CN102181404A - Recombinant adenovirus expressing E0, E2 gene of classical swine fever virus - Google Patents

Abstract

The invention provides recombinant adenovirus expressing E0, E2 genes of classical swine fever virus, which is characterized in that the E0 gene of classical swine fever virus is inserted into Bgl II and Kpn I enzyme sites of a multiple cloning site at downstream of adenovirus CMV promoter, and a adenovirus CMV promoter and a CMV-E2 segment of the E2 gene of classical swine fever virus are inserted into Xho I and Xba I enzyme sites. The invention solves the problems of low yield and weak protective effect of the fusion tandem expression of the E0, E2 genes, allows an immunized swine to obtain an antibody against classical swine fever virus only by immunization once, and the production cost is half of that of conventional vaccines; the recombinant adenovirus belongs to non-replicative virus, and does not propagate in swine; the phenomenons of virus atavism and virulence enhancement due to the long-term inoculation of attenuated vaccine are solved. The recombinant adenovirus only comprises main protective genes of classical swine fever virus and does not comprises other genes of the virus, so no recombination with field strains occurs. After a swine is immunized by vaccine prepared by the recombinant adenovirus, the protective effect against live virus infection of classical swine fever virus with a lethal dose of 100 times of TCID 50 is 100%.

Description

A kind of recombinant adenovirus of expressing Pestivirus suis E0, raq gene

Technical field

The present invention relates to a kind of recombinant adenovirus, the especially a kind of recombinant adenovirus that can express Pestivirus suis E0 gene and raq gene in same adenovirus belongs to biological technical field.

Background technology

Swine fever is the very strong transmissible disease of a kind of infectivity, often causes crushing loss to pig industry.At present, immunization is still the major measure of swine fever control, and is to eradicate the swine fever disease and stop the necessary means of wild poison biography to the sick swinery of no swine fever.No matter in the past, still in the future now swine Fever Vaccine all has important role to control and elimination swine fever disease, and the various countries scholar never abandons the development and the exploitation of swine Fever Vaccine.Traditional swine Fever Vaccine has inactivated vaccine and attenuated vaccine, wherein the deactivation vaccine immune efficacy is low, protection period is short, it is slow that excitating organism produces the speed of immunizing power, up to the present, the output of tissue culture swine fever poison and viral effective antigenic loss that inactivated vaccine caused are still the two big obstacles of making inactivated vaccine; The widespread use of swine fever attenuated vaccine, played keying action for the popular of control swine fever, but also there is following deficiency in attenuated vaccine: very big variation has taken place in the epidemic characteristic of swine fever at first in recent years, present periodically, corrugated provincialism is distributed and mildness swine fever disease, clinical symptom after the morbidity has nameless high-fever, atypical symptom, persistent infection, the birth piglet is congenital trembles and sow reproductive ability obstacle, usually cause immuning failure with the traditional vaccine immunity, even being increased to 750 rabbit precursor reactants (150 rabbit precursor reactants of national regulation), dosage can not prevent its infection, and have the tendency to spread, in the early stage vaccination of gestation vaccine virus also can take place by the placental infection fetus, cause stillborn foetus or weak son and become the persistence carrier, produce new epidemic disease source; Secondly, attenuated vaccine was continued to use nearly 40 years, wide variation have taken place in the popular form of swine fever, illustrate that the antigenicity of Pestivirus suis may exist variation, and particularly the domestic and international application monoclonal antibody detects the C-strain in recent years, discovery has different reaction patterns, show that the C-strain has produced variation through the domestication on different cells for many years, the domestic and international situation basically identical of the variation tendency of this epidemic isolates, popular poison is often escaped immunity, causes the serious consequence of immuning failure; The 3rd, the inoculation attenuated vaccine also can have influence on international trade, European Union has banned use of the swine fever attenuated vaccine, and the strict health and epidemic prevention system of employing, slaughter the positive pig of swine fever infected pigs and serology and control and eliminate swine fever, the employing policy of slaughtering needs great amount of manpower and material resources and a huge sum of money, and developing country is difficult to bear, and this comes this disease of prevention and control with regard to impelling people to research and develop novel swine Fever Vaccine.In recent years, the fast development of genetic engineering technique, for the research and development of novel swine Fever Vaccine provides instrument, at present, the development of hog cholera genetic engineering bacterin has become new research direction and focus.

