downstream primer P1/P2, moisturizing to 25 μ L；It is carried out after mixing brief centrifugation PCR amplification.PCR amplification program is 95 DEG C of 5 min；94 DEG C of 1 min, 53 DEG C of 45 s, 72 DEG C of 1 min, 35 circulations；72℃ 10 min; 4℃10 min.After reaction, take 5 μ L PCR products with 10 g/L Ago-Gels (containing 0.5 mg/L EB) Carry out the result of electrophoresis detection PCR.

The separation and culture of 1.4 viruses

By the pathological material of disease homogenate of PCR test positive, 10 min are centrifuged through 8000 r/min, are added in supernatant double Anti-, 4 DEG C of 12 h of effect are inoculated with ST cell respectively after 0.22 μm of bacterial filter filtration sterilization, abandon after 37 DEG C of 2 h of absorption Clearly, suitable RPMI-1640 maintaining liquid for containing 2% fetal calf serum is added to continue to harvest virus after cultivating 72 h.And on ST cell Continuous culture reached for the 6th generation.The cell culture for not connecing poison is set up in incubation simultaneously as negative control.Succeeding generations In, using PRV specific primer P1/P2, qualitative detection has PRV in the ST cell of vacuole lesion.

1.5 PRV gEThe detection of gene

By the PRV cell toxicant of harvest, freeze thawing 3 times with lytic cell, 6000 r/min are centrifuged 6 ~ 8 min, supernatant are taken to extract DNA, method is the same as 1.3.Using the cell toxicant DNA of extraction as template, PCR amplification is carried out with primer P3/P4.In PCR reaction system, Template DNA each 0.5 μ L of 3 μ L, upstream and downstream primer P3/P4,1 μ L, Ex Taq Mixture 12 of dimethyl sulfoxide (DMSO) μ L, moisturizing to 25 μ L；PCR amplification program is 94 DEG C of 5 min；95 DEG C of 30 s, 53 DEG C of 30 s, 72 DEG C of 1 min, 35 are followed Ring；72 DEG C of 8 min, 4 DEG C of 10 min.After reaction, take 5 μ L PCR products with 10 g/L Ago-Gels (containing 0.5 mg/L EB the result of electrophoresis detection PCR) is carried out.

1.6 virus titer TCID₅₀Measurement

The ST cell suspension inoculation that digestion is dispelled is into 96 orifice plates, every 200 μ L of hole, after cultivating 24 h, takes PRV respectively The continuous doubling dilution 10 of the 7th generation cell culture of different strains^-1~10^-10, every hole is inoculated with 100 μ L, in cell incubator After being incubated for 1 h, every hole is added RPMI-1640 maintaining liquid of the 100 μ L containing 2% fetal calf serum and continues to cultivate, and each dilution respectively makees 8 A repetition observes cytopathy situation day by day, and calculates virus titer TCID according to Reed-Muench method₅₀。

The test of 1.7 PRV infecting mouses

20 6 week old female KM mouse are divided into 2 groups, one group of the 7th generation cell culture Mice Inoculated by separation strains, 500 μ L/, intramuscular injection, blank control group mouse inoculating cell culture solution, 500 μ L/, intramuscular injection.Day by day it observes small The morbidity and death condition of mouse.

1.8 PRV/HN2012 plants of Study on Physico-chemical

The EP pipe for taking 8 sterilizings, is separately added into the 7th generation cell liquid of 500 μ L PRV/HN2012 strain in super-clean bench.1st The final concentration of 4.8% pure chloroform mixing oscillation of analysis is added in pipe, 4 DEG C of 30 min of effect is placed in, then with 500 rpm/min's Speed is centrifuged 5 min, then draws supernatant liquid；The hydrochloric acid tune pH to 3.0 of 2nd effective 0.1 mol/L is acted in 37 DEG C of water-baths 1 h, then recalled to pH value to 7.2 with the NaOH solution of 0.1 mol/L；The NaOH solution tune pH of 3rd effective 0.1 mol/L is extremely 11,1 h is acted in 37 DEG C of water-baths, then recalled to pH value to 7.2 with the hydrochloric acid solution of 0.1 mol/L；4th pipe is placed in 56 DEG C of water-baths 1 h of middle effect；5th pipe is added isometric trypsase and sets after 37 DEG C of water-baths act on 1.5 h, whole with 4mL inactivated fetal bovine serum Only it is acted on；6th pipe is added the analysis pure formaldehyde mixing concussion of volume ratio 1%, 37 DEG C of 2 d of effect, with the speed of 2000 rpm from 20 min of the heart, then draws lower liquid；7th pipe ultraviolet irradiation, 30 min；8th pipe is not handled as normal control.Then divide Not Jie Zhong ST passage cell measure its TCID according to a conventional method₅₀。

1.9 PRV/HN2012 plants of morphological observation

Take after the 8th net multigelation of generation cell culture of 200 mL PRV/HN2012 strain at 4 DEG C, 8000 r/min from Heart 1h takes at 4 DEG C of supernatant, and 30000 r/min are centrifuged 2 h, and precipitating is resuspended with 10 mLpH7.2,0.01 mol/L PBS, then 4 At DEG C, 8000 r/min are centrifuged 1 h, take at 4 DEG C of supernatant, and 30000 r/min are centrifuged 2 h, final 20 μ L pH7.2 0.01 Precipitating is resuspended in mol/L PBS.It takes 10 μ L re-suspension liquids and 10 g/L acetic acid uranium dye liquors to drip on sealed membrane, will there is the copper for supporting film Net is first placed on sample drop, is picked up after 3-5 min with tweezers, sucks liquid from copper mesh edge with clean filter paper, slightly dry postposition Dye liquor is sucked in dyeing 3 min on dyeing liquor, then with filter paper, morphology of virus is observed under Hitachi TEM transmission electron microscope and is clapped According to.

2 results

The PCR testing result of PRV DNA in 2.1 tissue pathological material of diseases

Suspension is made in the sterile grinding of pathological material of disease that doubtful PRV infects, extracts DNA.Using the DNA of extraction as template, PCR is carried out Amplification.5 μ L of pcr amplification product is taken, electrophoresis detection is carried out with 10 g/L Ago-Gels (containing 0.5 mg/L EB), amplifies 1 The band (Fig. 1) of 350 bp of item or so is consistent with expected amplified fragments size.

The cell culture of 2.2 PRV and cytopathy are observed

By the dual anti-processing of Henan pathological material of disease tissue homogenate of PCR test positive, ST cell is inoculated with after aseptic filtration, and It is passed on.There is the typical cell vacuole lesion of PRV in each generation, and utilizes PRVgESpecific primer, to cell toxicant DNA carries out PCR amplification, can detect PRVgESpecific target fragment, and normal ST cellular nucleic acid is that template carries out PCR expansion Increase, result is feminine gender.Cytopathy is stablized after connecing malicious 3rd generation, meets 24 h of poison and virus plaques occurs, plaque peripheral cell becomes Circle, falls off, and part cell fragment is adhered in culture bottle wall (Fig. 2).Cell detachment is up to 75% or more after meeting 72 h of poison.Show Virus has adapted to ST cell, and can be proliferated on ST cell.Isolated PRV is named as PRV/HN2012.

