CN104046597A - HVT (Herpesvirus of turkey) recombinant for expressing chlamydia psittaci pmpD-N gene and construction thereof - Google Patents

Abstract

The invention provides a recombinant HVT (Herpesvirus of turkey) virus which can be used as a vaccine. According to the HVT recombinant for expressing a chlamydia psittaci pmpD-N gene, a chlamydia psittaci pmpD-N gene is inserted into an HVT genome on the basis of a bacterial artificial chromosome of HVT, and then the recombinant HVT virus is obtained by virtue of a virus rescue technology. The virus can be used for ensuring that chlamydia psittaci pmpD-N protein can be expressed in infected chicken embryo fibroblasts, so that a bivalent poultry vaccine which can be used for preventing and controlling chicken Marek's diseases and effectively resisting chlamydia psittaci can be prepared; the vaccine can be used for overcoming the interference of a maternal antibody because HVT is transferred among cells, and can be used for immunization of one-day-old chicks, and even for eighteen-day-old chick embryos; meanwhile, because single protein is used as an immunogen, the vaccine also can be suitable for differential diagnosis of vaccine immunization and wild virus infection in the future.

Description

Express HVT recombinant chou and the structure thereof of chlamydia psittaci pmpD-N gene

Technical field

The present invention relates to express chlamydia psittaci pmpDthe HVT recombinant chou of-N gene, the invention still further relates to preparation method and the application of this recombinant virus.

Background technology

Chlamydia psittaci is a kind of Natur al foca pathogenic agent, and host range is extensive, can cause the various diseases of people, Mammals and birds.Its natural host is bird, lower animal and wildlife, mainly in birds and poultry, propagates.Poultry is caused multisystem after infecting, or fatal disease, and clinical manifestation is conjunctivitis, apocleisis, lose weight, suffer from diarrhoea, yellow dysentery, sinusitis paranasal sinusitis, sneeze, shed tears and have difficulty in breathing.After cuing open inspection, find that pathological change main manifestations is hepatic necrosis, hepatosplenomegaly, fiber disposition airsacculitis, peritonitis and pericarditis etc.Chlamydia psittaci broke out the earliest between 1929 ~ nineteen thirty, was being very popular in Europe and America of being caused by the green parrot of Amazon.In 30 years of past, in the U.S., China, Australia, turkey duck field and the chicken house in Europe all have popular.

Chlamydial infection is often stealthy form, and it parasitizes the inside of infected cell, causes the pathological change of carrying out property, non-reversibility, is difficult to by the clear cause of disease infecting of microbiotic.Therefore, the effective vaccine of searching prevention choamydiae infection is the long-term objective of chlamydozoan research.So far, existing multiple fowl obtained research with chlamydia vaccine, comprised full bacterium inactivated vaccine, attenuated vaccine, subunit vaccine, DNA vaccination and live vector vaccine etc.Deactivation vaccine is to kill chlamydozoan by physics, chemical process, but still keeps its immunogenic a kind of biotechnological formulation.But dead seedling can not stimulate Specific CTL Cells to produce, and cellullar immunologic response plays a crucial role in final removing choamydiae infection.Therefore deactivation vaccine discomfort is fit to do chlamydia vaccine immunity.Attenuated live seedling can effectively prevent chlamydial infection, but living vaccine has reply virulence, causes chlamydozoan contaminate environment and causes the potential risk that immunopathogenesis damages in theory.Therefore, the development prospect of chlamydozoan living vaccine is restricted.For removing the side effect material of chlamydozoan whole-bacterial-vaccine, avoid the danger of attenuated live vaccine reverse mutation, since the eighties in 20th century, subunit vaccine becomes the emphasis of chlamydozoan research.MOMP is major structural protein and the immunodominance antigen of chlamydozoan adventitia, can induce body to produce specificity neutralizing antibody and the cell-mediated immunne response of T, because of but the optimal selection of chlamydia vaccine target antigen.But; the antigenic determinant variation of various chlamydozoan MOMP is larger; a kind of vaccine is difficult for that various choamydiae infection is all had to protectiveness, and the native configurations of MOMP is closely related with its protectiveness effect, therefore subunit vaccine also very large obstacle of existence in clinical application.DNA vaccination is the of great value method of prevention choamydiae infection.Vanrompay etc. are by the gene of A type chlamydia psittaci coding MOMP ompAbe cloned on eukaryon expression plasmid pcDNA1, by the pcDNA1/MOMP plasmid DNA that is coated with bronze by gene muscle or mucosal immunity inoculation turkey, the restructuring MOMP that is similar to natural MOMP structure in turkey, detected, and detect and produced good ctl response by corresponding antibody simultaneously.But the stability of DNA vaccination and genetically modified risk have all limited the prospect of this vaccine in poultry is used.

Recombinant live-vector vaccine refers to and utilizes genetic engineering technique, protective antigen gene (goal gene) is transferred in corresponding carrier, by giving expression to the living vaccine of protective antigen protein after recombinating with virogene, this vaccine can pass through 1 immunoprophylaxis various diseases, obviously reduce the cost of vaccine, simplify immune programme for children, increase Immunization coverage rate, protective antigen composition in this vaccine is single simultaneously, be conducive to set up immunologic surveillance method, therefore utilizing the vaccine virus of living is an important directions of following vaccine as study on the carrier recombinant live-vector vaccine.Zhou etc. utilize the gland virus expression MOMP of restructuring, have also obtained good immune effect after immune chicken, illustrate that preparing vaccine method with live vector can become an important means of chlamydia vaccine research.

