CN100503816C - Recombinant gene engineered strain of aujeszkys disease-porcine reproductive and respirator syndrome virus, and its uses - Google Patents
Recombinant gene engineered strain of aujeszkys disease-porcine reproductive and respirator syndrome virus, and its uses Download PDFInfo
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Abstract
The invention relates to a construct, vaccination preparation and application of recombination infectious bulbar paralysis-pig breeding and breathing complex gene project toxic strain in the field of animal virus gene engineering technology. The recombination infectious bulbar paralysis-pig breeding and breathing complex gene project toxic strain is Pseudorabies virus HzauAVL-PRprrsvV-GP5mM which is preserved in CCTCC with the number: CCTCC-V200509. The gene project toxic strain can be used to prepare for infectious bulbar paralysis-pig breeding and breathing complex gene project vaccine.
Description
Technical field
The invention belongs to the animal virus gene engineering technology field.Be specifically related to a kind of reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain, with the recombinant vaccine and the application of this gene engineered strain preparation.
Background technology
Pseudoabies is by Pseudorabies virus (pseudorabies virus, be called for short PrV) acute infectious disease that causes of multiple domestic animal such as infected pigs, ox, sheep, dog, cat, rabbit, small white mouse, mink, fox and wildlife, pig is the storage person and the circulator (Mettenleiter etc. of this virus, Comp Immunol Microbiol Infect Dis.1991,14:151-163).Other animal except that pig is the form of distributing, and has heating after the morbidity usually, very itches and pattern of fever symptom such as encephalomyelitis, is lethal infection.This disease is eruption and prevalence to pig, and harm mainly causes sow breeding difficulty syndrome and newborn piglet mass mortality.PrV is generally through the pharynx nasalis infection animal, and infecting partial mucous membrane internal breeding, divide a word with a hyphen at the end of a line through lymphokinesis or nerve fiber then, it is generally acknowledged that virus arrives brain and causes the nervus centralis dysfunction through nerve pathway, or at olfactory bulb, set up in the trigeminal nerve latent infection (Nauwynck etc., In proceeding of PRRVS and Aujeszky ' s Disease.1999,321-322).
The genome of Pseudorabies virus is the wire double-stranded DNA, be about 150kbp, G+C content is up to 74%, whole genome 70-100 the albumen of encoding at least, wherein thymidine kinase (Thymidine Kinase, TK) gene is positioned at UL district, total length 963bp, 320 amino acid of encoding, catalytic deoxidation thymidine phosphorylation.The TK gene is relevant with the virulence of PrV, is the main virulence gene of Pseudorabies virus, relevant (KitS etc., Am J Vet Res.1985,46:1359-1367 with the latent infection of virus; Nunberg etc., J Virol.1989,63:3240-3249).Except the TK gene, the gene relevant with the Pseudorabies virus virulence also has gG, gE etc.TK, gE and gI gene all are that virus multiplication institute is nonessential, disappearance virus strain and wild malicious the same can normally propagation, and do not exist virulence return strong danger (Yokyama etc., Virol.1991,185:55-65).The mutated viruses strain has good immunogenicity; can produce protective immunological reaction behind the inoculation animal; but do not produce the antibody of anti-gE; therefore can be used as a sign distinguishing immune animal and wild virus infection animal; help setting up the swinery that no pseudoabies infects, and finally provide useful instrument for eradicating this disease.Hua Zhong Agriculture University is at the strong malicious Ea strain (Chen Huanchun etc. of pseudorabies virus of autonomous isolation identification, journal of animal science and veterinary medicine, 1998,29 (2), on basis 151-156), by the DNA recombinant technology with main virulence TK gene, nonessential glycoprotein gG or gE, gI genetically deficient, elimination (Liu Zhengfei etc., microorganism journal, 2002 that a series of recombination mutation strain is used for pseudoabies have been made up, 42 (3), 370-374; Liu Zhengfei etc., journal of animal science and veterinary medicine, 2004,35 (1), 70-73; Xu Xiao cuckoo etc., the biotechnology journal, 2004,20 (4), 532-535).
Porcine reproductive and respiratory syndrome is by porcine reproductive and respiratory syndrome virus (porcine reproductive andrespiratory syndrome virus, be called for short PRRSV) a kind of new transmissible disease that causes, mainly with breeding difficulty such as sow heating, apocleisis, premature labor, miscarriage, stillborn foetus, weak son and various age pig respiratory system disease and high mortality be feature.Owing to the harm at present of this disease seriously and prevents that preferably the system measure causes that global pig industry and investigator more and more note.Existing commercialization attenuation porcine reproductive and respiratory syndrome vaccine comes out, but has safety issue.And the immune efficacy of inactivated vaccine is unsatisfactory, because having antibody, porcine reproductive and respiratory syndrome virus relies on enhancing (ADE) effect (Chou Huaji etc., Chinese animal doctor's science and technology, 1999 simultaneously, 29 (7), 19-20), make conventional vaccine be difficult to prove effective (Chou Huaji etc., Chinese animal doctor's journal, 2000,20 (1), 100-103), therefore be necessary the development of new vaccine.The glycoprotein GP5 of porcine reproductive and respiratory syndrome virus ORF5 genes encoding is a topmost protective antigen among the PRRSV, can induce humoral immunization and cellular immunization, therefore is preferred object gene (the Dea S etc. of design PRRS new generation vaccine; Arch Virol; 2000a, 145,659-688).But be difficult to bring out higher immunne response (Pirzadeh B etc., J Gen Virol, 1998a, 79,989-999 based on the gene constructed vaccine of natural ORF5 separately; Ostrowski M etc., J Virol, 2002,76,4241～4250), based on this consideration, the applicant modifies described ORF5 gene, and the Nucleotide that is about to the coding helper T cell epi-position of synthetic inserts between the neutralizing epitope and covering epi-position of GP5, the modification type ORF5M gene that makes up has better immunogenicity (Jiang Yunbo etc., China animal doctor journal, 2005 (1), 1-3).(Balasuriya R etc. such as Balasuriya R, J Virol, 2000,74,10623-10630) when research and PRRSV generic equine arteritis virus (EAV) live vector vaccine, find, the E albumen of EAV its small peptide-MHC complex body when vivoexpression is very low to the efficient of golgi body transhipment from endoplasmic reticulum, therefore be difficult to excite very high antibody, and when ORF5 and ORF6 gene co-expressing, the existence that the two encoded protein forms this heterodimer of heterodimer greatly promotes E albumen small peptide-MHC complex body to transport to golgi body from endoplasmic reticulum, helps bringing out body and produces very strong immune response.But the research of PRRSV related fields does not appear in the newspapers as yet at present.