2.3 PRV gEThe PCR amplification result of gene

By the PRV cell culture of harvest, freeze thawing 3 times with lytic cell, 6000 r/min are centrifuged 6-8 min, take supernatant DNA is extracted, amplifies 1 about 450 bp from PRV genomic DNA as template with primer P3/P4 using the cell toxicant DNA of extraction Segment (Fig. 3), it is consistent with expected amplification.

2.4 virus titer TCID₅₀As a result

TCID is carried out to the 7th generation virus liquid of PRV/HN2012₅₀Measurement, is observed, 3 d are waited for after connecing poison by inverted microscope After the apparent CPE such as be rounded, fall off occurs in cell, the TCID of strain is counted and calculated by Reed-Muench method₅₀, result is 10^9.0。

2.5 virus infected mice test results

After attacking 24 h of poison, there are nervous symptoms in PRV/HN2012 plants of infected group mouse, roll up spasm, and sting rotten injection point Skin, cause local coat to fall off, dermatorrhagia.After attacking 48 h of poison, PRV/HN2012 plants of infected group mouse are all dead；Its Successively there are nervous symptoms after attacking malicious 72h in his 8 virus strain infection group mouse, attack all death after 120 h of poison.Blank control group is small Mouse injects still health survival after 144 h.

2.6 PRV/HN2012 plants of Study on Physico-chemical result

As it can be seen from table 1 viral PRV/HN2012 plants of separation acquisition sensitive to pure chloroform is analyzed, belonging to has cyst membrane sick Poison；There is stronger resistance to acid, alkali, the hydrochloric acid of pH3.0,1 h of NaOH processing of pH11.0 make its inactivation；Have to heat and supports more by force Drag, 56 DEG C of 1 h of water bath processing could be inactivated；Sensitive to trypsase, 30 min of UV treatment influences not its infectivity Obviously.These physicochemical properties meet the physicochemical property of porcine pseudorabies virus.

The testing result of 1 physicochemical property of table

2.7 morphology of virus observe result

After PRV/HN2012 plants of cell toxicants are done appropriate purification process, electric microscopic observation is set.Size can be seen in the visual field Uniform, approximate circle virion, about 110 ~ 150 nm of size, cyst membrane surface have the fibre being arranged radially to dash forward (Fig. 4), with Reported PRV form is coincide substantially, has no the virion of other forms.

3 conclusions and discussion

gEGene is one of major virulence gene of PRV, gEGene-deleted vaccine quilt in terms of Virus entry nervous system Think that glycoprotein missing seedling more necessary than other single expenses is safer, simultaneouslygEGene delection will not influence the duplication and neutralization of virus The generation of antibody, therefore it is defined as by World Organization for Animal Health the missing gene of gene-deleted vaccine.And due togEIt is all A kind of glycoprotein that street strain expresses, therefore this research, from the doubtful pathological material of disease that pig farm inspection is immunized in Henan PR, separation PRV is simultaneously Secondary culture is carried out, marker gene is passed throughgEAmplification, identifying separated virus is street strain.PRV gEThe G+ of gene C content is up to 73%, has complicated secondary structure, thereforegEThe design of primers and PCR amplification difficulty of gene are larger.The present invention With reference to the Ea strain announcedgEGene order devises 1 pair of primer, using the DNA of separation strains as template, expands gE Gene Partial piece Section (425bp) establishes and distinguishes the specific PCR amplification method that strain and street strain is immunized in pseudorabies virus vaccine.

Homogenate is made in the Henan pathological material of disease tissue grinder of PCR test positive, dilution, is followed by kind of a ST cell with processing, Isolate PRV/HN2012.Cytopathy is stablized after connecing malicious 3rd generation, meets 24 h of poison and virus plaques, plaque peripheral cell occurs It is rounded, falls off, part cell fragment is adhered in culture bottle wall (Fig. 2 B), and cell detachment is up to 75% or more after meeting 72 h of poison.It mentions Cell toxicant DNA is taken, PRV is utilizedgESpecific primer carries out PCR amplification to cell toxicant DNA, can detect PRVgESpecifically Property target fragment, and normal ST cellular nucleic acid is that template carries out PCR amplification, and result is feminine gender.6 are inoculated with by PRV/HN2012 plants There is typical PR symptom, roll up spasm, and sting the skin of rotten injection point in all female mices, cause local coat to fall off, skin Skin bleeding etc., with Peng Jinmei etc., newly to separate HeN1 plants of Mice Inoculated test results similar by PRV after 2011 of reports for this.

The present invention reached for the 7th generation for PRV/HN2012 plants, and had carried out virus titer TCID to the 7th generation virus₅₀Measurement, As a result TCID₅₀It is 10^9.0.And by PRV/HN2012 plants of Mice Inoculateds, after attacking 24 h of poison, PRV/HN2012 plants of infected group mouse are There are nervous symptoms, after attacking 48 h of poison, mouse is all dead, shows that the strain has very strong toxicity.Based on this, by right PRV/HN2012 plants of Study on Physico-chemical, the results showed that pure chloroform is sensitive, and belonging to has togavirus to analyzing for the strain；To acid, Alkali has stronger resistance, and the hydrochloric acid of pH3.0,1 h of NaOH processing of pH 11.0 make its inactivation；There is a stronger resistance to heat, 56 DEG C 1 h of water bath processing could be inactivated；Sensitive to trypsase, 30 min of UV treatment influences unobvious (table to its infectivity 1).These physicochemical properties meet the physicochemical property of porcine pseudorabies virus.It is observed through Electronic Speculum, it can be seen that size is uniform, approximate circle The virion of shape, about 110 ~ 150 nm of size, cyst membrane surface has the fibre being arranged radially prominent, with reported PRV form Substantially it coincide.Therefore, the present invention is successfully separated out PRV/HN2012 plants, and has carried out physicochemical property examination to PRV/HN2012 plants It tests and morphological observation, the prevalence for control porcine pseudorabies in Henan provides theoretical foundation.

Two, porcine pseudorabies virus PRV/HN2012 plantsgEThe clone of full genome and sequence are analyzed

1 materials and methods

1.1 material

1.1.1 strain and carrier

Competent cell DH5 α, cloning vector pMD^®18-T is purchased from Dalian treasured bioengineering Co., Ltd.

1.1.2 toolenzyme and other reagents

Prime GXL enzyme, rTaq enzyme, T₄It is DNA ligase, DNA Marker DL 2000, IPTG, X-gal, restricted Restriction endonucleaseEcoRⅠ、PstI is equal purchased from precious bioengineering (Dalian) Co., Ltd；DNA gel QIAquick Gel Extraction Kit, plasmid extract reagent Box is purchased from hundred Tyke Bioisystech Co., Ltd of Beijing；Proteinase K is purchased from Huamei Bio-Engrg Co.,；Agarose is purchased from Promega company；Disodium ethylene diamine tetraacetate (EDTA), lauryl sodium sulfate (SDS), saturated phenol, chloroform, isoamyl alcohol are purchased from Shanghai bioengineering Co., Ltd.

1.1.3 Strain

PRV/HN2012 plants are separated identification by this laboratory and are saved.