Along with molecular biological development, viral molecular locus and structure that some genomes are huge are progressively made clear, thereby have established the status of these viruses in recombinant live-vector vaccine research.In the research of poultry diease vaccine, mainly contain for the virus vector that builds recombiant vaccine: bird pox virus (FPV), aviadenovirus and simplexvirus, comprise avian infectious laryngotracheitis virus (ILTV) and chicken Marek's disease virus (MDV).Current widely used virus live vector is poxvirus vector, poxvirus has many good qualities as carrier, as insert foreign gene group and greatly, can not be integrated into that host chromosome, immunization method are simple, production of vaccine low cost and other advantages, therefore recombinant Borrel vaccine has been widely applied in production practice.But poxvirus is to cause support effect as most important shortcoming of carrier, thereby poxvirus vector vaccine can not be used for inoculating the fowl of bovine vaccine, and because the anti-poxvirus antibody of body generation also can be eliminated vector virus, thereby poxvirus vector vaccine also cannot overcome the interference of maternal antibody.

Herpes turkey virus (Herpesvirus of turkey, HVT) is chicken Marek's disease serum virus III type.HVT does not have pathogenic to chicken group, be typically used as vaccine and prevent the generation of commercial chicken tumour, immunosuppression and the fatal disease that are caused by MDV.HVT and MDV are the simplexviruss in serology with dependency, and the immune response being produced by HVT induction has obvious cross protection to MDV.HVT can induce generation congenital and adaptive immunity reaction.Raise natural killer cell reactive behavior, produce body fluid and the cell immune response of virus-specific.The feature of HVT genome structure with and continue the characteristic existing at chicken body, become the good carrier for building restructuring epiornitic seedling.At present, the existing infectious bursal disease of vaccine, newcastle disease, highly pathogenicity avian influenza etc. for the carrier fowl based on HVT.But there is no at present the relevant report of the genetically engineered bigeminy vaccine of chlamydozoan and HVT.

Past; build the method for recombinant hvt normally; first the protective antigen gene of cause of disease and marker gene (as galactosidase gene Lacz or green fluorescence protein gene EGFP) are built to transfer vector with HVT nonessential region; then the chick embryo fibroblast that this transfer vector transfection has been infected to HVT; again by many wheel plaque selects and the positive recombinant virus of purifying; so not only waste time and energy, and be difficult to obtain 100% recombinant virus.

Summary of the invention

First object of the present invention is to provide a kind of chlamydia psittaci of expressing for above-mentioned deficiency pmpD-Nthe HVT recombinant chou of gene;

Second object of the present invention is to provide above-mentioned expression chlamydia psittaci pmpD-Nthe construction process of the HVT recombinant virus of gene;

The 3rd object of the present invention is to provide above-mentioned HVT recombinant virus in the purposes of preparation bigeminy fowl in vaccine.

For achieving the above object, the present invention builds first respectively and can under CMV and EF-1 α promoter regulation, express chlamydia psittaci PmpD-N protein expression box, then by the method for homologous recombination, its expression cassette is inserted to HVT bacterial artificial chromosome (Bacteria artificial chromosome, BAC) in, obtain the HVT BAC of restructuring PmpD-N gene, again BAC DNA transfection chick embryo fibroblast is saved out to recombinant virus, and evaluate in vitro the correlation properties of these two kinds of recombinant viruses.Then by analyzing with the protection effect for MDV and chlamydia psittaci after the recombinant hvt immunity SPF chicken of CMV promoter expression chlamydia psittaci pmpD-N gene, confirm that this virus can be used as the candidate vaccine of opposing MD and chlamydia psittaci.

Accordingly, technical scheme of the present invention is as follows:

A kind of recombinant virus, it is by chlamydia psittaci pmpD-N gene is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

Preferably, above-mentioned recombinant virus, it is by chlamydia psittaci pmpDthe expression cassette that-N gene, CMV promotor and BGH polyA transcription termination signal form is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

Preferably, above-mentioned recombinant virus, it is by chlamydia psittaci pmpDthe expression cassette that-N gene, pEF-1 α promotor and BGH polyA transcription termination signal form is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

The construction process of above-mentioned recombinant virus, comprise the following steps: structure can be expressed chlamydia psittaci PmpD-N protein expression box under CMV or EF-1 α promoter regulation, then by the method for homologous recombination, its expression cassette is inserted in HVT bacterial artificial chromosome, obtain the HVT BAC of restructuring PmpD-N gene, again BAC DNA transfection chick embryo fibroblast is saved out to recombinant virus, obtain HVT recombinant virus.

Preferably, be pVAX1 or pEF6/V5-His for the plasmid of construction expression chlamydia psittaci PmpD-N protein expression box.More having the plasmid that is selected to construction expression chlamydia psittaci PmpD-N protein expression box is pVAX1.

The present invention utilizes BAC system successfully to save rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpDtwo kinds of recombinant viruses, Western blot shows that the target protein expression level under the control of CMV promotor is higher than the expression level under the control of EF-1 α promotor, and rHVT-CMV- pmpDcompared with wild-type HVT, duplication characteristic does not change.

The present invention utilizes HVT BAC construction expression chlamydia psittaci pmpDthe recombinant hvt of-N, viral complete genome DNA can be copied in intestinal bacteria, both facilitated the operations such as foreign gene insertion, can utilize again virus rescue technology, on eukaryotic cell, directly obtaining the recombinant virus of 100% purity, is the revolution of the traditional technology method of the above-mentioned recombinant virus of preparation.