Some patent applications related to the present invention, for example patent application of pseudoabies, the application number that has Hua Zhong Agriculture University to propose is 03156706.1 (a kind of pseudoabies TK
-/ gE
-/ gI
-Genetically deficient sign living vaccine and preparation method), the described a kind of genetically deficient mutant strain of this application patent is at artificial constructed pseudoabies TK
-/ gE
-/ LacZ
+Disappearance LacZ gene in the mutant strain, a strain of structure does not have the pseudoabies TK of foreign gene
-/ gE
-/ gI
-The mutant strain of three genetically deficients, this application only relates to this strain and prepares the application of pseudoabies genetic marker vaccine on the single disease of anti-system pseudoabies simultaneously.
Other patent applications related to the present invention, for example the patent application of porcine reproductive and respiratory syndrome sees following document: the application number that Hua Zhong Agriculture University proposes is 200410000238.0, and (a kind of porcine reproductive and respiratory syndrome " Suicidal DNA Vaccine) and application number are the patent application of 200410009838.3 (a kind of porcine reproductive and respiratory syndrome virus ORF5 gene and application of modification), and above-mentioned two patent applications only relate to the anti-system to the single disease of porcine reproductive and respiratory syndrome.
With the closely-related one piece of patent documentation of the present invention be 03132417.7 patent application of (expressing recombinant pseudorabies virus and the application of porcine reproductive and respiratory syndrome virus GP5) of China national Department of Intellectual Property disclosed a application number on July 21st, 2004, it is parent plant that this application adopts a strain gE gene-deleted vaccine Bartha-K61 strain, utilizes homologous recombination technique to make up a strain pseudorabies virus mutant strain.This strain is a strain low virulent strain, does not produce functional gE/gI albumen, causes the excalation of TK gene simultaneously, imports porcine reproductive and respiratory syndrome virus ORF5 gene and intestinal bacteria tilactase (i.e. the Chang Yong LacZ reporter gene-usefulness of marking).The purpose of its invention is to utilize this strain to prepare divalence or multivalent genetic engineering live vector vaccine, but from disclosed specification sheets and claims, this application does not propose the related biological experimental data of this invention effect of any explanation, can not allow the people understand the effect of its enforcement, not having embodiment to how utilizing the detail file of this invention and reproduction invention effect yet, being difficult to apply.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists, obtain the gene engineered strain of the better and pseudoabies-porcine reproductive and respiratory syndrome that security is stronger of a kind of immunogenicity;
Second purpose of the present invention is to utilize this gene engineered strain to prepare pseudoabies-porcine reproductive and respiratory syndrome recombinant vaccine;
The 3rd purpose of the present invention is the application of pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain on preparation pseudoabies disease-porcine reproductive and respiratory syndrome recombinant vaccine.
The present invention is achieved through the following technical solutions:
A kind of reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain Pseudorabies virusHzauAVL-PRprrsvV-GP5mM is deposited in CCTCC, deposit number: CCTCC-V200509.
A kind of reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain, the TK/gE that described gene engineered strain relates to
-/ LacZ
+Parent plant is the exquisite weak genetically engineered mutant strain of a strain people, it is derived from PRV (Pseudorabies virus) Ea strain, described Ea strain is separated, is identified by Hua Zhong Agriculture University animal virus research department, be that the titre height is bred in a strain on cell culture, the virulent strain that immunogenicity is strong 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), 151-156).Its main virulence gene TK of described reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain has lacked 205bp, causes virulence to reduce greatly; The disappearance of 1247bp takes place in the gE/gI gene, causes not producing structural glycoprotein gE/gI, can duplicate in noble cells, but duplicating in undifferentiated neurocyte is prevented from or limits, even latent infection takes place in the inoculation back, also be difficult for being activated, thereby have very high security.
Sill construction process of the present invention is: with the modification type main immunogenic gene ORF5M and the ORF6 gene of porcine reproductive and respiratory syndrome virus, be that CMV-ORF5M-polyA and CMV-ORF6-polyA are inserted into Pseudorabies virus TK with complete independent expression cassette (comprising human cytomegalic inclusion disease virus CMV strong promoter and polyA signal)
-/ gE
-/ LacZ
+Between the genome gE and gl gene of vaccine strain (and application number be 03132417.7 patent application foreign gene be to insert in the TK gene), make it can independently express the GP5m and the M albumen of approximate natural porcine reproductive and respiratory syndrome virus, so GP5m that expresses and the proteic biological characteristics of M and immunogenicity do not change, gE albumen still can not get expressing simultaneously.Can be used for preparing anti-application of making on pseudoabies-porcine reproductive and respiratory syndrome recombinant vaccine by a large amount of biological experiment digital proof recombinant strains of the present invention.
