CN101240289A - Fowl infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2 - Google Patents

Abstract

The invention provides a DNA vaccines Pires-S1/IL2 expressed by poultry infectivity bronchitis virus S1 gene and poultry IL-2 gene in animals medicine biological engineering study field. After thepIBVS1, pIRES-EGFP/DsRed articles are cut by SacI and XhoI enzyme, S1 gene is recovered and coupled with carrier fragment to construct eukaryon expressing article pIRES-S1/DsRed. Poultry source IL-2 gene is obtained by PCR using Pdnail-18 article as template. After the IL-2, pIRES-S1/DsRed articles are cut by BamHI and NotI enzyme, IL-2 gene is recovered and coupled with carrier fragment to construct DNA vaccines Pires-S1/IL18 expressed by poultry infectivity bronchitis virus Chinese separation plant SAIBk S1 gene and poultry IL-2 gene. The vaccines can be used to prevent poultry infectivity bronchitis.

Description

A kind of avian infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2

[technical field] the present invention relates to animal medical bioengineering field, is a kind of avian infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2.

[background technology] infectious bronchitis of fowl (Avian Infectious Bronchitis, IB) be the transmissible disease of a kind of acute, the height contact of the chicken that causes of the avian infectious bronchitis virus of coronaviridae coronavirus genus, it is characterized by that respiratory symptom appears in the disease chicken, the quantity of laying eggs and quality descends and renal lesions etc.Avian infectious bronchitis virus can infect the chicken of all ages in days, causes growth retardation, death, weightening finish and the price of deed to reduce; Also usually cause secondary infection such as Mycoplasma polyinfection and colibacillosis and improve chicken group's mortality ratio that giving raises chickens produces and bring massive losses.At present, this disease is the world and is widely current, and is one of serious infectious diseases of serious harm world aviculture.

Avian infectious bronchitis virus is the representative species of coronaviridae coronavirus genus, and its antigenicity is complicated unusually, and isolating in the world at present clinical strain has surpassed strain more than 100, adheres to serotype more than 30 kinds separately.Avian infectious bronchitis virus belongs to the RNA viruses of sub-thread normal chain, genome total length 27.6kb, the three kinds of structural protein of mainly encoding: spike protein (S), membranin (M), nucleoprotein (N).Many studies show that, S protein cleavage are S1 and two subunits of S2, and wherein S1 albumen can be induced and be produced neutralizing antibody and hemagglutination inhibition antibody, is the main immunogens of IBV.N albumen is carrying t cell epitope, may play a role in immunity and protection.The proteic immunogenicity of M is lower, also have on the report M albumen and may exist antigenic determinant with important immunization, the immune response of energy inducing cell mediation (referring to: Yin Zhen, Liu Jinghua etc. animal virology (second edition). Beijing: Science Press, 1997).

The different strains of avian infectious bronchitis virus exist very big-difference in virulence, pathogenic and tissue tropism, between the different serotypes strain a little less than the cross protection power.On anti-system, conventional single strain vaccine often has the report of immuning failure, and polyvalent vaccine can better solve the problem of control.The inactivated vaccine security is good, does not exist to disseminate cause of disease and virulence is returned strong problem, and can excite the good humoral immune response.Its weak point is to use dosage big, needs to cooperate adjuvant, the preparation more complicated, thereby cost is higher.Inactivated vaccine can not bring out body generation cellular immunization in addition, and a series of studies confirm that in the immunoprotection of IBV, humoral immunization and cellular immunization occupy critical role (referring to: Wang Hongning chief editor. the fowl respiratory system disease. Beijing: agricultural science and technology press, 2002; BW Ka Ernike chief editor. disease of poultry (the 9th edition). Beijing: press of Beijing Agricultural University, 1991,407-418; Kusters J G, Niesters, H G M, et al.Virology, 1989,169:217-221; AvellanedaG E, Villeges P, Jackwood M W, et al.Avian Dis, 1994,38:589-597).Along with progressively going deep into of researchs such as avian infectious bronchitis virus epidemiology survey, molecular biology research, immunologic mechanism, make up the carrier for expression of eukaryon of this viral primary structure protein gene, it is significant that the development dna vaccination is used for this disease prevention.