In addition the rHVT-CMV-that, the present invention builds pmpDafter recombinant live-vector vaccine immunity chicken, can obtain good cellular immunization and Humoral.This vaccine is attacked protection can be provided completely MDV, and can obviously alleviate the relevant diseases of chlamydia psittaci, reduces the discharge of bacteria of chlamydia psittaci and residual at target organ, can be used as candidate's bigeminy vaccine of opposing chlamydia psittaci and MDV.

Brief description of the drawings

Fig. 1 is the western blot qualification result of HVT recombinant chou, and wherein M:fermentas dyes Marker in advance; 1:rHVT-EF-1 α- pmpDthe CEF cell that recombinant chou infects; 2:rHVT-CMV- pmpDthe CEF cell that recombinant chou infects; 3: the CEF cell that wild-type HVT infects.

Fig. 2 is the IFA qualification result of recombinant hvt, wherein A:rHVT-EF--1 α- pmpDrecombinant chou infects CEF 3 d; B:rHVT-CMV- pmpDrecombinant chou infects CEF 3 d.

What Fig. 3 showed is that wild-type HVT and recombinant hvt infect the pathology situation producing after CEF cell, wherein A: the HVT of wild-type infects the plaque (100x) that CEF produces; B:rHVT-EF-1 α- pmpDrecombinant chou infects the plaque (100x) that CEF produces; C:rHVT-CMV- pmpDrecombinant chou infects the plaque (100x) that CEF produces.

Fig. 4 is rHVT-CMV- pmpDone step growth with wild-type HVT.

Fig. 5 is PCR method qualification rHVT-CMV- pmpDgene stability, wherein M:DL2000; 1:CEF negative control; 2: third generation rHVT-CMV- pmpDcell toxicant pcr amplification product; 3: the five generation rHVT-CMV- pmpD4: the ten generation rHVT-CMV-of cell toxicant pcr amplification product pmpDcell toxicant pcr amplification product; 5: the 15 generation rHVT-CMV- pmpDcell toxicant pcr amplification product; 6: the 20 generation rHVT-CMV- pmpDcell toxicant pcr amplification product.

Fig. 6 is IFA method qualification rHVT-CMV- pmpDthe stability of PmpD-N protein expression, A: the 5th generation rHVT-CMV- pmpDinfect CEF cell 3 d; B: the tenth generation rHVT-CMV- pmpDinfect CEF cell 3 d; C: the 15 generation rHVT-CMV- pmpDinfect CEF cell 3 d; D: the 20 generation rHVT-CMV- pmpDinfect CEF cell 3 d.

What Fig. 7 showed is to attack poison latter 4 days, respectively organizes the pathology average situation of chicken internal organs.

What Fig. 8 showed is the average result of larynx swab discharge of bacteria.

What Fig. 9 showed is the bacterial loads average result of internal organs.

Embodiment

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.Do not deviating under the prerequisite of the present invention's spirit and essence, amendment made for the present invention or retouching all belong to scope of the present invention.

If no special instructions, the experimental technique in the present invention is ordinary method, and molecule manipulation method is carried out with reference to the associated viscera of " molecular cloning experiment guide " third edition.

related experiment material is as follows:

1. bacterial strain, virus, serum and cell

Chlamydia psittaci CB7 strain and CB3 strain: purchased from China Veterinery Drug Inspection Office, the kind daughter bacteria after breeding is kept at this laboratory.

The FC126 strain of turkey bleb vaccine virus and chicken serum thereof, be purchased from China Veterinary Drugs Supervisory Inst.'s virus; Attack the poison strong malicious RB1B strain of MDV, for preserving Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute; Chick embryo fibroblast: get according to a conventional method 9 ~ 11 age in days SPF chicken embryos (purchased from Beijing's Experimental Animal Center), preparation chick embryo fibroblast individual layer, propagation recombinant virus is used.

2. plasmid

Containing the recombinant plasmid pET-pmpD of chlamydia psittaci pmpD, complete people by the present invention and built before experiment, and be stored in this laboratory.Concrete operation step is as follows:

The DNA that extracts chlamydia psittaci CB7 strain, it increases to adopt following primer pair: PmpDF(upstream primer): 5 '-CCG gAATTCaTGGGATCCAATGTGTTGATTTCTGGAA-3 ' (EcoRI restriction enzyme site indicates with underscore); PmpDR(downstream primer): 5 '-CCG cTCGAGtCAAACAGCCCCACCTGTAGGAGCA-3 ' (XhoI restriction enzyme site indicates with underscore).Adopt EcoRI/XholI to carry out after double digestion it, be cloned into prokaryotic expression carrier pET-30a carrier, be accredited as after the positive through PCR and EcoRI/XholI double digestion, be converted in BL21, after IPTG induction, adopt western blot to identify it.

This research is respectively pVAX1 and pEF6/V5-His for the eucaryon plasmid of construction expression box; All purchased from Invitrogen.They are respectively expression cassette human cytomegalic inclusion disease virus (CMV) promotor, elongation growth factor gene (pEF-1 α) promotor and BGH polyA transcription termination signal are provided.