Major advantage of the present invention is:
1, the used solid support material of the present invention is pseudorabies virus Ea TK/gE
-/ LacZ
+The genetically engineered attenuated vaccine strain; this vaccine strain is not only fool proof effectively to growing and fattening pigs, piglet; even it is also fool proof to newborn piglet; can produce strong protection; and can be used for the leading immunity of piglet; have excellent prevention and result of treatment, this just provides wide prospect for it being developed to a kind of good viral lived vaccine carrier.
2, one of expressed exogenous gene of the present invention ORF5M gene is the modification type of natural ORF5 gene, and applicant's research data shows that this gene has better immunogenicity, and then has showed its wide development application prospect.
3, the present invention utilizes the dna homology recombinant technology successfully to replace the LacZ gene, and 03132417.7 application only is a kind of supposition, how on earth implement, 03132417.7 patent application does not have clear and definite solution, the present invention is then for further preparation pseudoabies and porcine reproductive and respiratory syndrome vaccine have proposed concrete technical scheme.
4, the present invention has expressed porcine reproductive and respiratory syndrome virus ORF5M and ORF6 gene with two independently complete Expression elements (comprising human cytomegalic inclusion disease virus CMV strong promoter and polyA signal), and this has just guaranteed the high-caliber non-interfering expression of foreign gene that imports.
5, the present invention is for deeply estimating the application of this reorganization porcine reproductive and respiratory syndrome-pseudorabies virus gene engineered strain on preparation anti-system pseudoabies and porcine reproductive and respiratory syndrome vaccine; this strain is prepared into recombinant vaccine has carried out the immune effect of mouse and pig and immunoprotection research, compare with 03132417.7 patent application and have obvious practicability.
The present invention and application number are that the comparative analysis of 03132417.7 major technique feature and implementation result is as shown in table 1:
The major technique difference of table 1 the present invention and prior art
| Item compared | Major technique feature of the present invention | 03132417.7 the technical characterictic of patent application | The present invention is with respect to the outstanding effect of 03132417.7 patent application |
| Parent's strain | Lack main virulence gene TK, the pseudorabies virus Ea TK of nonessential replicator gE/gI -/ gE -/ LacZ +Genetically engineered sudden change low virulent strain | Lack the Pseudorabies virus Bartha-K61 genetically engineered sudden change low virulent strain of nonessential replicator gE/gI | Aspect security, both are close; But aspect immunogenicity; because the strain of deriving of the present invention is from China's Local Isolates; have local preferably specificity with respect to the strain of deriving from external in the contrast application, the protection that is provided also can be higher. |
| The exogenous antigen gene | Comprise ORF5M (the modification type gene of natural ORF5) and ORF6 gene | Single ORF5 gene | The ORF5M gene that relates among the present invention has better immunogenicity with respect to the natural ORF5 gene of contrast application, stimulates body to produce higher immunne response; And the M albumen of ORF6 coded by said gene is the main immunogenic antigen of porcine reproductive and respiratory syndrome virus equally, the ORF6 protective antigen gene is inserted in the vector virus can produce at the porcine reproductive and respiratory syndrome virus better protection. |
| External rubbish gene | The gene engineered strain that obtains among the present invention does not contain any rubbish gene (being the LacZ gene) | Contain external rubbish gene LacZ | Show that by data the existence of external rubbish gene may have negative impact to organism itself; Moreover, the biotechnological formulation that is used for organism be do not allow at will to insert the rubbish gene (Jin Shengzao etc., Scientia Agricultura Sinica, 2002,35 (1), 89-93).And contrast the external LacZ rubbish gene that contains in the application, and can only be used for studying dynamic (dynamical) a kind of means of virus infection and propagation, can not come into the market as commodity. |
| Practicality | The gene engineered strain that obtains among the present invention has carried out the biological Characteristics Study evaluation, and utilizes the recombinant vaccine of this strain preparation to carry out immune effect and the immunoprotection experiment of mouse and pig | Any biological experiment data and data are not provided | The present invention has and the parent plant similar biological through the constructed gene engineered strain of biological characteristics experiment confirm.Recombinant vaccine immunization experiment animal with this strain preparation can produce good immune effect and provide porcine reproductive and respiratory syndrome and two kinds of disease good immune protection of pseudoabies. |
| Creative | The present invention has fully proved beneficial effect of the present invention with full and accurate data and specific embodiment | The biological experimental data of any this beneficial effect of the invention of proof is not provided | The present invention imports two antigen genes aspect novelty, and the contrast application is one; Has better technology openly from the complicacy of technology and helpfulness comparison the present invention of invention. |
Description of drawings
Fig. 1: be pIECMV-5m6 transferring plasmid collection of illustrative plates of the present invention.CMV i.e. is the human cytomegalic inclusion disease virus immediate early promoter among the figure, and polyA is the Trobest transcription termination signal, and gD, gI, gE, 11K and 28K are from the genomic homology arm sequence of pseudorabies virus.
Fig. 2: be pIECMV-5m6 transferring plasmid qualification result of the present invention.
The pcr amplification of ORF5M gene among the pcr amplification 5:pIECMV-5m6 of ORF5M gene among M:DNA marker (DL15000) the 1:pIECMV-5m6/BamH I2:pIECMV-5m6/Kpn I3:pIECMV-5m6/Xba I 4:pIECMV-5m6 among the figure.
Fig. 3: be the schema that pIECMV-5m6 transferring plasmid of the present invention makes up.
Fig. 4: be reorganization PrV virus TK of the present invention
-/ gE
-/ GP5m
+/ M
+Make up flow process.