People-5 type adenovirus carrier is compared with other animal virus vectors, have many advantages: at first be safety, adenovirus toxicity is low, substantially not pathogenic or only cause slight symptom, after the adenovirus reorganization, virulence further reduces, and has overcome retroviral vector and may cause inserting the potential risk of sudden change because of random integration; Next is that host range is wide, and adenovirus can infect various kinds of cell and comprise division and Unseparated Cell; The 3rd is stable, and adenovirion is very firm, is difficult for sudden change, and a large amount of easily preparation and purifying, obtains the virus of high titre, obtains 10 external ¹¹Pfu/mL; The 4th studies more clearlyly for the 26S Proteasome Structure and Function of adenoviral gene group exactly at present, and genetic manipulation is convenient; The 5th is that insertion foreign gene capacity is big, make up the adenovirus carrier that adopts disappearance E1 and E3 district gene in the recombiant vaccine mostly in gene therapy, usually replication-defective adenoviral can hold the foreign gene of about 7kb～8kb, and is more a lot of greatly than the capacity of retrovirus and adeno-associated virus; Be exactly that immunization route is easy in addition, adenovirus can be in digestive tube and respiratory tract propagation, and vaccine inoculation is easy, can be oral or aerosol suck and need not inject, and can produce local mucosal immune response after the oral administration, better than other vaccine on immunization method, promote the use of easily.

The non-replicating adenovirus is not only safer and more effective, and produces antigen protein and lysing cell not in the mode of low dosage, so the time of antigen expressed is more lasting, helps exciting secular immune response.At present, there has been multiple product to go on the market, for example: " An Kerui---recombinant human 5 type adenovirus injection liquids "; " recombinant human gamma-interferon adenovirus injection liquid (E10B) "; " Gendicine (reorganization human P 53 adenovirus injection liquid) "; " research of safe evaluation method before the human and bird fluenza recombinant adenovirus vaccine is clinical " achieves initial success; " recombinant adenovirus-thymidine kinase gene preparation "; " porcine reproductive and respiratory syndrome recombinant adenovirus and vaccine " applied for a patent by Agricultural University Of Nanjing in addition.

Summary of the invention

Problems such as reorganization take place for solving long-term viral reversion, virulence enhanced phenomenon and the attenuated vaccine strain and the wild poison that cause inoculated of swine fever attenuated vaccine; and Pestivirus suis E0, raq gene yielding poorly of merging that tandem expression exists and protect the weak problem of effect, the invention provides a kind of recombinant adenovirus that can in same adenovirus, express Pestivirus suis E0, raq gene.

The present invention is to provide a kind of like this recombinant adenovirus of expressing Pestivirus suis E0, raq gene, it is characterized in that in the Bgl II of the CMV of adenovirus promotor downstream multiple clone site and KpnI restriction enzyme site, inserting Pestivirus suis E0 gene, in Xho I and XbaI enzyme cutting site, insert the CMV-E2 fragment of adenovirus CMV promotor and Pestivirus suis raq gene.

Described Pestivirus suis is conventional commercial virus, and the accession number of E0 gene in GenBank of Pestivirus suis is: AF058715.1, the accession number of the raq gene of Pestivirus suis in GenBank is: AY775178.2

Described adenovirus is conventional commercial people-5 type non-replicating adenovirus.

Described recombinant adenovirus of expressing Pestivirus suis E0, raq gene in adenovirus simultaneously respectively comprises following construction step:

A. from commercial Pestivirus suis and Pestivirus suis, amplify E0 and raq gene respectively;

B. the Pestivirus suis E0 gene that steps A is obtained is inserted among the adenovirus shuttle vector pAdTrack by BglII and KpnI restriction enzyme site, forms adenovirus shuttle vector pAdTrack-E0;

C. the Pestivirus suis raq gene that steps A is obtained is inserted among the adenovirus shuttle vector pAdTrack by Kpn I and Not I restriction enzyme site, forms adenovirus shuttle vector pAdTrack-E2;

D. the adenovirus shuttle vector pAdTrack-E2 PCR method routinely with step C amplifies the CMV-E2 gene that contains the CMV promotor, be inserted into by Xhol I and Xba I restriction enzyme site among the Xhol I and Xba I restriction enzyme site among the adenovirus shuttle vector pAdTrack-E0 of step B, form recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2;

E. with the recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2 of step D behind the PmeI linearization for enzyme restriction, be transformed in the intestinal bacteria BJ5183 competent cell that contains adenovirus skeleton carrier pAd-easy-1, form recombinant adenovirus skeleton carrier pAd-easy-E0-CMV-E2;

F. with recombinant adenovirus skeleton carrier pAd-easy-E0-CMV-E2 after the PacI linearizing, among the transfection human embryonic kidney cell HEK293, pack out recombinant adenovirus rAd-E0-CMV-E2.