1.2 design of primers

Referring to what is delivered in GenBankgE(AF171937) gene order devises 1 pair using primer-design software and draws Object, for expand includegEThe segment of gene complete sequence, length are 1855 bp, and primer is by the raw limited public affairs of work bioengineering in Shanghai Department's synthesis.

P1:5'- GGTGTGGCGTTTTATCT-3'

P2:5'- TTTGGGCATGTCGGAATG-3'

The extraction of 1.3 viral DNAs andgEThe amplification of full genome

By the PRV cell toxicant of harvest, freeze thawing 3 times with lytic cell, 6000 r/min are centrifuged 6-8 min, take supernatant, use egg White enzyme K method extracts DNA.Using the cell toxicant DNA of extraction as template, PCR amplification is carried out with primer P1/P2.In PCR reaction system In, each 1 μ of 0.5 μ L, Primer STAR GXL DNA Polymerase of template DNA 0.5 μ L, upstream and downstream primer P1/P2 5 μ L, dNTP Mixture of L, 5 × Primer STAR GXL Buffer, 2 μ L, moisturizing to 25 μ L；PCR amplification program is 95℃3 min；98 DEG C of 10 s, 55 DEG C of 15 s, 68 DEG C of 2 min, 35 circulations；4℃10 min.After reaction, 5 μ L are taken PCR product carries out (containing 0.5 mg/L EB) result of electrophoresis detection PCR with 10 g/L Ago-Gels.

1.4 PRV gEThe clone of full genome and identification

It is positive product recovery purifying by PCR testing result, PRV/HN2012 plants of genetic fragment is carried out to add A tail After processing, it is connected to cloning vector pMD^®In 18-T, the extraction of conversion, recombinant plasmid through connection product, recombinant plasmid PCR The processes such as identification, digestion identification carry out sequencing to the positive colony of identification, are completed by Shanghai bioengineering Co., Ltd.

1.5 PRV gEThe sequence of full genome is analyzed

With softwares such as MEGA4, DNAStar to the PRV of clone gE18 plants downloaded in complete genome sequence and GeneBank PRV gEComplete genome sequence is compared, to PRVgEThe amino acid sequence of full genome and gE glycoprotein is compared point Analysis.All Reference strains (see Table 2)s are the typical strain included on GenBank.

2 Reference strains accession number of table

2 results

2.1 PRV gEThe amplification of full genome

To extract PRV/HN2012 plants of cell toxicant DNA as template, PCR amplification, amplified fragments warp are carried out with primer P1/P2 Agarose gel electrophoresis detection is crossed, the band (Fig. 5) of 1 treaty, 1900 bp occurs, the length of amplified fragments is consistent with expection.

2.2 PRV gEThe clone of full genome and the PCR identification of recombinant plasmid

After PCR product recovery purifying, enzyme used is Primer GXL when due to amplification full genome, which is high-fidelity Enzyme, with 3 ' to 5 ' exonucleases activity, cause PCR react after will not tailing, influence the company of target gene and carrier T Efficiency is connect, therefore is connected in carrier T after adding A, connection product is converted, extracts recombinant plasmid, PCR mirror is carried out to recombinant plasmid Fixed, there is the band (Fig. 6) of 1 treaty, 1900 bp in electrophoresis, is consistent with expected size.

2.3 PRV gEThe digestion of full genome recombinant plasmid is identified

Recombinant plasmid warpEcoRⅠ、PstI double digestion, electrophoresis occur 2 bands: wherein a band be vector plasmid, about 2 600 Bp, another 1 band are the PRV that is cloned gEFull genome segment, about 2 000 bp(Fig. 7), it is consistent with expected results.

2.4 PRV gEThe sequencing result of full genome

It is accredited as positive recombinant plasmid to PCR and digestion to be sequenced, the PRV/HN2012 strain of sequencer addressgEGene Nucleotide sequence is 1855 bp, includes a complete open reading frame as shown in SEQ ID No:1.gEGene is PRV A kind of glycoprotein gene, be located at the area US, long 1740 bp encode 579 amino acid, and G+C content is 73.91%.

GTGGCGTTTTATCTCCGTCCGCGCCGTTTTAAACCTGGGCACCCCCGCGAGTCTCGCACACACCGGGGT TGAGACCATGCGGCCCTTTCTGCTGCGCGCCGCGCAGCTCCTGGCGCTGCTGGCCCTGGCGCTCTCCACCGAGGCCC CGAGCCTCTCCGCCGAGACGACCCCGGGCCCCGTCACCGAGGTCCCGAGTCCCTCGGCCGAGGTCTGGGACGACCTC TCCACCGAGGCCGACGACGATGACCTCAACGGCGACCTCGACGGCGACGACCGCCGCGCGGGCTTCGGCTCGGCCCT CGCATCCCTGAGGGAGGCGCCCCCGGCCCATCTGGTGAACGTGTCCGAGGGCGCCAACTTCACCCTCGACGCGCGCG GCGACGGCGCCGTGCTGGCCGGGATCTGGACGTTCCTGCCCGTCCGCGGCTGCGACGCCGTGTCGGTGACCACGGTG TGCTTCGAGACCGCGTGCCACCCGGACCTGGTGCTGGGCCGCGCCTGCGTCCCCGAGGCCCCGGAGATGGGCATCGG CGACTACCTGCCGCCCGAGGTGCCGCGGCTCCGGCGCGAGCCGCCCATCGTCACCCCGGAGCGGTGGTCGCCGCACC TGAGCGTCCTGCGGGCCACGCCCAACGACACGGGCCTCTACACGCTGCACGACGCCTCGGGGCCGCGGGCCGTGTTC TTTGTGGCGGTGGGCGACCGGCCGCCCGCGCCGGCGGACCCGGTGGGCCCCGCGCGCCACGAGCCCCGCTTCCACGC GCTCGGCTTCCACTCGCAGCTCTTCTCGCCCGGGGACACGTTCGACCTGATGCCGCGCGTGGTCTCGGACATGGGCG ACTCGCGCGAGAACTTTACCGCCACGCTGGACTGGTACTACGCGCGCGCGCCCCCGCGGTGCCTGCTGTACTACGTG TACGAGCCCTGCATCTACCACCCGCGCGCGCCCGAGTGCCTGCGCCCGGTGGACCCGGCGTGCAGCTTCACCTCGCC GGCGCGCGCGCGGCTGGTGGCGCGCCGCGCGTACGCCTCGTGCAGCCCGCTGCTCGGGGACCGGTGGCTGACCGCCT GCCCCTTCGACGCCTTCGGCGAGGAGGTGCACACGAACGCCACCGCGGACGAGTCGGGGCTGTACGTGCTCGTGATG ACCCACAACGGCCACGTCGCCACCTGGGACTACACGCTCGTCGCCACCGCGGCCGAGTACGTCACGGTCATCAAGGA GCTGATGGCCCCGGCCCGGGCCCCGGGCACCCCGTGGGGCCCCGGCGGCGGCGACGACGCGATCTACGTGGACGGCG TCACGACGCCGGCGCCGCCCGCGCGCCCGTGGAACCCGTACGGCCGGACGACGCCCGGGCGGCTGTTTGTGCTGGCG CTGGGCTCCTTCGTGATGACGTGCGTCGTCGGGGGGGCCATCTGGCTCTGCGTGCTGTGCTCCCGGCGCCGGGCGGC CTCGCGGCCGTTCCGGGTGCCGACGCGGGCGCGGACGCACATGCTCTCTCCGGTGTACACCAGCCTGCCCACGCACG AGGACTACTACGACGGCGACGACGACGACGACGAGGAGGCGGGCGTCATCCGCCGGCGGCCCGCCTCCCCCAGCGGA GACAGCGGCTACGAGGGGCCGTACGCGAGCCTGGACCCCGAGGACGAGTTCAGCAGCGACGAGGACGACGGGCTGTA CGTGCGCCCCGAGGAGGCGCCCCGCTCCGGCTTCGACGTCTGGTTCCGCGATCCGGAGAAACCGGAAGTGACGAATG GACCCAACTATGGCGTGACCGCCAACCGCCTGTTGATGTCCCGCCCCGCTTAAATACCGGGAGAACCGGTCCGCCCG CATTCCGACATGCCC