Fig. 5: be recombinant virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+The electrophoretogram of pcr amplification ORF5M gene identification.1:PK-15 cell contrast 2:H among the figure
2O contrast 3: plasmid pIECMV-5m6 contrast 4: parent plant PrVTK
-/ gE
-/ LacZ
+Contrast 5-10: recombinant virus PrV TK
-/ gE
-/ GP5m
+/ M
+The contrast of M:DL2000DNA molecular weight
Fig. 6: be recombinant virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+The electrophoretogram of pcr amplification ORF6 gene identification.1:H20 contrast 2:PK-15 cell contrast 3 among the figure: parent plant PrV TK
-/ gE
-/ LacZ
+Contrast 4: plasmid pIECMV-5m6 contrasts 5-8: recombinant virus PrV TK
-/ gE
-/ GP5m
+/ M
+The contrast of M:DL2000DNA molecular weight
Fig. 7: be recombinant pseudorabies virus PrVTK of the present invention
-/ gE
-/ GP5m
+/ M
+The Southern-blotting analytical results.A among the figure: the electrophoresis on 0.8% sepharose; B: with ORF5M is the result of probe hybridization; C: with ORF6 is the result of probe hybridization; 1, Sal I enzyme is cut PrV TK
-/ gE
-/ GP5m
+/ M
+The genome of strain; 2, Sal I enzyme is cut TK
-/ gE
-/ LacZ
+The genome of parent plant; 3, Bgl II enzyme is cut PrV TK
-/ gE
-/ GP5m
+/ M
+The genome of strain; 4, Bgl II enzyme is cut TK
-/ gE
-/ LacZ
+The genome of parent plant; The contrast of M:DL15000 dna molecular amount
Fig. 8: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+Porcine reproductive and respiratory syndrome virus GP5m and the proteic Western-blotting analytical results of M are expressed in strain.
Among the figure 1: infect TK
-/ gE
-/ LacZ
+The PK-15 cell of strain; The 1:TK-/gE-/GP5m+/M+ strain; 2: infect TK
-/ gE
-/ GP5m
+/ M
+The PK-15 cell of strain; M: protein Marker.
Fig. 9: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE/GP5m
+/ M
+Produce the neutralizing antibody of specificity behind the immune swine and after attacking poison at PRRSV.
Figure 10: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+Produce the lymphopoiesis of specificity behind the immune swine among the PBMC at PRRSV.
Figure 11: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+Produce the efficient that specific killing infects the porcine alveolar macrophage of PRRSV behind the immune swine among the PBMC.
Figure 12: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+Temperature of pig body changes behind the immune swine PRRSV strong virus attack.
Figure 13: be recombinant pseudorabies virus PrV TK of the present invention
-/ gE
-/ GP5m
+/ M
+Pathological section micro-variations behind the immune swine PRRSV strong virus attack in the pig lung tissue (HE dyeing, 40 times of magnifications).
A:TK among the figure
-/ gE
-/ LacZ
+Parent plant contrast immune group; B: negative control group; C: deactivation vaccine control group (KV control); D: recombinant pseudorabies virus PrV TK
-/ gE
-/ GP5m
+/ M
+Immune group.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description
1, design of primers (being used for gene clone and Molecular Detection)
ORF5M and ORF6 gene order (Jiang Yunbo etc. according to the porcine reproductive and respiratory syndrome virus of having delivered, China animal doctor journal, 2005 (1), 1-3) design primer, the amplified fragments size is about 650bp and 540bp respectively, and its two ends are designed BamH I, XbaI and EcoR I, SmaI restriction enzyme site respectively.Design ORF7 gene order primer simultaneously, the amplified fragments size is about 430bp.Human cytomegalic inclusion disease virus CMV promotor, MCS multiple clone site and polyA signal segment in Pcmv and PpolyA primer amplification pCI-neo (the Promega company product) carrier, size is about 1450bp.Above-mentioned primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.Primer sequence is as follows:
Porf5ml：5′--GAAGGATCCAGTATGTTGGGGAAATGCTTGACC--3′
Porf5m2：5′--TTTTCTAGAGAGACCCCATTGTTGTTCCGC--3′
Porf61：5′--TTTGAATTCACAATGGGGTCGTCTCTAG--3′
Porf62：5′--TTTGATATCTTATTTGGCATATTTG--3′
Porf71：5′--TAGGTGACTTAGAGGCACAGT--3′
Porf72：5′--TAAATAAGCCAAATAACAAC--3′
Pcmv：5′--GATAAGCTTGATCAGATCTTCAATATTGGCCATTA--3′
PpolyA：5′--CGTAATAGCGAAGAGGCCCGCACGGA--3′
2, the structure of pIECMV-5m6 transferring plasmid (seeing shown in the accompanying drawing 1,2,3)
To contain PRRSV YA strain (Fang Liurong etc., the clone and the sequential analysis of porcine reproductive and respiratory syndrome virus YA strain ORF5 gene, Hua Zhong Agriculture University's journal, 2002,21 (6), 501-505) the plasmid pMD-ORF5M of complete ORF5M gene is a template with the pMD-ORF6 that contains complete ORF6 gene, Porf5ml/Porf5m2, Porf61/Porf62 is that primer carries out pcr amplification, the amplification condition of ORF5M and ORF6 gene two portions sequence is: enter circulation after 95.0 ℃ of 5min sex change, loop parameter is: 95.0 ℃ of 1min, 57.0 ℃ of 1min, 72.0 ℃ 1min, 35 circulations are extended 10min for back 72.0 ℃.Reaction finishes the back and obtains the big or small ORF5M gene fragment of 650bp and the ORF6 gene fragment that size is about 550bp of being about.