Recombinant hvt transfer vector: the UL45 of the Nonessencial region of HVT is connected with pacI enzyme with UL46 gene, is then cloned in pGEM-T plasmid called after pUL45-46.Construction step is as follows: the first design primer of corresponding UL45 and UL46 gene fragment in HVT genome that increases respectively, wherein 3 ' end of 5 ' of the downstream primer of UL46 end and UL45 upstream primer is with the sequence of pacI restriction enzyme site, and between them, being reverse complemental, sequence is as follows: HVT-UL46-FP:5'-TAAACGTAGATTGAAAAGGGCGAAGC-3'; HVT-UL46-RP:5'-TCAGCTTAATTAACTGTCCTGCTCCTT-3 '; HVT-UL45-FP:5'-AAGGAGCAGGACAGTTAATTAAGCTGA-3'HVT-UL45-RP 5'-CACAAAATCGCTGGGGTATATCATA-3'

Then utilize UL46 and UL45 upstream and downstream primer separately to amplify respectively corresponding fragment, then after the purified product 1:1 of above-mentioned fragment mixes as template, with HVT-UL46-FP and HVT-UL45-RP primer carry out overlapping PCR obtain in the middle of with pacthe gene fragment of I restriction enzyme site, finally this gene fragment according to a conventional method with pGEM-T(promega company product) be connected and transform intestinal bacteria TOP10 competent cell, thereby obtain the clone who contains transfer vector pUL45-46.

3. HVT -galK BAC

Utilize existing HVT BAC system, galactokinase gene (galK) electricity is transformed into the intestinal bacteria SW105 competent cell containing HVT BAC, filter out again the bacterium colony of selecting substratum growth at galK forward taking galK gene as object fragment PCR, obtain in the UL45-46 of HVT gene regions and insert the serve as a mark positive colony of gene of galK, for the present invention, to complete people prepared at british animal health research.Li, et al. Vaccine, the main construction step of 2011:8257-8266. is as follows: first primer (the HVT-UL45-galK-F:5'-CCAGTGTCCAAGGTAGAACATATTTTACGTAGGCTGA AAGTGTCCAGCGAATAATTAACCTGTTGACAATTAATCATCGGCA-3' with the UL45 with HVT and UL46 partial sequence; HVT-UL46-galK-R:5'-GGATCTTTATTTCATATAATGTATTATACAGACCGCG CATCGCGTGTGTTTATCAGCACTGTCCTGCTCCTT-3'), then according to the Recombineering protocol #3(S ren Warming of Invitrogen company) carry out homologous recombination and pick out positive colony.

4. main agents

1 × M9 nutrient solution: take 6 g Na ₂hPO ₄, 3 g KH ₂pO ₄, 1 g NH ₄cl and 0.5 g NaCl are dissolved in 800 mL ddH ₂in O, fully after stirring and dissolving, add ddH ₂solution is settled to 1 L by O, and 121 DEG C of autoclaving 20 min are placed on 4 DEG C and save backup.

5 × M63 nutrient solution: take 10 g (NH ₄) ₂sO ₄, 68 gKH ₂pO ₄with 2.5 mgFeSO ₄7H ₂o, is dissolved in 800 mL ddH ₂in O, fully, after stirring and dissolving, with KOH adjusting pH value to 7.0, add ddH ₂solution is settled to 1 L by O, and 121 DEG C of autoclaving 20 min are placed on 4 DEG C and save backup.

0.2 mg/mL vitamin H: the vitamin H that takes 10mg is dissolved in 50 mLddH ₂in O, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, preserve 4 DEG C for subsequent use.

20% 2-deoxidation-semi-lactosi: the 2-deoxidation-semi-lactosi that takes 4 g is dissolved in 20 mL ddH ₂in O, after dissolving completely, 121 DEG C of autoclaving 20 min are placed on 4 DEG C and save backup.

20% glycerine: measure 20 mL glycerine and 80 mL ddH ₂o, after being mixed, 121 DEG C of autoclaving 20 min are placed on 4 DEG C and save backup.

10 mg/mL L-Leus: take 500 mg L-Leus and be dissolved in 50 mL ddH ₂o, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, preserve 4 DEG C for subsequent use.

10 mg/mL ILEs: take 500 mg ILEs and be dissolved in 50 mL ddH ₂o, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, preserve 4 DEG C for subsequent use.

10 mg/mL Valines: take 500 mg Valines and be dissolved in 50 mL ddH ₂o, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, preserve 4 DEG C for subsequent use.

50 mg/mL paraxin: take 2g paraxin and be dissolved in 40 mL ethanol, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, be distributed into tubule, preserve-20 DEG C for subsequent use.

C25+LB liquid nutrient medium: in LB liquid nutrient medium, add 50 mg/mL paraxin, after mixing, preserve 4 DEG C for subsequent use.

1M MgSO ₄7H ₂o: take 2.46 g MgSO ₄7H ₂o is dissolved in 10 mL ddH ₂o, completely dissolve after, after the membrane filtration degerming with 0.22 μ m, preserve 4 DEG C for subsequent use.

1000 × MM1: the ZnSO that adds successively 0.5 M ₄, CuSO ₄, Na ₂moO, CoCl ₂, MnSO ₄, CrCl ₃and NiCl ₂, each reagent adds completely and adds lower one after dissolving again, all adds after it dissolves completely, and 121 DEG C of autoclaving 20min are placed on 4 DEG C and save backup.

DOG culture plate: in the bottle of 2 L, take 15 g agar and be dissolved in 800 mL ddH ₂o, after dissolving completely, 121 DEG C of autoclaving 20 min.After cooling a little, add the autoclaved 5 × M63 nutrient solution of 200 mL and 1 mL MgSO ₄7H ₂o, uses ddH ₂o adjusts volume to 1 L.When temperature drops to approximately 50 DEG C of left and right, add successively 5 mL 0.2 mg/mL vitamin Hs, 4.5 mL 10 mg/mL L-Leus, 4.5 mL 10 mg/mL ILEs, 4.5 mL 10 mg/mL Valines, 10 mL 20% 2-deoxidation-semi-lactosis, 10 mL 20% glycerine, 1 mL 1000 × MM1 and 500 μ L 50mg/mL paraxin.After fully mixing, start a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, the quantity of a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is 25 ~ 40.