Many in recent years investigators attempt to study the dna vaccination of avian infectious bronchitis virus, and have obtained certain progress.Step, CHIGO etc. was (referring to step CHIGO, river state holder etc. Chinese animal doctor's journal, 1998,18 (6): be immunogen gene 527-530) with the fine prominent glycoprotein S1 gene of the local epidemic strain IBV of domestic class M41, made up the dna immunization expression plasmid, and its immunogenicity has been carried out initial analysis.The result shows that this expression plasmid can induce body to form cellular immunization and humoral immune reaction at IBV simultaneously.Chen Hongyan etc. (referring to: Chen Hongyan, the holder of river state, Yang Qiwei etc. Chinese Preventive Veterinary Medicine newspaper, 1999,21 (4): 250-253) avian infectious bronchitis virus kidney type T strain S1 gene cDNA is connected in pcDNA3 and has made up eukaryon expression plasmid, serum IgG antibody raises gradually behind intramuscular injection immunity SPF chicken, peak to 35 ages in days, but serum IgG antibody rising amplitude is not as good as the IB oil seepage.Immunity is attacked the attack that 40% chicken can the strong poison of anti-mistake behind the poison.Liu Siguo etc. (referring to Liu Siguo, Kang Lijuan, river state holder etc. Chinese animal doctor's journal, 2001,21 (1): 14-16) with the N gene subclone of chicken infectious bronchitis HB strain to pcDNA3, made up eukaryon expression plasmid pcDNA-N.Carry out challenge test with twice immune SPF chick of this plasmid after one week, protection ratio is 40% as a result, shows that the protein mediated cellular immunization of N brought into play effect in anti-infective.(Kapczynski D R such as Kapczymki DR; Hilt D A, Shapiro D, et al.Avian Dis; 2003; the proteic dna vaccination of expression S1 that 47:272-285) will make up is by inoculation or intramuscular injection in the embryo, and the result shows that the chicken embryo of 18 ages in days is used earlier this vaccine immunity; after hatching for 2 weeks, chick uses weak malicious seedling booster immunization again; can make chicken obtain 100% protection, and single with inoculation or weak malicious seedling intramuscular injection chick in the dna vaccination embryo, protection ratio is all less than 80%.Liu Zhaoqiu etc. (Liu Zhaoqiu, Jiang Shijin, Chang Weishan etc. Chinese animal doctor's journal, 2005,25 (3): 241-243) with IBV S1 gene constructed carrier for expression of eukaryon pcDNA-S1, studies show that the dna vaccination of structure can induce the chicken body to produce antigen-specific immune responses, the strong poison of opposing infects.The dna vaccination infection prevention is bronchitic to be discovered, there are shortcomings such as immunogenicity immune protective rate poor, that induce body to produce is low in single antigenic dna vaccination.Therefore, enhance immunity reply efficiency how, the immune effect that strengthens dna vaccination becomes the key issue of current vaccine research.