BamH I and Xba I enzyme are cut ORF5M gene PCR amplified production, directly be inserted into transfer vector pIECMV (Fang Liurong, Hua Zhong Agriculture University, Ph D dissertation (a kind of papers written by postgraduates that can open inquiry in the Hua Zhong Agriculture University Library), 2003) BamH I in and Xba I restriction enzyme site obtain the pIECMV-ORF5M transfer vector.
Utilize primer Pcmv/PpolyA amplification from CMV promotor, MCS multiple clone site and polyA signal segment in pCI-neo (the Promega company product) carrier, obtain the PCR product that size is about 1450bp.This PCR product is carried out Hind III and BamH I enzyme is cut, be inserted in pBluescript SK+ (the Stratagene company product) carrier of cutting with Hind III and BamH I enzyme, obtain a kind of artificial constructed carrier for expression of eukaryon pCMVPA.
The ORF6 gene PCR amplified production that EcoR I and SmaI enzyme are cut is inserted into EcoR I and the SmaI site of artificial constructed carrier for expression of eukaryon pCMVPA, obtains intermediate carrier pCMVPA-ORF6.Further pCMVPA-ORF6 is carried out Bgl II and BamH I enzyme is cut, obtain the CMV-ORF6-PolyA expression cassette that size is about 2.5kb, the CMV-ORF6-PolyA expression cassette is carried out Klenow I klenow fragment mend location, flat back and be inserted into and utilize CIAP dephosphorylation enzyme to carry out the Stu I site of dephosphorylized transfer vector pIECMV-ORF5M, at T
4The dna ligase effect connects down, will connect product then and transform DH
5 αIntestinal bacteria, a small amount of prepares plasmid, enzyme is cut evaluation, thereby obtains transferring plasmid pIECMV-5m6.Its structural simulation figure sees Fig. 1.Qualification result confirms to make up correct (see figure 2), and it makes up schema as shown in Figure 3.
3, recombinant pseudorabies virus TK
-/ gE
-/ GP5m
+/ M
+Structure
Propagation PrV TK on the PK-15 cell
-/ gE
-/ LacZ
+Parent plant, extract its genomic dna (extracting method is with reference to Wang Liu etc., biotechnology, 1994,4 (4), 33-35).Because PrV TK
-/ gE
-/ LacZ
+Genomic dna in only have an EcoR I restriction enzyme site, and be arranged in the LacZ gene.With EcoR I with PrV TK
-/ gE
-/ LacZ
+The genomic dna enzyme be cut into two sections, (method is with reference to Zhou Fuchun etc. to use liposome-mediated rotaring dyeing technology, journal of animal science and veterinary medicine, 2001,32 (2), 129-133) with itself and pIECMV-5m6 transfer vector cotransfection PK-15 cell, by take place the homologous recombination exchange with two of CMV-ORF5M-polyA and CMV-ORF6-polyA independently the expressed intact box import between PrV genome gE and the gI gene locus (Fig. 4), displace the LacZ gene simultaneously, wait to produce and receive poison after the cytopathy.Viral liquid is inoculated in the PK-15 cell, keeps liquid with the DMEM (for example U.S. GIBCO company product) that contains 1% low melting-point agarose and cover, in 37 ℃ of 5%CO
2Condition under cultivate, when just cytopathy occurring, toluylene red dyeing 1h with 0.01%, because sick cell can not be painted, and normal cell is dyed redness, thereby the round point shape plaque occurs, the single plaque inoculation of picking PK-15 cell enlarged culturing, utilize the pcr amplification detection method of ORF5M and ORF6 gene to identify positive recombinant virus (PCR the results are shown in Figure 5 and Fig. 6), so carry out purifying three times, utilize the PCR detection method of LacZ gene can not detect PrV TK at last
-/ gE
-/ LacZ
+The existence of strain, thus determine only to contain recombinant pseudorabies virus TK in the viral liquid
-/ gE
-/ GP5m
+/ M
+It makes up flow process such as Fig. 2.
4, recombinant pseudorabies virus (TK
-/ gE
-/ GP5m
+/ M
+) biological characteristics and the evaluation thereof of strain
Recombinant pseudorabies virus (TK
-/ gE
-/ GP5m
+/ M
+) strain Southern-blotting and Western-blotting evaluation with reference to (Sa Mubulu I, Ritchie E and Manny A Disi T. work not, " molecular cloning experiment guide " second edition. Jin Dongyan etc. translate the school, Science Press, 1998) in method carry out.
Southern-blotting identifies: extract recombinant pseudorabies virus TK
-/ gE
-/ GP5m
+/ M
+The genomic dna of mutant strain (reference: Wang Liu etc., biotechnology, 1994,4 (4), 33-35) cut with Bg1II and Sal I enzyme after, at 30 volts of electrophoresis on 0.8% the sepharose after 6 hours, according to a conventional method (Sa Mubulu I, not Ritchie E and Manny A Disi T. are outstanding, " molecular cloning experiment guide " second edition. and Jin Dongyan etc. translate the school, Science Press, 1998) carrying out Southern-blotting identifies.Carry out the genomic dna of electrophoretic mutant strain after enzyme cut and transfer on the nitrocellulose filter, standby behind 80 ℃ of roasting films.The dna fragmentation of pcr amplification complete ORF 5M and ORF6 gene, after pressing DNA recovery test kit (worker bio-engineering corporation is given birth in Shanghai) operation instructions recovery target DNA, with digoxigenin labeled and detection kit (Roch company product) mark (concrete grammar is pressed the test kit specification sheets operation of Roch company), as the dna probe of ORF5M and ORF6 gene, place-20 ℃ of preservations standby.With the dna probe of ORF6M and ORF6 gene and standby nitrocellulose filter hybridization, the result as shown in Figure 7 at last.Test-results shows, the TK that Bgl II and Sal I enzyme are cut
-/ gE
-/ GP5m
+/ M
+Pnca gene group DNA swimming lane has tangible specific hybrid band, is consistent with expection, and TK
-/ gE
-/ LacZ
+Pnca gene group DNA swimming lane does not then have, and confirms that ORF5M and ORF6 expression casette correctly have been inserted among the parent plant genome gE/gI.