2.5 PRV separation strainsgEComplete genome sequence homology analysis

Using DNAStar software, 18 will be chosen in strain sequence that isolated PRV/HN2012 strain is included with GenBank Strain is carried out from the PRV strain of country variant and areagEGene sequencing, from figure 8, it is seen that PRV/HN2012 is separated Nucleotide sequence homology is higher between strain and all PRV strains, between 97.5% ~ 99.6%, wherein with Ea plants Chinese (AF171937) homology highest (99.6%), and it is minimum (97.5%) with 00V72 plants Belgian (FJ605133) homology.

2.6 PRV separation strainsgEFull genome derives the sequence analysis of amino acid

Using DNAStar, the PRV/HN2012 separation strains that we are separatedgEThe amino acid sequence and river that full genome derives Southern strain (EU561349, HNJZ) and represent Ea plants, P-PrV plants, Yangsan plants, Becker plants of strain etc. totally 18 plants both at home and abroad PRV'sgEFull genome derive amino acid sequence carry out homology analysis, the results show that PRV/HN2012 separation strains and both at home and abroad The homology of other strains is between 95.3% ~ 99.1%(Fig. 9), wherein PRV/HN2012 separation strains with South Korea Yangsan plants (AY249861) homology highest is 99.1%；With (FJ605135) NS374 plants Belgian and 00V72(FJ605132) same Source property is minimum, is 95.3%.With external Becker(AY368490), Yangsan(AY249861), CL/15(JF460026), 00V72(FJ605132)、75V19(FJ605133)、89V87(FJ605134)、NS374 (FJ605135)、NIA-1 (FJ605136), NIA-3 (EU502923) is compared, and PRV/HN2012 separation strains have to be changed at 27, at 48 and 494 1 aspartic acid is inserted into position.It is compared with other domestic and international strains, PRV/HN2012 separation strains are sent out at 448 and 510 Raw amino acid replacement (V⁴⁴⁸I, G⁵¹⁰S), and in 385 generation amino acid replacements (T → M) (table 3).

2.7 PRV separation strainsgEFull genome phylogenetic evolution relationship analysis

Using in software MEGA5.0 Boot-strapped Neigh-bor-Joining draw PRV gE full genome into Change tree, can be seen that PRV/HN2012 and domestic Ea strain (AF171937), GDSH plants from PRV gE full genome chadogram (EF552427) affiliation is nearest, in the same biggish branch that evolves (Figure 10)；With SH plants (AF207700) etc. it is domestic its His 5 strains and P-PrV plants of Malaysia (FJ176390), South Korea Yangsan plants (AY249861) adhere to same big evolution separately Branch, genetic affinity relative close；And with American-European separation strains 00V72 plants (FJ605132), 89V87 plants of (FJ605134), 78V19 Strain (FJ605133), NS374 plants (FJ605135), Becker plants (AY368490), CL15 plants (JF460026), NiA-3 plants (EU502923) and NiA-1 plants (FJ605136) are adhered to separately different evolutionary branchings, and affiliation is farthest.

3 conclusions and discussion

Porcine pseudorabies are the most serious to piglet and brood sow harm, and the death rate is 100% after Infection in Piglets, sow sense Dye causes miscarriage, produces stillborn foetus.Porcine pseudorabies virus gene-deleted vaccine has played important function in porcine pseudorabies, so that the disease It is effectively controlled.Especially since 2011, pseudorabies case presents growth trend in China again, and diseased region is fast Speed expands, this more causes great attention of the veterinarian for the disease.Therefore current porcine pseudorabies virus is understood at me The hereditary variation situation of state be it is vital, can the epidemic status to porcine pseudorabies virus accurately held, for The prevention and control of the disease are of great significance.

Pseudorabies virus belongs to herpetoviridae (Herpesuiridae) Alphaherpesviridae.The virulence of PRV is main By gE, gD, gI and TK Collaborative Control, whereingEGene is one of major virulence gene of PRV and World Organization for Animal Health The missing gene of defined gene-deleted vaccine.GE glycoprotein is about 1.7kb, contains 577 amino acid residues, belongs to typical I Type transmembrane protein.GE is the nonessential glycoprotein of virus replication, can the intercellular diffusion of mediate retroviral, to PRV in nerveous system The invasion and propagation of system play an important role.

PRV gEThe G+C content of gene is up to 73%, has complicated secondary structure, thereforegEThe design of primers of gene and PCR amplification difficulty is larger.This research is referring to delivering in GenBankgE(AF171937) gene order, it is soft using design of primers Part devises 1 pair of primer.By PCR amplification, clone, PRV/HN2012 separation strains are obtainedgEGene order.Strain complete sequence into Row tetraploid rice and phylogenetic analysis, homology is up between this PRV/HN2012 separation strains and other separation strains both domestic and external 95.3% ~ 99.1%, this showsgEGene has stringent conservative, while also illustrating that PCR method expands PRVgEGene is reliable Property is good.By comparing PRV/HN2012 separation strains and domestic and international other strainsgEThe amino acid sequence discovery that gene derives, this The amino acid of PRV/HN2012 separation strains gE albumen has to be changed at 27, in 48 and 494 insertions, 1 aspartic acid, In 448 generation amino acid replacement (V⁴⁴⁸I), and in 385 and 510, only PRV/HN2012 plants of generation amino acid replacement (T³⁸⁵M, G⁵¹⁰S).From the chadogram of building as can be seen that PRV/HN2012 separation strains and domestic Ea strain, GDSH plants and Yangsan plants of affiliations are nearest, and with the Becker strain of source America and Europe and NiA3 plantsgEGene difference is larger, affiliation Farther out, this illustrates that PRV/HN2012 separation strains have closer affiliation in recent years domestic isolated strain.This is for understanding The popularity of Henan area porcine pseudorabies virus provides the reference frame of science.

Three, porcine pseudorabies virus henan strainsgCThe clone of full genome and sequence are analyzed

1 materials and methods

1.1 material

1.1.1 strain and carrier

1.1.2 toolenzyme and other reagents

1.1.3 Strain

1.2 design of primers

Referring to what is delivered in GenBankgCGene order devises 1 pair of primer using primer-design software, for expanding IncludegCThe segment of gene complete sequence, length are 1576 bp, and primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.