For chick embryo fibroblast cultivate, the nutritive medium M199, foetal calf serum of growth and digestion and 0.25% pancreatin be all purchased from Hyclone company.

structure and the qualification of embodiment 1 recombinant hvt virus

one, experimental technique

1. pmpDthe clone of-N gene

according to the sequence of the chlamydia psittaci of having delivered in GenBank, design and synthesize pmpDthe a pair of expression primer of-N, its upstream primer 5 ' end comprises KpnI restriction enzyme site and kozak sequence, downstream 5 ' end comprises EcoRV restriction enzyme site sequence. pmpDEFK：5′-CGG GGTACC GCCGCCACCATGGGATCCAATGTGTTGATTTCTGGAA-3′

pmpDER：5′-AAA GATATCTCAAACAGCCCCACCTGTAGGAGCA-3′

Utilize above-mentioned primer and high-fidelity pfu DNA synthetic enzyme, go out to contain kozak sequence taking CB7 DNA as template pcr amplification pmpD-N, and by this gene clone in pZeroBack carrier.Adopt KpnI/EcoRV double digestion, said gene fragment is cloned into respectively in eukaryon expression plasmid pVAX1 and pEF6/V5-His, build recombinant expression plasmid, called after pV- pmpDk and pE- pmpDk.

2. the structure of recombinant hvt transfer vector

According to pV- pmpDk and pE- pmpDthe gene order of K recombinant plasmid, designs and synthesizes respectively pV- pmpDk and pE- pmpDthe CMV-of the eukaryotic expression box of K pmpD-BGH and EF- pmpDtwo pairs of amplimers of-BGH gene fragment, these two pairs of primers contain PacI restriction enzyme site sequence simultaneously.Wherein CMV- pmpD-BGH gene fragment 5 ' end has CMV promotor, and 3 ' end has BGH PolyA tail; EF- pmpD-BGH gene fragment 5 ' end has EF-1 α promotor, and 3 ' end has BGH PolyA tail.Primer sequence is respectively: CMV- pmpD-BGH(CMVF:5 '-GTTCCGCG tTAATTAAcTTACGGTA-3 ', CMVR:5 '-TG tTAATTAAgGTTCGCTTGCTGT-3 '); EF- pmpD-BGH(EFF:5 '-GG tTAATTAAcCTTCTAGGTCTTGAAAGGAGTGGGA-3 ', EFR:5 '-GG tTAATTAAgCCTCAGAAGCCATAGAGCCCACC-3 ') italics mark PacI restriction enzyme site sequence.

First above-mentioned amplified fragments is cloned in pZeroBack carrier, then utilizes pacI restriction enzyme site that above-mentioned fragment is connected with dephosphorylized pUL45-46/pacI, construct recombinant transfer vector called after pHVT-UL45-CMV-respectively pmpD-uL46 and pHVT-UL45-EF – pmpD-uL46.

3. contain the structure of the recombinant hvt BAC of pmpD gene

Utilize restriction enzyme PvuII and NruI to transfer vector pHVT-UL45-CMV- pmpD-uL46 and pHVT-UL45-EF – pmpD-uL46 carries out respectively double digestion, then the endonuclease bamhi electricity of purifying is transformed to (conversion condition: 1800kv, 200 Ω, 25 μ F) and contains HVT – galK BAC intestinal bacteria SW105 competent cell, makes UL45-CMV- pmpD-BGH-UL46 and UL45-EF-1 α- pmpDthere is homologous recombination to replace galK gene with HVT-BAC-galk respectively in-BGH-UL46, then utilizes PCR to screen and identify the bacterium colony of growing in oppositely selecting substratum DOG, obtains recombinant C MV- pmpDwith EF – pmpDthe HVT BAC of expression cassette.Adopt the Maxi prep of Qiagen company test kit to extract and identify that correct recombinant hvt BAC DNA is ready for use on the rescue of recombinant virus.

4. express rescue and the vitro characteristics qualification of the recombinant hvt of pmpD gene

Adopt the Lipofectamine LTX of invitrogen company test kit, above-mentioned recombinant hvt BAC DNA transfection is grown up to 80% ~ 90% chick embryo fibroblast.After transfection the 4th day, with the cell of transfection under 0.25% trysinization, and is inoculated into off-the-shelf growth and has in 80% the T75 cell bottle of chick embryo fibroblast.After about 3 ~ 4 d, can see obvious HVT cytopathic effect, set it as first-generation seed poison P1.And the expansion that continues to go down to posterity is malicious, and to P3, for obtaining, needed kind is malicious to be measured, and in liquid nitrogen, saves backup.

4.1 express gene structure and the repeated pruning of the recombinant hvt of pmpD gene

The DNA of the recombinant hvt extracting with QIAgen DNA test kit, adopts respectively pmpDthe above-mentioned DNA of-N primer carries out pcr amplification, and identifies that with same method P5, P10, P15, P20 are for seed poison pmpDthe existence of-N, determines that this gene inserts the stability of HVT thus.

4.2 recombinant hvt expressthe qualification of pmpD albumen

Adopt the goat-anti chicken IgG of the anti-chlamydozoan bacterial strain of the chicken 6BC serum of the 1:100 dilution of having prepared and the HRP mark of 1:5000 dilution, respectively to rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpDthird generation cell toxicant carries out western blot and analyzes in the chick embryo fibroblast that is subject to recombinant hvt infection pmpDthe expression of albumen.In addition, utilize the anti-PmpD-N mouse serum of 1:100 dilution and anti-HVT chicken serum to clean to growing in the cellmediated immunity fluorescent test (IFA) that is subject to above-mentioned recombinant virus on cover glass.With confocal microscopy pmpDprotein expression and distribution situation.Further P5, P10, P15, P20 are carried out to IFA qualification pmpDthe stability of protein expression.