Western-blotting identifies: with recombinant pseudorabies virus TK
-/ gE
-/ GP5m
+/ M
+Strain is inoculated in the PK-15 cell that just grows up to individual layer, collects sick cell after 16 hours.According to a conventional method (Sa Mubulu I, not Ritchie E and Manny A Disi T. are outstanding, " molecular cloning experiment guide ", second edition. Jin Dongyan etc. translate the school, Science Press, 1998) the preparation sample, carry out SDS-PAGE and Western-blotting and identify that the result as shown in Figure 8.Analyze through Western-blotting, infect TK
-/ gE
-/ GP5m
+/ M
+The PK-15 cell swimming lane of strain, find that it can react with the porcine reproductive and respiratory syndrome virus positive serum, find two specific hybrid bands at about 26kD and 19kD place, then do not have in two contrast swimming lanes, show that the GP5m of porcine reproductive and respiratory syndrome virus and M albumen have obtained expression.
Recombinant pseudorabies virus strain TK
-/ gE
-/ GP5m
+/ M
+Be the present invention constructed with the dual-gene disappearance strain of Pseudorabies virus TK
-/ gE
-/ LacZ
+Be virus vector, the modification type main immunogenic gene ORF5M and the ORF6 gene of porcine reproductive and respiratory syndrome virus is inserted into the dual-gene disappearance strain of Pseudorabies virus TK
-/ gE
-In the genome of/LacZ, the artificial constructed divalence attenuated vaccine strain that obtains.Its genetic stability and biological characteristics and parent's strain TK
-/ gE
-/ LacZ
+There is not difference.It is double-stranded DNA that recombinant virus has cyst membrane, nucleic acid, and the virion diameter is 120-180nm, to ether, chloroform and trypsinase sensitivity, and can complete inactivation in 56 ℃ of 30min.TCID on the PK-15 cell
50Still can reach 10
-8.4/ mL.Recombinant virus was uploaded for 30 generations at the PK-15 cell continuously, and its propagation titre does not have obvious variation, the GP5m of expression porcine reproductive and respiratory syndrome virus that can be stable and M albumen.
This recombinant virus need be gone up propagation and detect survival condition at pig kidney subculture cell (PK-15), the substratum of PK-15 cell is Dulbecco ' the s Modified Eagle Medium (DMEM) (U.S. GIBCO company product) that contains 10% calf serum, the pH scope is 6.8-7.2, in 37 ℃ of cultivations.Can adopt very low temperature to freeze and/or lyophil preservation.
5, the preparation of Pseudorabies virus-porcine reproductive and respiratory syndrome virus recombinant vaccine
With the recombinant pseudorabies virus TK that obtains
-/ gE
-/ GP5m
+/ M
+Identify that its genetic stabilities are found in the back of going down to posterity for 30 times, foreign protein can stably express, and has the candidate strain of the Pseudorabies virus-porcine reproductive and respiratory syndrome virus gene engineered strain of good biologic activity as recombinant vaccine.The propagation titre of this vaccine strain on pig kidney passage cell reaches 10
84TCID
50, through screening, adopting the easy chick embryo fibroblast propagation vaccine virus of preparation, its propagation titre reaches 10
7.0TCID
50With vaccine kind poison by 1/10 volume ratio inoculated into chick embryo inoblast; collect qualified viral liquid after the cytopathy; add by viral liquid: gelatin is that the volume ratio adding gelatin protective material of 7:1 is (in every 100ml deionized water with sucrose 40g; gelatin 8g after fully melting, puts 121 ℃ of sterilizations down); in sterilization freeze-drying bottle, press the packing of 2.5mL/ bottle; put freeze-drying in the freeze drier, freeze-drying 36-40h rear pressing cover, it is standby to put-20 ℃ of preservations.
6, the safety evaluation of Pseudorabies virus and porcine reproductive and respiratory syndrome virus genetically engineered bivalent vaccine
The healthy Balb/C small white mouse of 30 pseudoabies serology feminine genders is divided into three groups at random, wherein inoculates 10 respectively for two groups
6TCID
50The TK of virus quantity
-/ gE
-/ GP5m
+/ M
+Vaccine strain and PrV Ea strain, another is organized as the blank group, observes 14 days.Small white mouse spirit appetite vaccinated and control group is all normal, no abnormal performance; And the small white mouse of the strong poison of inoculation (PrV Ea strain) is dead from inoculating beginning in back 72 hours, and is all dead by 144 hours, shows that this genetic vaccine is safe (table 2) to small white mouse.
Table 2 Pseudorabies virus of the present invention-porcine reproductive and respiratory syndrome virus recombinant vaccine is to the security of Balb/C small white mouse
With TK
-/ gE
-/ GP5m
+/ M
+Vaccine is by 10
7TCID
50Virus quantity inoculate the piglet of 25 ages in days, slight one exothermic reaction of crossing property appears in part, recovers normal two days later, the spiritual appetite of piglet is normal during this period, no abnormality seen changes, and can detect the Pseudorabies virus neutralizing antibody.The piglet of PrV Hubei Province A strain (virulent strain) inoculation all presented exothermic reaction in second day, continues a week, and a death is arranged, and analyse and find to have typical pseudoabies pathological change, and control group had not both had fervescence, did not have unusual clinical manifestation yet.By 10
7TCID
50Virus quantity inoculation TK
-/ gE
-/ GP5m
+/ M
+Vaccine and nonvaccinated pregnant sow, the nest litter size is suitable substantially, stillborn foetus, mummy tire etc. all do not occur, confirms that this vaccine is safe to pregnant sow.