P₁: 5 '-GGC GTT TCC TGA TTC AC-3 '

P₂: 5 '-GCC CTC GCT GTC GTT TAT TG-3 '

The extraction of 1.3 viral DNAs andgCThe amplification of full genome

By the PRV/HN2012 strain cell toxicant of harvest, freeze thawing 3 times with lytic cell, 6000 r/min are centrifuged 6-8 min, Supernatant is taken, extracts DNA with proteinase-K pathway.Using the cell toxicant DNA of extraction as template, PCR amplification is carried out with primer P1/P2.? In PCR reaction system, each 0.5 μ L, Primer STAR GXL DNA of template DNA 2 μ L, upstream and downstream primer P1/P2 15 μ L, dNTP Mixture of μ L, 5 × Primer STAR GXL Buffer of Polymerase, 2 μ L, moisturizing to 25 μ L； PCR amplification program is 94 DEG C of 3 min；98 DEG C of 10 s, 60 DEG C of 15 s, 68 DEG C of 100s, 35 circulations；4℃10 min.Reaction After, take 5 μ L PCR products to carry out the result of electrophoresis detection PCR with 10 g/L Ago-Gels (containing 0.5 mg/L EB).

1.4 PRV gCThe clone of full genome and identification

It is positive product recovery purifying by PCR testing result, PRV/HN2012 plants of genetic fragment is carried out to add A tail After processing, it is connected to cloning vector pMD^®In 18-T, the extraction of conversion, recombinant plasmid through connection product, recombinant plasmid PCR The processes such as identification, digestion identification carry out sequencing to positive clone is accredited as, and by Shanghai, bioengineering Co., Ltd is complete At.

1.5 PRV gCThe sequence of full genome is analyzed

With softwares such as MEGA4, DNAStar to the PRV of clone gC18 plants downloaded in complete genome sequence and GeneBank PRV gCComplete genome sequence is compared, to PRVgCThe amino acid sequence of full genome and gC glycoprotein is compared point Analysis.All Reference strains are the typical strain included on GenBank, are shown in Table 4.

4 Reference strains accession number of table

2 results

2.1 PRV gCThe amplification of full genome

Using the cell toxicant DNA of extraction as template, PCR amplification is carried out with primer P1/P2, amplified fragments pass through Ago-Gel , there is the band (Figure 11) of 1 treaty, 1700 bp in electrophoresis detection, and the length of amplified fragments is consistent with expection.

2.2 PRV gCThe PCR of full genome recombinant plasmid is identified

It by PCR product recovery purifying and is connected in carrier T, after the conversion of connection product, extracts recombinant plasmid, it is right Recombinant plasmid carries out PCR identification, and the band (Figure 12) of 1 treaty of electrophoresis showed, 1700 bp is consistent with expected amplification size.

2.3 PRV gCThe digestion of full genome recombinant plasmid is identified

Recombinant plasmid warpEcoRⅠ、PstI double digestion, electrophoresis occur 2 bands: wherein a band be vector plasmid, about 2 600 Bp, another 1 band are the PRV that is cloned gCFull genome segment, about 1600 bp(Figure 13), it is consistent with expected results.

2.4 PRV gCThe sequencing result of full genome

Digestion and PCR are accredited as positive recombinant plasmid and carry out sequencing, the PRV/HN2012 strain of sequencer addressgCBase It include a complete open reading frame (1464 bp) as shown in SEQ ID No:2 because nucleotide sequence is 1576 bp, (G+C) % is 75.14%, encodes the polypeptide of 487 amino acid, relative molecular mass 51961 altogether.gCThe amino that gene derives Acid sequence has 7 potential N- glycosylation sites, there is 10 cysteines related with disulfide bond formation.

GTTTCCTGATTCACGCCCACGCCCGTGTCGTTTTTAAAACCGCGATGGGGGGACGGGGGGCCATTCGCA CGCGCCATGGCCTCGCTCGCGCGTGCGATGCTCGCGCTGCTGGCGCTCTACACGGCGGCCATCGCCGCGGCGCCGTC GTCCACGACGGCGCTCGGCACGACGCCCAACGGGGGCGGGGGCGGCAACAGCAGCGCGGGCGAGCTCTCGCCCTCGC CGCCCCCGACGCCCGAGCCCGTCTCGGGGACGACGGGGGCCGCGGCCTCCACGCCCGCCACCGTCTCGACGCCCCGG GTCCCGCCGCCCTCGGTCTCGCGCCGGAAGCCCCAGCGGAACGGCAACAGGACGCGCGTCCACGGCGACAAGGCCAC CTCGCACGGGCGCAAGCGCATCGTGTGCCGCGAGCGGCTGTTCTCGGCGAGGGTGGGGGACGCGGTCAGCTTCGGGT GCGCCGTCGTCCCGCGCGCCGGGGAGACCTTCGAGGTCCGCTTCTGCCGCCGCGGGCGCTTCCGCTCGCCCGACGCC GACCCCGAGTACTTTGACGAGCCCCCGCGCCCGGAGCTCCCGCGGGAGCGGCTCCTCTTCAGCTCCGCCAACGCCTC CCTCGCCCACGCGGACGCGCTCGCCTCCGCCGTCGTCGTCGAGGGCGAGCGCGCGACCGTCGCCAACGTCTCGGGCG AGGTGTCCGTGCGCGTGGCCGCGGCGGACGCCGAGACCGAGGGCGTCTACACGTGGCGCGTGCTGTCCGCCAACGGC ACCGAGGTCCGCAGCGCCAACGTCTCGCTCGTCCTGTACCACCAGCCCGAGTTCGGCCTGAGCGCGCCGCCCGTCCT CTTCGGCGAGCCCTTCCGGGCGGTGTGCGTCGTCCGCGACTACTACCCGCGGCGCAGCGTGCGCCTGCGCTGGTTCG CGGACGAGCACCCGGTGGACGCCGCCTTCGTGACCAACAGCACCGTGGCCGACGAGCTCGGGCGCCGCACGCGCGTC TCCGTGGTGAACGTGACGCGCGCGGACGTCCCGGGCCTCGCGGCCGCGGACGACGCGGACGCGCTCGCGCCGAGCCT GCGCTGCGAGGCCGTGTGGTACCGCGACAGCGTGGCCTCGCAGCGCTTCTCCGAGGCCCTGCGCCCCCACGTCTACC ACTCGGCGGCGGTCTCGGTGCGCTTCGTCGAGGGCTTCGCCGTCTGCGACGGCCTCTGCGTGCCCCCGGAGGCGCGC CTCGCCTGGTCCGACCACGCCGCCGACACCGTCTACCACCTCGGCGCCTGCGCCGAGCACCCCGGCCTGCTCAACGT GCGGAGCGCCCGCCCGCTGTCGGACCTCGACGGGCCCGTCGACTACACCTGCCGCCTCGAGGGCATGCCCTCGCAGC TGCCCATCTTCGAGGACACGCAGCGCTACGACGCCTCCCCCACGTCCGTGAGCTGGCCCGTCGTGACCAGCATGATC ACCGTCATCGCCGGCATCGCCATCCTAGCCATCGTGCTGGTCATCATGGCGACGTGCGTCTACTACCGCCGGTCCGC GCTGTGACGCCCCCGCCCGCCCCGAATCAATAAACGACAGCGAG

2.5 PRV separation strainsgCComplete genome sequence homology analysis

Using DNAStar software, 19 plants are chosen in the strain sequence that isolated PRV/HN2012 and GenBank is included and is come It is carried out from the PRV strain in country variant and areagCNucleotide sequences homologous between gene sequencing, and the PRV strain of analysis Property is between 94.2% ~ 99.9%(Figure 14), wherein with BJ plants Chinese (EU719644) homology highest (99.9%), and with Japan S-81 plants of Yamagata (D49435) homologys are minimum (94.2%).