The mensuration of 4.3 recombinant hvt virus titers and preservation period are determined

To the rHVT-CMV-preserving under-80oC condition pmpDcell toxicant, according to 10 ^-3, 10 ^-4with 10 ^-5after three extent of dilution inoculation CEF cell 4 d, then the cell through PBS rinsing with the acetone of precooling, carry out after the each 1h of immune response with the goat-anti chicken IgY of the anti-HVT serum of chicken of 1:100 and the HRP mark of 1:5000 again, utilize AEC test kit to dye, observe plaque size, plaque form, and count PFU.Further, in the time of three months, six months, nine months and 1 year, measure the titre of recombinant virus in different time by calculating PFU quantity, to determine its preservation period.

two, experimental result

1. the western blot qualification result of recombinant hvt

By rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpDthird generation cell toxicant carries out western blot qualification.Western blot qualification result shows two HVT recombinant chou (rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpD) all there is object band at about 43 kDa places, size conforms to expection, and control cells does not have (Fig. 1).

2. the IFA qualification result of recombinant hvt

By rHVT-CMV-pmpD and rHVT-EF-1 α-pmpD third generation cell toxicant inoculation CEF cell, after 3 d, adopt IFA to identify and in conjunction with confocal microscopy.Result shows: obvious fluorescence appears in the CEF of rHVT-CMV-pmpD and rHVT-EF-1 α-pmpD recombinant virus infection, and the PmpD albumen of expressing is mainly distributed in the surface and cytoplasm of cell (Fig. 2).

3. recombinant hvt causes the mensuration of the morphologic observation of CEF plaque and titre

For observing more intuitively the cytopathic effect of recombinant hvt and the malicious valency of detection HVT recombinant chou, the generation that needs are detected is according to 10 ^-3, 10 ^-4with 10 ^-5after three extent of dilution inoculation CEF cell 4 d, carry out plaque test and detect.The plaque form (Fig. 3) that HVT recombinant chou and wild-type HVT produce is basically identical.Wild-type HVT, rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpDthe malicious valency in the 4th generation is basically identical, is respectively 1 × 10 ⁶pFU/mL, 8 × 10 ⁵pFU/mL and 1.2 × 10 ⁶pFU/mL.

4. the one step growth of rHVT-CMV-pmpD

By rHVT-CMV- pmpDinoculate CEF cell with wild-type HVT with 400 PFU simultaneously, collect the cell of metainfective 12 h, 24 h, 48 h, 72 h, 96 h and 120 h, extract cell DNA, the method for the fluorescent quantitation of setting up according to chapter 3 detects the viral level of different time.Detected result (Fig. 4) shows, rHVT-CMV- pmpDclose with the rate of propagation of wild-type HVT, in the time of 48 h, start to be increased significantly, be increased logarithmic phase at 72 h during to 96 h, until 120 h are raising always.

5. the stability test of rHVT-CMV-pmpD

By rHVT-CMV- pmpDon CEF cell, carry out 20 generations of continuous passage, in every 5 generations, adopt pmpD-N Auele Specific Primer carries out PCR testing goal gene.PCR qualification result (Fig. 5) shows, in the process going down to posterity pmpD-N gene is not lost, all-the-time stable heredity to 20 generation.The result (Fig. 6) that every five generations adopt IFA to detect shows, rHVT-CMV- pmpDin the time passing to for 20 generation, albumen still can be expressed.

6. the preservation period of rHVT-CMV-pmpD test

The frozen third generation rHVT-CMV-at-80 DEG C pmpDcell toxicant takes out respectively one and carries out plaque test in the time of three months, six months, nine months and 1 year, calculates PFU.Result (table 1) shows, the malicious valency titre that can decline after a year.

Table 1 is kept at the third generation rHVT-CMV-of-80 DEG C pmpDcell toxicant is preserved the malicious valency of different time and is measured

Table 5-6 Titration of rHVT-CMV- pmpD working virus stored at -80°C for different time

Above result shows to utilize BAC system successfully to save rHVT-CMV- pmpDwith rHVT-EF-1 α- pmpDtwo kinds of recombinant viruses, Western blot shows that the target protein expression level under the control of CMV promotor is higher than the expression level under the control of EF-1 α promotor, and rHVT-CMV- pmpDcompared with wild-type HVT, duplication characteristic does not change.

the immunoprotection experiment of embodiment 2 recombinant hvt viruses

one, experimental technique

1. recombinant hvt is attacked malicious immune protective test for MDV

The SPF chicken of one age in days is divided into 3 groups at random, 10 every group.First group is control group, immune CEF cell, and cell count is 1.3 × 10 ⁵/ only.Distinguish immune wild-type HVT and rHVT-for second group and the 3rd group pmpD, dosage is only 8000PFU/.Immunization route is all that back neck is subcutaneous.Latter 8 days of immunity, malicious RB1B-BAC cell toxicant is attacked in abdominal cavity, and attacking toxic agent amount is 1000 PFU/.After attacking poison, observe at any time the clinical symptom that MDV is relevant, if there is dead chicken to cut open at any time inspection, observe pathological change midway; attacking 60 d after poison; all not dead chickens are all cutd open inspection, record the pathological change that MDV is relevant, calculate the protection efficiency of vaccine.