7, the immune efficacy of Pseudorabies virus and porcine reproductive and respiratory syndrome virus genetically engineered bivalent vaccine and protection detect
1) immunity of pig and attack malicious program
With mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) freeze-dried live vaccine made injects the negative piglet of wean of 25 ages in days, 5 every group, every pig immunity 10 respectively through musculi colli
6TCID
50, immunity is 2 times altogether, at interval 4 weeks; Establish parent plant TK simultaneously
-/ gE
-/ LacZ
+, not immunity (neg control) contrast of PRRSV deactivation vaccine (KV control) (herding biological products stock company product in the Chengdu) and feminine gender.And exempt from then to carry out the strong malicious YA strain of PRRSV 5 weeks in two and attack.Experiment pig is taken a blood sample through precaval vein, carries out ELISA antibody and the neutralizing antibody of specificity at PRV behind the separation of serum respectively, and at the neutralizing antibody level detection of PRRSV (method is with reference to Fang Liurong etc., viral journal, 2004,20 (3), 249-254).Gather anticoagulation simultaneously, (method is with reference to Pirzadeh B etc. to separate the porcine alveolar macrophage efficient that peripheral blood mononuclear cell (PBMC) detection specificity infects PRRSV at lymphopoiesis and the specific killing of PRRSV, J Gen Virol, 1998a, 79,989-999), to assess the cellullar immunologic response level that it induces body to produce.Experiment pig is attacked the poison back and is observed its clinical symptom every day, and the take temperature variation, keeps for three weeks.In attacking poison back 0,7,10,14,17, utilizing ORF7 Auele Specific Primer Porf71, the Porf72 of PRRSV to carry out RT-PCR in 21 days detects the content of PRRSV in serum, nose swab and the tonsilla swab (method is with reference to Pirzadeh B etc., J Gen Virol, 1998a, 79,989-999), the RT-PCR reaction conditions is: 50 ℃ of reverse transcription 30min; 94 ℃ of pre-sex change 5min; Enter the PCR circulation, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 35 circulations, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1.0% agarose gel electrophoresis.Carried out pig only weighing in back 12 days and 21 days in attacking poison, observe the influence of attacking behind the poison body weight.In attacking malicious back 21 days with all experiment pig sacrificed by exsanguination, the general pathology of observing tonsilla, lung, hilar lymph node, mesenteric lymph nodes, kidney respectively changes, and carries out the pathology section examination of these six tissues simultaneously.
2) the pseudoabies immune effect detects
Head exempts from back 3 all mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) can specificly detect ELISA and neutralizing antibody at PrV, and in two exempt from the back 2 weeks reached the highest, serum ELISA antibody horizontal under the extension rate of 1:40 reaches the highest, its antibody titers reaches 1:20480, and neutralizing antibody reaches 33.6 ± 17.6 and parent plant TK
-/ gE
-/ LacZ
+Trend that immune group showed and actual value there are no significant difference (table 3) shows mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) can produce the immunne response of specificity preferably at PrV.
Table 3 recombinant virus immune mouse brings out the antibody horizontal detection of specificity at PRV
Illustrate: a:SN is meant neutralizing antibody:
The ELISA antibody titer of b:PrV is meant the ELISA antibody titers at PrV totivirus particle;
C: be meant the mouse quantity that can detect at the PrV neutralizing antibody.
3) the porcine reproductive and respiratory syndrome neutralizing antibody detects
The employing document (Pirzadeh B etc., J Gen Virol, 1998a, 79,989-999) Bao Dao neutralizing antibody detection method, specificity is at the neutralizing antibody level of PRRSV in the detection serum.The result shows, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group exempts from the back at head and can detect the neutralizing antibody (〉=1:4) of specificity at PRRSV 7 weeks, and be tending towards rising.Attack poison back in it and the antibody horizontal fast rise, reached the highest in back 17 days in attacking poison, wherein have the neutralizing antibody of 3 pigs all to reach 1:256, and deactivation vaccine contrast immune group detects substantially less than neutralizing antibody before attacking poison, the neutralizing antibody level of attacking behind the poison is not high yet, with mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group compares extremely significantly (P＜0.01 of difference, t-test), the blank group does not all detect the neutralizing antibody of specificity at PRRSV in whole experiment, parent plant contrast immune group had 1 pig to show in back 21 days can to detect neutralizing antibody (1:4) attacking poison.Test-results such as Fig. 9.
4) peripheral blood mononuclear cell (PBMC) lymphopoiesis and specific killing
Gathering immune swine head respectively exempted from the back 42,63 days and attacked the back 14 days anticoagulation of poison, (method is with reference to PirzadehB etc. to separate PBMC, J Gen Virol, 1998a, 79,989-999), detection specificity is at the lymphopoiesis of PRRSV, the result shows, is that differential stimulus is former with the PRRSV of uv irradiating deactivation, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group compares the deactivation vaccine control group and show significant multiplication effect, it is more obvious particularly to attack this species diversity of poison back, shows the memory T cell immune response.Test-results such as Figure 10.
The porcine alveolar macrophage that infects with PRRSV is a target cell, detect the specific cell cytotoxic activity that immune swine head exempts from back 63 days specific killing lymph T cell (method is with reference to Pirzadeh B etc., J Gen Virol, 1998a, 79,989-999).The result shows, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group compares the deactivation vaccine control group and show the porcine alveolar macrophage effect that significant specific killing infects PRRSV, shows mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group has higher specific killing cytotoxic activity (CTL), can better suppress virus duplicating in vivo.Test-results such as Figure 11.