2.6 PRV separation strainsgCFull genome derives the sequence analysis of amino acid

By comparing PRV/HN2012 separation strains and domestic and international other strainsgCGene derives amino acid sequence discovery, from figure 15 as can be seen that PRV/HN2012 separation strains and domestic and international other strainsgCGene derives amino acid sequence homology in 90.9%- Between 99.8%, wherein be up to 99.8% with BJ/RD plants Chinese (KF017273) homology, and with come from Hungary Bartha plants (EU719641) homology minimum 90.9%.PRV/HN2012 separation strains exist with BJ/RD, BJ, DG, Ea, Fa and P-PrV plants 7 amino acid (AAASTPA) (table 5) are inserted at 63-69, there is also many places amino acid to change for PRV/HN2012 separation strains, Wherein with HS, SN, SCZ, SQ, SS plant Chinese, South Korea NYJ plants, S-81 plants of Japan Yamagata and American-European separation strains Becker Strain (M12778), NIA3 plants (D49437), S plants of Indiana (D49436), Bartha plants (EU719641) compare, PRV/ HN2012 separation strains co-exist in the variation of amino acid sites at 41, and entirely different with all Reference strains, PRV/HN2012 Strain is in 280 generation amino acid replacements (F → L).Compared with BJ, DG and Ea, PRV/HN2012 separation strains are in 194 and 280 Amino acid replacement (G occurs¹⁹⁴E, F²⁸⁰L).

5 PRV of tablegCThe comparison for the amino acid sequence that gene derives

2.7 PRV separation strainsgCFull genome phylogenetic evolution relationship analysis

PRV is drawn using the Boot-strapped Neigh-bor-Joining in software MEGA5.0gCFull genome into Change tree, from PRVgCFull genome chadogram can be seen that PRV/HN2012 separation strains and domestic BJ-RD strain (KF017273) parent Edge relationship is nearest, in same evolutionary branching；With other domestic several strains such as Ea plants (AF158090) and Malaysia P-PrV Strain (EU915280) adheres to same big evolutionary branching (Figure 16), genetic affinity relative close separately；And with South Korea separation strains PRV/ HN2012J plants (GQ325659), S-81 plants of Yamagata of Japan (D49435) and American-European Becker plants of separation strains (M12778), NIA3 plants (D49437), S plants of Indiana (D49436), Bartha plants (EU719641) are adhered to separately different evolution Branch, affiliation are farthest.

3 conclusions and discussion

About 150 Kb of PRV Genome Size, G+C content are up to 73%, have complicated secondary structure and higher structure, because This is rightgCGene carries out design of primers and PCR amplification difficulty is larger.The present invention is with reference to the Ea strain announcedgCGene order, design 1 pair of primer is established using the DNA of separation strains as templategCThe specific PCR amplification method of full genome.Due to expanding full base Because enzyme used in being is Primer GXL, which is high fidelity enzyme, and the activity with 3 ' to 5 ' exonucleases causes PCR to react Later will not tailing, make troubles for subsequent TA clonogenic assay, therefore the present invention carries out PRV/HN2012 plantsgCFull genome gram Target gene recycling segment is 72 DEG C of 20 min of incubation using rTaq enzyme before grand to handle, to improve target gene and carrier T Joint efficiency.

This PRV/HN2012 separation strainsgCHomology is up between gene nucleotide series and other domestic and international separation strains 94.2% ~ 99.9%, this showsgCGene has stringent conservative, while also illustrating that PCR method expands PRV gCGene is reliable Property is good.Position in homology analysis and chadogram can be seen that PRV/HN2012 separation strains and domestic BJ/RD strain parent Edge relationship is nearest, in same evolutionary branching；It is adhered to separately with P-PrV plants of other domestic several strains such as Ea plants and Malaysia same Big evolutionary branching, genetic affinity relative close；And with separation strains PRV/HN2012J plants of South Korea, Japanese separation strains Yamagata S-81 plants and American-European Becker plant of separation strains, NIA3 plants, S plants of Indiana, Bartha plants adhere to different evolutionary branchings separately, it is close Edge relationship is farthest.The domestic recently popular strain in Henan has in recent years domestic isolated strain since this explanation autumn in 2012 Closer affiliation is referred to other prevalences of other country's separation to the further research of isolated strain biological characteristics Strain.

The present invention passes through to PRVgCGene sequence comparison discovery, although difference PRV straingCGene nucleotide and amino Acid sequence homology is relatively conservative, but there are still different degrees of differences respectively 94.2% and 90.9 or more.It is this Difference is mainly reflected in 7 amino acid of insert continually or missing after the 63rd amino acids caused by height missing region sequence (AAASTPA) and by other parts nucleotide point mutation lead to the difference of coded amino acid.Show that different regions gC albumen is deposited In apparent regional disparity.Currently, existing research proves that preceding 1/3 part of herpetoviridae gC glycoprotein homologue N-terminal is protected Keeping property is far below C-terminal.It is confirmed again in this research, because by PRV gCGene nucleotide series it was found that, Variation occurs in the section before the 162nd amino acids at the 21 of different PRV strain gC Argine Monohydrochlorides, accounts for total variation 52.5%, and first 1/3 section of gC albumen is major function area, in the not altogether clear situation of epitope of gC, if meeting PRV is influenced to the functions such as the absorption of host cell and the release of virion, all needs further to study verifying.

In addition, Bartha plants of PRV be Hungary scholar Bartha in 1961 with pig source pseudoabies it is virulent by pig kidney, One vaccine strain of chicken embryo Attenuation repeatedly.From Bartha plants of PRV puppet it is mad dog Bartha-K61 plants be prevention and control PR warp Allusion quotation attenuated vaccine strain has at home and abroad been continued to use for many years.Pseudo- mad dog Bartha-K61 was once used from China's many of spring in 2012 PR case occurs for the immune pig farm of strain.It is isolated from the PRV/HN2012 separation strains in HenangCGene derive amino acid sequence and its He is compared discovery at domestic and international separation strains, and the homology with Bartha plants is minimum, and only 90.9%.As can be seen from Table 5, Amino acid change all has occurred in PRV/HN2012 plants and Bartha plants of gC protein amino acid sequence at 16,25,43,52 etc. 19 Change, shows PRV/HN2012 separation strains compared with Bartha plants, PRV/HN2012 separation strains gC albumen morphs.This may be Pig farm uses the pseudo- mad dog Bartha-K61 plants immune reasons that PR still occurs at present.