2. recombinant hvt is attacked malicious immune protective test for chlamydia psittaci CB7

The SPF chicken of one age in days is divided into 3 groups at random, and first group 10 is control group, immune CEF cell, and cell count is 1.3 × 10 ⁵/ only.Second group 10, immune wild-type HVT, dosage is 8000PFU/.The 3rd group 15, immune rHVT- pmpD, dosage is 8000 PFU/.Immunization route is all that back neck is subcutaneous.The 36th d after immunity, attacks poison to the chicken of all groups, and every chicken larynx is inoculated 5 × 10 of 0.1 mL ^8.5the chlamydia psittaci CB7 strain of TCID50.Attack the rear pathological change of observing clinical symptom and dead chicken every day of poison, at the 4th day, all chickens are carried out to euthanasia, observe pathological change, according to recording score situation shown in table 1.Within the 4th day, gather the larynx swab of all chickens, in BGM cell, cultivate, adopt the direct fluorescent dye test kit of Dako to dye, calculate toxin expelling score situation.The method of calculation of larynx swab chlamydozoan toxin expelling situation score are: 400 × microscope under 5 visuals field of random counting, if do not have chlamydial inclusion body to appear as 0 point, observing 1-2 inclusion body is 1 point, observing 3 ~ 5 inclusion bodys is 2 points, and observing >6 inclusion body is 3 points.Attacking poison latter the 4th day, gather spleen and the lungs of all groups, carry out cell cultures, calculate the carrying capacity of chlamydozoan in internal organs, the same swab of method of calculation of score situation.

Table 2 lesion tissue situation grade form

Table1 Scoresystem for Chlamydia psittaci gross lesions in chickens

Tissue	0 point (-)	1 point (+)	2 points (+)	3 points (+)
					Lung	Normally	Slight congested	Serious congested	Serious hyperemia+grey inflammatory lesions
Chest bladder	Normally	Diffusivity muddiness	A small amount of fibrin deposition	Serious fiber disposition airsacculitis
					Belly bladder	Normally	Diffusivity muddiness	A small amount of fibrin deposition	Serious fiber disposition airsacculitis
Spleen	Normally	Slight enlargement	Moderate swelling	Serious enlargement

two, result

1. recombinant hvt is attacked malicious immune protective test for MDV

Carry out the situation of protection ratio judges by observing clinical symptom, pathological change and the death condition relevant to MDV.Result (table 3) shows, immunity wild-type HVT and rHVT- pmpDgroup, all chickens are all healthy attacking 60 d after poison, do not have clinical symptom that MDV is relevant and pathological change to occur.And CEF control group has two chickens to occur dead, there is typical clinical symptom in three chickens, cuts open inspection and find that 10 chickens all have typical organ damage and tumour to occur.Show rHVT- pmpDafter immunity chicken, can produce protection completely to the attack of strong malicious MDV, the protection ratio (100%) with wild-type HVT to chicken is the same.

The respectively immune CEF contrast of table 3, wild-type HVT and rHVT- pmpDafter the strong malicious RB1B strain protest test result of MDV of carrying out

Vaccine	There is chicken number/total chicken number of tumour	Organ damage/total chicken number	Clinical symptom/total chicken number	Dead chicken number/total chicken number	Protection ratio
						rHVT- pmpD	0/10	0/10	0/10	0/10	10/10(100%)
Wild-type HVT	0/10	0/10	0/10	0/10	10/10(100%)
						CEF contrast	10/10	10/10	3/10	2/10	0/10 (0%)

2. recombinant hvt is attacked malicious immune protective test for MDV

The 36th d after immunity, attacks malicious chlamydia psittaci CB7 strain to the chicken of all groups.Attack the rear pathological change of observing clinical symptom and dead chicken every day of poison, at the 4th day, all chickens are carried out to euthanasia, observe pathological change.Score situation result (Fig. 7) shows, lungs, air bag and the spleen of all chickens of CEF control group and wild-type HVT group have obviously serious pathological change, rHVT- pmpDthe saccus abdominalis of all chickens of group and spleen are all normal, and the score of lungs and torso bag has obvious minimizing, and wherein to have the lungs of 3 chickens be normal, and the torso bag of 4 chickens is normal; And the score of CEF control group and wild-type HVT group does not have notable difference.Therefore, rHVT- pmpDvaccine has good protection for chicken.

Attack the rear larynx swab that gathers all groups of chickens on the 4th day of poison, adopt BGM cell to carry out separation and Culture.The score calculated case result (Fig. 8) of larynx swab shows, the chicken discharge of bacteria situation of CEF control group and wild-type HVT group does not have notable difference, rHVT- pmpDthe chicken discharge of bacteria situation that the chicken toxin expelling of group is compared CEF control group and wild-type HVT group obviously reduces.

Attack after malicious 4 d, carry out the bacterial loads analysis of lung and spleen, analytical results (Fig. 9) shows, rHVT- pmpDthe average of the bacterial loads of group has obvious minimizing with respect to CEF control group and wild-type HVT group.

Above result shows, the rHVT-CMV-that the present invention builds pmpDafter recombinant live-vector vaccine immunity chicken, can obtain good cellular immunization and Humoral.This vaccine is attacked protection can be provided completely MDV, and can obviously alleviate the relevant diseases of chlamydia psittaci, reduces the discharge of bacteria of chlamydia psittaci and residual at target organ, can be used as candidate's bigeminy vaccine of opposing chlamydia psittaci and MDV.