5) attack poison afterwards clinical symptom and body temperature and the body weight change of pig
Do not observe the obvious clinical symptom of attacking malicious pig in the malicious experimentation whole attacking.Attacking poison preceding 2 days and attacking in malicious back 21 days, carry out the measurement of bldy temperature of all immune swines every day, and it the results are shown in Figure 10, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) and the deactivation immune group in attacking poison back one 40.0 ℃ of the heatings (〉 of crossing property appearred on the the 3rd and the 4th day respectively), keeps and recover normal after one day, and parent plant and negative control group performance persistent fever, and fluctuation is bigger, the results are shown in Figure 12.
When attacking poison back 12 days and 21 days, carry out the measured body weight of all pigs, calculate per day weightening finish.The result shows, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group compares parent plant and the per day weightening finish of negative control group is higher, has significant difference, is attacking poison in 12 days early stages especially, this species diversity is bigger, but does not have the significance difference opposite sex with deactivation vaccine.The results are shown in Table 4.
Table 4 is attacked the body weight change of poison back pig
Unit: kilogram
6) viremia and toxin expelling situation
Utilize ORF7 Auele Specific Primer Porf71, the Porf72 of PRRSV carry out RT-PCR detect attack poison after, 7,10,14,17,21 days serum is attacked poison back 7,14,21 days nose swabs and the content of attacking PRRSV in 10,17, the 21 days tonsilla swabs in poison back, specifically undertaken, to determine viremia and toxin expelling situation by RT-PCR test kit (TAKARA company product) specification sheets.The RT-PCR reaction conditions is: 50 ℃ of reverse transcription 30min; 94 ℃ of pre-sex change 5min; Enter the PCR circulation, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 35 circulations, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1.0% agarose gel electrophoresis.The result shows, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) immune group compares the generation that deactivation vaccine can more effectively suppress viremia, and can remove the existence of virus in serum fast.Simultaneously, by detecting the content of virus in nose swab and the tonsilla swab, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) the outside toxin expelling situation of immune group (comprise discharge virus content and time) also significantly is better than the deactivation vaccine immune group.The results are shown in Table 5.
Table 5 is attacked ORF7 gene RT-PCR result in poison back porcine blood serum, nose swab, the tonsilla swab
Illustrate :-: the RT-PCR amplification is negative; +: there is 1 RT-PCR amplification positive in 5 pigs; ++: have 2 RT-PCR amplifications positive in 5 pigs: +++: there are 3 RT-PCR amplifications positive in 5 pigs; ++ ++: there are 4 RT-PCR amplifications positive in 5 pigs; ++ +++: there are 5 RT-PCR amplifications positive in 5 pigs.
7) the immune swine histopathology of attacking behind the poison changes
In attacking malicious back 21 days with all experiment pig sacrificed by exsanguination, the general pathology of observing its tonsilla, lung, hilar lymph node, mesenteric lymph nodes, kidney respectively changes, the pathology section examination of carrying out these six tissues simultaneously is (with reference to the bright volume of Zhao De, veterinary pathology, the China Agricultyre University Press, 1998).Found that there be extravasated blood and the blutpunkte that is dispersed in parent plant and negative control group lung, while hilar lymph node and mesenteric lymph nodes oedema, severe haemorrhage, and other organizes the general pathology variation not obvious.Deactivation vaccine and mutant strain (TK-/gE-/GP5m
+/ M
+) immune group lung do not have the considerable pathological change of obvious naked eyes, but that hilar lymph node also shows is heavier hemorrhage.Organizing micro-pathological change to observe finds, parent plant and negative control group lung have the serious interstitial pneumonia of typical case and change, alveolus wall thickens, in segmental bronchus and bronchiole wall thickening and the pipe lymphocyte and macrophages infiltration are arranged, hilar lymph node significant quantities of fat cell deposition, hemorrhage oedema, and because the iron xanthematin deposition that the macrophage phagocytic red corpuscle causes, the mesenteric lymph nodes oedema, severe haemorrhage, kidney shows the oedema of uriniferous tubules and the epithelial damage formation cast that comes off, and tonsilla and spleen are not seen typical pathological change.The interstitial pneumonia of the lung performance moderate gentleness of deactivation vaccine immune group pig, the micro-pathological change of its hetero-organization and control group basically identical.Compare parent plant, negative control and deactivation vaccine immune group, mutant strain (TK
-/ gE
-/ GP5m
+/ M
+) notable difference that immune group shows be lighter interstitial pneumonia, alveolar and bronchial wall do not have significantly substantially and thicken.Acini lienalis enlarges, and germinal center is obvious, and this may be due to the immune response that brings out.But the micro-pathological change of its hetero-organization does not have evident difference yet.Wherein the micro-pathological change difference of lung tissue is seen Figure 13.
Claims (3)
1, a kind of reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain (Pseudorabies virus) HzauAVL-PRprrsvV-GP5mM, be deposited in Chinese typical culture collection center (CCTCC), deposit number: CCTCC-V200509, it has following feature:
1) the main virulence gene TK of disappearance pseudorabies virus does not produce functional glycoprotein gE/gI;
2) porcine reproductive and respiratory syndrome virus ORF5M and ORF6 gene are the form insertions with the expression cassette of two independent completions;
3) express the GP5m and the M proteantigen of porcine reproductive and respiratory syndrome virus simultaneously.
2, the vaccine that comprises the reorganization pseudoabies-porcine reproductive and respiratory syndrome gene engineered strain preparation of claim 1.
3, the application of the described reorganization pseudoabies of claim 1-porcine reproductive and respiratory syndrome gene engineered strain on preparation pseudoabies-porcine reproductive and respiratory syndrome recombinant vaccine.
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