Four, porcine pseudorabies virus PRV/HN2012 plants of Analysis of Immunogenicity

1 materials and methods

1.1 material

1.1.1 Strain and cell strain

PRV/HN2012 plants of porcine pseudorabies virus separate identification and purifying by this laboratory, and ST cell is purchased from Chinese veterinary drug Product supervise institute, are frozen by this laboratory.

1.1.2 experimental animal

6 week old female KM mouse are purchased from experimental animal center of henan province.

1.1.3 main agents

1640 culture medium, fetal calf serum and trypsase are purchased from Solarbio company；Formaldehyde is purchased from Yantai City's chemical industry in pairs Co., Ltd；Freund's complete adjuvant and incomplete Freund's adjuvant are SIGMA Products.

1.2 method

1.2.1 the proliferation of PRV virus

ST cell is conventionally cultivated, using 1640 culture medium and 10% fetal calf serum is added as the complete of ST cell Culture solution；It is inoculated with the 7th generation of PRV/HN2012 strain after cell passes on 24 h, is placed in 5%CO₂, it cultivates in 37 DEG C of cell incubators, It changes 1640 maintaining liquids containing 2% fetal calf serum into, continues to cultivate 48 h, PRV/HN2012 plants are harvested when there is about 80% CPE 8th generation.By cell culture multigelation 3 times, 6000 rpm are centrifuged 10 min and remove cell fragment, save backup in -80 DEG C.

1.2.2 the TCID of virus₅₀Measurement

The ST cell suspension inoculation that digestion is dispelled is into 96 orifice plates, every 200 μ L of hole, after cultivating 24 h, takes PRV PRV/ HN2012 plants of the continuous doubling dilution 10 of the 8th generation cell culture^-1~10^-10, every hole is inoculated with 100 μ L, in cell incubator After being incubated for 1h, every hole is added RPMI-1640 maintaining liquid of the 100 μ L containing 2% fetal calf serum and continues to cultivate, and each dilution respectively makees 8 A repetition observes cytopathy situation day by day, and calculates virus titer TCID according to Reed-Muench method₅₀。

1.2.3 viral inactivation and its inactivation are examined

1.2.3.1 inactivation time

Formaldehyde is added in virocyte liquid, until final concentration of 0.3%, 24 h are acted under the conditions of 37 DEG C, during inactivation, Every 4~6 h shakes primary.

1.2.3.2 the detection of inactivation of viruses liquid

By the virus liquid of inactivation and control group synchronize be inoculated on ST cell, during which in continuous 3 generation of blind passage, observes cytopathy Situation, and detect culture solution hemagglutination activity, with judgement inactivating efficacy.

Inactivation of viruses liquid is inoculated in ordinary broth and ordinary nutrient agar, 37 DEG C of 24~48 h of culture, observation whether there is or not Bacterial growth.

1.2.4 the preparation of porcine pseudorabies virus emulsifier

5 mL Freund's complete adjuvants and 5 mL incomplete Freund's adjuvants are broken into oily emulsus with dispersion refiner respectively, dropwise Isometric inactivation of viruses liquid is added, is allowed to mix and formed the oil emu type of Water-In-Oil.

1.2.5 the inspection of porcine pseudorabies virus emulsifier

It is examined by veterinary biologics quality standard.

(1) appearance detects

Visually observe the appearance of porcine pseudorabies virus emulsifier.

(2) cold water meter interview is tested

A cleaning suction pipe is taken, emulsifier is drawn and slowly drips in cold water, whether observation oil droplet spreads.

(3) viscosity is examined

Taking bore is that the l mL suction pipe of 1.2 mm is filled l mL emulsifier at room temperature, natural when with suction pipe in vertical state Time needed for flowing out 0.4 mL oil emu is as criterion.

(4) stability test

Emulsifier is filled it up with into the small centrifuge tube of 5 mL, 3000 rpm are centrifuged 15 min, with not stratified to have good stability；37℃ 21 d are placed, have seen whether demulsification, lamination.

(5) steriling test

Manufactured emulsifier is inoculated in ordinary broth, ordinary nutrient agar, blood agar culture-medium, 37 DEG C of culture observations 3 D, asepsis growth are qualification.

1.2.6 mouse immuning test

(1) grouping and animal experiment

60 6 week old female KM mouse are divided into 4 groups, every group 15, i.e. PRV/HN2012 plants of injection group, Bartha- K61 vaccine injection group, 98 vaccine injection group of Hubei and negative control group.The every mouse injection 0.5 of PRV/HN2012 plants of injection groups ML PRV/HN2012 strain virus liquid and Freund's complete adjuvant emulsifier, every mouse injects 0.5 mL PRV/HN2012 after two weeks Strain virus liquid and Freund non-fully adjuvant emulsion agent inject 0.5 mL after every mouse after two weeks and inactivate PRV/HN2012 plants of diseases Venom；Every Bartha-K61 vaccine injection group, 98 vaccine injection group of Hubei mouse inject the vaccine that 0.5 mL has diluted, negative Every mouse of control group injects 0.5 mL1640 culture medium, is immunized again with same dose once, with PRV/HN2012 every two weeks The immunization time of strain injection group is synchronous.It is divided into two groups for 4 groups every group of mouse after 32 d, one group (10) is made PRV/HN2012 plants Attack malicious processing, every mouse injects the 8th generation cell culture of 0.5 mL PRV/HN2012 strain, daily observation mice clinical table It is existing, record mouse diing time；Another group of (5) blood samplings, separate serum, carry out serum and make neutralization test.

(2) PRV/HN2012 plants of serum neutralization tests

A. the processing of serum to be checked: 56 DEG C of 30 min water-bath of serum to be checked carries out inactivation of complement；500 μ L are taken to inactivate Serum does 2 times of serial dilutions；

The processing of b.PRV/HN2012 strain virus liquid: by PRV/HN2012 the 8th generation of strain cell culture multigelation 3 times, 6000 rpm are centrifuged 10 min and remove cell fragment, take supernatant, are diluted to 200 TCID₅₀；

C. sense is made: taking the serum of quantitative virus liquid and isometric different dilutions to mix well 37 DEG C of senses and makees 1 h；

D. it is inoculated with: after feeling work, serum virus mixture being inoculated with the ST cell monolayer on 96 orifice plates rapidly, is placed in 5%CO₂, cultivate in 37 DEG C of cell incubators, observe CPE day by day, sufficiently occur infecting effect to it, be sentenced with Reed-Muench method Half protective number (PD that is fixed and calculating neutralization test₅₀).

2 results

2.1 virus titer TCID₅₀Measurement result

Routine culture ST cell is observed by inverted microscope after connecing poison, occurs being rounded to cell, fall off, forms vacuole After apparent CPE, the TCID of strain is counted and calculated by Reed-Muench method₅₀, as a result obtain PRV/HN2012 plants TCID₅₀It is 10^-9.0。

2.2 inactivation of virus validity checks

2.2.1 lesion is observed

With 0.3% formalin-inactivated virus, after 10 times of dilution, 3 generation of blind passage on ST cell, without generating cytopathy.