Sequence table

<110> Beijing City Agriculture and Forestry Institute

<120> expresses HVT recombinant chou and the structure thereof of chlamydia psittaci pmpD-N gene

<130> PN1306369-02

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 37

<212> DNA

<213> artificial sequence

<400> 1

ccggaattca tgggatccaa tgtgttgatt tctggaa 37

<210> 2

<211> 34

<212> DNA

<213> artificial sequence

<400> 2

ccgctcgagt caaacagccc cacctgtagg agca 34

<210> 3

<211> 26

<212> DNA

<213> artificial sequence

<400> 3

taaacgtaga ttgaaaaggg cgaagc 26

<210> 4

<211> 27

<212> DNA

<213> artificial sequence

<400> 4

tcagcttaat taactgtcct gctcctt 27

<210> 5

<211> 27

<212> DNA

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<400> 5

aaggagcagg acagttaatt aagctga 27

<210> 6

<211> 25

<212> DNA

<213> artificial sequence

<400> 6

cacaaaatcg ctggggtata tcata 25

<210> 7

<211> 82

<212> DNA

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<400> 7

ccagtgtcca aggtagaaca tattttacgt aggctgaaag tgtccagcga ataattaacc 60

tgttgacaat taatcatcgg ca 82

<210> 8

<211> 72

<212> DNA

<213> artificial sequence

<400> 8

ggatctttat ttcatataat gtattataca gaccgcgcat cgcgtgtgtt tatcagcact 60

gtcctgctcc tt 72

<210> 9

<211> 46

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cggggtaccg ccgccaccat gggatccaat gtgttgattt ctggaa 46

<210> 10

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<400> 10

aaagatatct caaacagccc cacctgtagg agca 34

<210> 11

<211> 25

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gttccgcgtt aattaactta cggta 25

<210> 12

<211> 24

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tgttaattaa ggttcgcttg ctgt 24

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<211> 36

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ggttaattaa ccttctaggt cttgaaagga gtggga 36

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<211> 34

<212> DNA

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ggttaattaa gcctcagaag ccatagagcc cacc 34

Sequence table

<110> Beijing City Agriculture and Forestry Institute

<120> expresses HVT recombinant chou and the structure thereof of chlamydia psittaci pmpD-N gene

<130> PN1306369-02

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 37

<212> DNA

<213> artificial sequence

<400> 1

ccggaattca tgggatccaa tgtgttgatt tctggaa 37

<210> 2

<211> 34

<212> DNA

<213> artificial sequence

<400> 2

ccgctcgagt caaacagccc cacctgtagg agca 34

<210> 3

<211> 26

<212> DNA

<213> artificial sequence

<400> 3

taaacgtaga ttgaaaaggg cgaagc 26

<210> 4

<211> 27

<212> DNA

<213> artificial sequence

<400> 4

tcagcttaat taactgtcct gctcctt 27

<210> 5

<211> 27

<212> DNA

<213> artificial sequence

<400> 5

aaggagcagg acagttaatt aagctga 27

<210> 6

<211> 25

<212> DNA

<213> artificial sequence

<400> 6

cacaaaatcg ctggggtata tcata 25

<210> 7

<211> 82

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<400> 7

ccagtgtcca aggtagaaca tattttacgt aggctgaaag tgtccagcga ataattaacc 60

tgttgacaat taatcatcgg ca 82

<210> 8

<211> 72

<212> DNA

<213> artificial sequence

<400> 8

ggatctttat ttcatataat gtattataca gaccgcgcat cgcgtgtgtt tatcagcact 60

gtcctgctcc tt 72

<210> 9

<211> 46

<212> DNA

<213> artificial sequence

<400> 9

cggggtaccg ccgccaccat gggatccaat gtgttgattt ctggaa 46

<210> 10

<211> 34

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<400> 10

aaagatatct caaacagccc cacctgtagg agca 34

<210> 11

<211> 25

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<400> 11

gttccgcgtt aattaactta cggta 25

<210> 12

<211> 24

<212> DNA

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<400> 12

tgttaattaa ggttcgcttg ctgt 24

<210> 13

<211> 36

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ggttaattaa ccttctaggt cttgaaagga gtggga 36

<210> 14

<211> 34

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ggttaattaa gcctcagaag ccatagagcc cacc 34

Claims (7)

1. a recombinant virus, it is by chlamydia psittaci pmpD-N gene is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

2. recombinant virus according to claim 1, is characterized in that, it is by chlamydia psittaci pmpDthe expression cassette that-N gene, CMV promotor and BGH polyA transcription termination signal form is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

3. recombinant virus according to claim 1, is characterized in that, it is by chlamydia psittaci pmpDthe expression cassette that-N gene, pEF-1 α promotor and BGH polyA transcription termination signal form is inserted into can expressing of obtaining in the genome of HVT pmpDthe recombinant hvt virus of-N albumen.

4. the recombinant virus described in claim 1 ~ 3 any one is in the application in vaccine for fowl of preparation bigeminy.

5. the construction process of recombinant virus described in claim 1, it comprises the following steps: structure can be expressed chlamydia psittaci PmpD-N protein expression box under CMV or EF-1 α promoter regulation, then by the method for homologous recombination, its expression cassette is inserted in HVT bacterial artificial chromosome, obtain restructuring pmpDthe HVT BAC of-N gene, then BAC DNA transfection chick embryo fibroblast is saved out to recombinant virus, obtain HVT recombinant virus.

6. construction process according to claim 5, is characterized in that, is pVAX1 or pEF6/V5-His for the plasmid of construction expression chlamydia psittaci PmpD-N protein expression box.

7. according to the construction process described in claim 5 or 6, it is characterized in that, is pVAX1 for the plasmid that builds chlamydia psittaci PmpD-N protein expression box.