Along with deepening continuously of pair cell factor research; many cytokines are found has tangible molecule adjuvant effect; the immunogenicity of energy specificity enhancement antigen or enhancing body are to antigenic immunoreactivity; and the networked interaction energy optimization host's of cytokine specific immune response, induce body to produce effective immune protective efficiency.IL-2 is most widely used in the cytokine adjuvant, and it can act on multiple effector cell, comprises T, bone-marrow-derived lymphocyte, scavenger cell and NK cell, can strengthen cellular immunization and humoral immunization simultaneously.Henke etc. have made up the co-expression carrier of expressing influenza A virus gene and IL-2 gene, can stop effectively behind the immune mouse influenza virus infection (Henke A, Rohland N, etal.Interviology, 2006,49:249-252).The plasmid that Barouch etc. have compared the plasmid of single expression IL-2 and HIV gp120DNA vaccine and expressed the IL-2/Ig fusion rotein is to the mouse immune effect, found that having only fusion rotein significantly to strengthen specific antibody produces and T cell proliferation, and the plasmid of single expression IL-2 (Barouch DH to no effect, Santra S, etal.Immunol, 1998,161:1875-1882).Discover, the plasmid of the Codocyte factor relevant with the dna vaccination injection, its inject time is very important.When coding GM-CSF plasmid 3d or injection simultaneously before the dna vaccination injection, and must can induce the strongest CTL to reply at same site injection.If but dna vaccination that will make up respectively and GM-CSF coding plasmid hybrid injection will have interference effect (Kamath AT, Hanke T, et al.Immuology, 1999,96:511 to genetic immunization; Hong Xiaowu, Dou Jun etc., practical tumour magazine, 2003,18:154).Barouch etc. have made up HIV gp120 and the GM-CSF co-expression plasmid with the bicistronic mRNA form, only just can induce to produce and only express the suitable antibody titers of coat protein plasmid, TH cell response and T cell proliferation (Barouch DH with 100 μ g with 1 μ g bicistronic mRNA plasmid PV1J-gp120/GM-CSF, Santra S, etal.Immunology, 2002,168:562-568).Wang etc. have made up the nucleic vaccine plasmid of WHV antigen gene and IFN-γ coexpression, behind the immune animal, effectively the duplicating of viral interference (Wang J, Gujar SA, et al.JVirol, 2007,81:903-916).

After [summary of the invention] the present invention used SacI and XhoI double digestion with pIBVS1, pIRES-EGFP/DsRed plasmid, glue reclaimed the S1 gene and is connected with carrier segments, makes up eukaryon expression plasmid pIRES-S1/DsRed.With the pDNAIL-2 plasmid is template, and PCR obtains fowl source IL-2 gene.Behind IL-2, pIRES-S1/DsRed plasmid usefulness BamHI and NotI double digestion, glue reclaims the IL-2 gene and is connected with carrier segments, has made up avian infectious segmental bronchus virus Chinese pathogenic strain SAIBk strain S1 gene and fowl IL-2 gene coexpression DNA bacterin.This vaccine can be used for preventing infectious bronchitis of fowl.

[embodiment]

1. eukaryon expression plasmid pIBVS1 used in the present invention, pDNAIL-2 and carrier for expression of eukaryon pIRES-EGFP/DsRed are made up and are provided by this laboratory; Host e. coli JM109 is preserved by this laboratory; Avian infectious bronchitis virus is attacked poison and is also preserved by laboratory qualification with strain SAIBk strain.

2. primer design and synthetic chicken interleukin-2 2 gene orders that are basis has been logined both at home and abroad in GenBank design behind multiple comparisons.According to the restriction enzyme site on the carrier for expression of eukaryon pIRES-EGFP/DsRed, upstream primer designs at the initiator codon place, and adds the BamHI restriction enzyme site; Downstream primer designs at the gene end, and adds the NotI restriction enzyme site.

Primer sequence is: upstream primer: 5 '-CCAGGATCCACCATGATGTGCAAAG-3 '; Downstream primer: 5 '-GAAGCGGCCGCAGATTAGTTAGC-3 '.

3. the structure of eukaryon expression plasmid pIRES-S1/DsRed, pIBVS1 is carried out double digestion with SacI and XhoI to be handled, glue reclaims the band of 1.8kb, with same method the pIRES-EGFP/DsRed plasmid is carried out double digestion and handles, and glue reclaims 5.3kb left and right sides dna fragmentation.

Get the S1 gene fragment that glue reclaims behind the 5 μ L double digestions respectively, add 10xT4DNA Ligase buffer 1 μ L, T4DNA ligase enzyme 1 μ L, enzyme cut and handle carrier pIRES-EGFP/DsRed 1 μ L, ddH2O 2 μ L, and 16 ℃ connect 18 hours.To connect product and transform the JM109 competent cell, contain 37 ℃ of cultivations on the LB flat board of kantlex.