2.2.2 the Sterility testing of inactivation of viruses liquid

Virus liquid after taking inactivation is inoculated in respectively in agar plate and nutrient broth, is observed after 37 DEG C of 24~28 h of culture Without bacterial growth, meet biological products regulatory requirements.

The identification of 2.3 PRV/HN2012 plants of emulsifier

2.3.1 appearance detects

Appearance is creamy white homogeneous emulsion, not stratified after standing.It meets the quality standard.

2.3.2 cold water meter interview is tested

The emulsifying of preparation is water-in-oil-in water.By the emulsifier drop of preparation in cold water surface, result is first indiffusion, It slowly disperses around again.

2.3.3 viscosity measurement

1 mL emulsification is filled at room temperature at 25 DEG C with l mL suction pipe (lower internal diameter 1.2mm, upper internal diameter are 2.7 mm) Agent, vertical 0.4 mL that releases need 2 S.Within the scope of 2~6 S, illustrates viscosity qualification, be suitable for injection.

2.3.4 Stability Determination

By emulsifier without layering and demulsifying phenomenon after 3000 rpm are centrifuged 15 min, after 37 DEG C of placements 21d also without be demulsified, Lamination illustrates it with certain stability.

2.4 PRV/HN2012 plants of challenge test result

Respectively from PRV/HN2012 plants of immune groups, Bartha-K61 vaccine immunity group, 98 vaccine immunity group of Hubei and feminine gender Random in control group mice respectively to extract 10 mouse, every mouse injects the 8th generation cell culture of 0.5mL PRV/HN2012 strain, Observation mice clinical performance daily, records mouse diing time.10 mouse of negative control group after inoculation in 72 h all Death, and 10 mouse of Bartha-K61 vaccine immunity group are being inoculated in 72 h, only 2 generations nervous symptoms, up to 120 h When dead 5 mouse, and 98 immune group of Hubei has 3 mouse nervous symptoms and the phenomena of mortality just occur after being inoculated with 120 h, respectively Group dead mouse and the death rate are shown in Table 6.

6 PRV/HN2012 plants of challenge test mortality statistics result of table

2.5 PRV/HN2012 plants of neutralization test result

Respectively from PRV/HN2012 plants of immune groups, Bartha-K61 vaccine immunity group, 98 vaccine immunity group of Hubei and feminine gender 5 mouse are randomly selected in the mouse of control group, docking blood sampling takes serum, and 56 DEG C of inactivations, 2 times dilute, and minimum concentration is diluted to 2^-9, with the 8th generation (TCID of PRV/HN2012 strain₅₀It is 10^-9/ mL) neutralization experiment is carried out, cytopathy situation is observed day by day, as a result Show that the serum 1:42 dilution of PRV/HN2012 plants of immune groups can protect 50% cell from there is CPE(table 7), and Bartha- The neutralization titer of 98 vaccine immunity group serum of K61 vaccine immunity group and Hubei is relatively low, and negative control cannot protect cell.

7 PRV/HN2012 plants of neutralization test result of table

3 conclusions and discussion

The purification scheme of the external mad dog of puppet is usually to use pseudo- mad dog live vaccine is immune wild malicious detection is added to eliminate the side for infecting pig Method, vaccine strain generally use the live vaccine of Bartha-k61 strain, since foreign countries are generally large-scale pig farm, are immunized Position, laboratory testing is timely, and bio-security is stringent, so making the pseudo- mad dog of external some national successful decontaminations.

The current pseudo- rabies vaccine used in domestic pig farm is essentially pseudo- mad dog live vaccine, and strain has Bartha-k61 and HB98 Two kinds, with the most of Bartha-k61 strain, import vaccine (Bo Linge, Cimmeria, Hai Bolai) and domestic vaccine (Pu Lai Ke, Guangdong Yongshun, big Hua Nong, Zhongmu Stocks Trading Co.) etc. enterprises be Bartha-k61 strain, import is either used on immune programme Vaccine and domestic vaccine, boar: preventing epidemic 4 times for general 1 year, interval March 1 time, 1 part/head；Growing and fattening pigs: it prevents epidemic within import seedling 10 weeks Once, 1 part/head；Serious pig is infected individually in 1 part/head of domestic vaccine 1-3 d collunarium, 6 week old intramuscular injection 1 time The 10 weeks booster immunizations in field are primary, and dosage is 1 part/head.

The country is for pseudo- mad dog clinical onset pig farm, general sample detecting gE antibody, however, it is determined that the positive can be true at present It examines, for making a definite diagnosis positive field generally with pseudo- rabies vaccine urgent immunity inoculation, 2 parts of piglet, 4 parts of boar, due to major part Pig farm wild virus infection rate is higher, now takes to detect and eliminates the less of wild virus infection pig.Vaccine is mainly taken to wild virus infection swinery The method of booster immunization.

Since spring in 2012, most of China pig farm starts to break out pseudoabies, and either vaccine immunity program is endless The reasonable extensive pig farm in kind middle and small scale pig farm and vaccine immunity program, either using import vaccine and domestic vaccine There is a morbidity phenomenon on pig farm, and sow produces weak young, stillborn foetus or the mummification of fetus, and nervous symptoms, death occurs in piglet, the death rate up to 10% ~ 50%, huge economic losses are caused to China's pig breeding industry.Individual purified pig farms also reappeared wild virus infection feelings in the past Condition, gE positive rate is most of all 50% or more, and analysis re-infects the reason of falling ill: pseudo- rabies vaccine does not conform to before 1. Reason, unordered be immunized cause wild poison to make a variation；2. country's circovirus, the infection rate of blue ear are very high at present, and both diseases Infection can destroy the immunocyte of body, decline the immune effect of vaccine；3. Mycotoxins in Feed is widely present The immune effect of vaccine can be caused to decline, pseudo- mad dog is made to fall ill again.

To determine whether these pig farms have occurred PRV wild virus infection, and new epidemic strain to porcine pseudorabies novel vaccine Examine whether have reference significance, the present invention is to PRV/HN2012 plants of progress immunogenicity researchs.By PRV/HN2012 plants, After Bartha-K61 plants and the immune mouse in 98 plants of Hubei, challenge test is carried out with PRV/HN2012 plants respectively, PRV/HN2012 plants The inoculation group death rate is significantly lower than Bartha-K61 plants and 98 strain vaccine injection group of Hubei；Using the serum dilution of 50%CPE as The criterion of highest neutralization titer, PRV/HN2012 plants with itself serum neutralization titer highest, be secondly Bartha-K61 Strain, is slightly below Bartha-K61 plants with 98 plants of the neutralization titer in Hubei, shows PRV/HN2012 plants and Bartha-K61 plants and lake There are certain antigenic cross properties between 98 plants of north, but antigenicity has differences.Peng Jinmei etc. also reports new separation PRV in 2011 HeN1 plants lower with k61 plants of inoculation Swine serums generation neutralizing antibody levels of Bartha, and is inoculated with Swine serum with HeN1 separation strains It is high to generate antibody level.But existing vaccine strain, which need animal experiment to PRV/HN2012 separation strains immune protective effect, to be come into one Step card.

Claims

1. a kind of porcine pseudorabies strain (Pseudorabies Virus), it is characterised in that: deposit number CCTCCNO: V201314。