4. the evaluation of eukaryon expression plasmid pIRES-S1/DsRed is containing the single bacterium colony of picking on the LB flat board of kantlex, and the extracting plasmid DNA is carried out double digestion and identified.The pIRES-S1/DsRed eukaryon expression plasmid is carried out double digestion with SacI and XhoI, enzyme is cut product and is observed at 0.8% agarose gel electrophoresis, produce two bands of about 1.8kb and 5.3kb behind the pIRES-S1/DsRed double digestion, prove that the S1 gene replaced the green fluorescence gene (EGFP) in the pIRES-EGFP/DsRed plasmid.

5. the structure of co-expression plasmid pIRES-S1/IL2 is a template with the pDNAIL-2 plasmid, and PCR obtains fowl source IL-2 gene, with the PCR product of IL-2 gene phenol chloroform extracting and purifying, and ethanol sedimentation, dissolve with TE dry back.The PCR purified product of IL-2 gene carries out double digestion with BamHI and NotI to be handled, and glue reclaims the band of 0.5kb.With same method the pIRES-S1/DsRed plasmid is carried out enzyme and cut processing, glue reclaims the band about 6.3kb.

Get the IL-2 gene fragment that glue reclaims behind the 5 μ L double digestions respectively, add 10xT4DNA Ligase buffer 1 μ L, T4DNA ligase enzyme 1 μ L, enzyme cut and handle carrier pIRES-S1/DsRed 1 μ L, ddH2O 2 μ L, and 16 ℃ connect 18 hours.To connect product and transform the JM109 competent cell, contain 37 ℃ of cultivations on the LB flat board of kantlex.

6. the evaluation of co-expression plasmid pIRES-S1/IL2 is containing the single bacterium colony of picking on the LB flat board of kantlex, and the extracting plasmid DNA is carried out double digestion and identified.The pIRES-S1/IL2 eukaryon expression plasmid is carried out double digestion with BamHI and NotI, enzyme is cut product and is observed at 0.8% agarose gel electrophoresis, produce two bands about about 0.5kb and 6.3kb behind the pIRES-S1/IL2 double digestion, prove successfully to have made up eukaryon expression plasmid pIRES-S1/IL2.

7. the development of avian infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2.Picking list bacterium colony from the LB solid medium is inoculated in the triangular flask that fills the 10ml liquid nutrient medium, 37 ℃ of concussion incubated overnight.The fermention medium that in the triangular flask of 1000ml, adds 100ml, the bacterial classification of access 5ml, 37 ℃, 200rpm/min cultivates 20h..Extract the alkaline lysis method of plasmid with routine and carry out a large amount of extractings of plasmid, carry out purifying with the diatomite adsorption method.Electrophoresis is identified the purity and the content of plasmid.Plasmid is diluted to 1mg/ml with the PBS damping fluid, is a kind of avian infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2.This dna vaccination carries out preventive vaccination with the method for intramuscular injection.

Claims (3)

1. avian infectious bronchitis virus S1 gene and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2, it is characterized in that avian infectious bronchitis virus Chinese pathogenic strain SAIBk strain S1 gene and fowl IL-2 gene being inserted among the carrier for expression of eukaryon pIRES-EGFP/DsRed with molecular biology method, structure can be expressed S1 gene and fowl IL-2 gene co-expressing plasmid, development dna vaccination pIRES-S1/IL2, this dna vaccination pIRES-S1/IL2 can be used to prevent infectious bronchitis of fowl.

2. described a kind of avian infectious bronchitis virus S1 gene of claim 1 and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2, it is characterized in that with avian infectious bronchitis virus Chinese pathogenic strain SAIBk strain S1 gene through SacI and XhoI double digestion, fowl IL-2 gene is behind BamHI and NotI double digestion, insert among the carrier for expression of eukaryon pIRES-EGFP/DsRed, make up eukaryon expression plasmid, development dna vaccination pIRES-S1/IL2.

3. described a kind of avian infectious bronchitis virus S1 gene of claim 2 and fowl IL-2 gene coexpression DNA bacterin pIRES-S1/IL2 are used to prevent infectious bronchitis of fowl.