The present invention has following advantage and effect:

The recombinant adenovirus rAd-E0-CMV-E2 that adopts such scheme to obtain; can in same adenovirus, express Pestivirus suis E0 albumen and Pestivirus suis E2 albumen respectively; having solved E0, raq gene merges tandem expression and yields poorly and protect the weak problem of effect; the present invention only just can make pig after the immunity obtain antibody to Pestivirus suis by one shot immunity; swine fever had stronger resistivity; production cost is half of conventional vaccine, greatly reduces the vaccine cost.Recombinant adenovirus rAd-E0-CMV-E2 provided by the invention belongs to non-replicating virus, does not breed in the pig body, has solved the viral reversion that the long-term inoculation of attenuated vaccine causes, virulence enhanced phenomenon.The main protecting group that recombinant adenovirus rAd-E0-CMV-E2 provided by the invention only contains Pestivirus suis because of, do not contain other gene of virus, therefore, can not recombinate between recombinant adenovirus provided by the invention and the street strain.The vaccine that the recombinant adenovirus that adopts such scheme to make up to obtain is made, behind the immune swine to 100 times of TCID of lethal quantity ₅₀The protection effect of Pestivirus suis strong virus attack be 100%.Virulence was returned strong problem after both having solved the wild malicious homologous recombination of swine Fever Vaccine and swine fever, had also solved the high problem of attenuated vaccine production cost.

Description of drawings

Fig. 1 is the level of the Pestivirus suis antibody of ELISA detection.

Embodiment:

The present invention will be further described below in conjunction with embodiment.

Embodiment

A. the amplification of Pestivirus suis E0 gene and raq gene

(1) design of pcr amplification primer

Add Bgl II and Kpn I restriction enzyme digestion sites in the upstream and downstream primer of Pestivirus suis E0 gene respectively, the upstream and downstream primer of described Pestivirus suis E0 gene is designed to respectively:

Upstream primer P1:5 '-AAA AGATCTATGGAAAATATAACTCAATGG-3 '

Downstream primer P2:5 '-CAC GGTACCGTAAGGCGATAGGGCATAG-3 '

Add Kpn I and NotI and restriction enzyme digestion sites in the primer of the upstream and downstream of Pestivirus suis raq gene respectively, the upstream and downstream primer of described Pestivirus suis raq gene is designed to respectively:

Upstream primer P1:5 '-AAA GGTACCATGAGGGGACAGATCGTGC-3 '

Downstream primer P2:5 '-CCC GCGCCCGCACCAGCGGCGAGTTGTTCTG-3 '

(2) recovery of the amplification in vitro of Pestivirus suis E0 gene fragment and amplified production and purifying

The pMD18-T-E0 plasmid that will contain Pestivirus suis E0 gene dilutes 100 times with distilled water, and getting 1 μ L is template, and with the proteic purpose fragment of primer amplification coding E0 of Pestivirus suis E0 gene, wherein the PCR reaction system is as follows:

Template 1 μ L

10 * PCR damping fluid, 5 μ L

2.5mmol/L?dNTP 5μL

E0 upstream region of gene primer (50 μ mol/L) 2 μ L

E0 gene downstream primer (50 μ mol/L) 2 μ L

Ex Taq archaeal dna polymerase (5U/ μ L) 1 μ L

Sterilization deionized water 34 μ L

Reaction totally is 50 μ L

PCR reaction conditions: 95 ℃ of pre-sex change 5min; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of storages; The PCR product is placed the 8g/L sepharose, behind 60V electrophoresis 20min, cut the fragment of purpose stripe size, reclaim the amplified production of Pestivirus suis E0 gene;

(3) recovery and the purifying of segmental amplification in vitro of Pestivirus suis raq gene and amplified production

The pMD18-T-E2 plasmid that will contain the Pestivirus suis raq gene dilutes 100 times with distilled water, and getting 1 μ L is template, and with the proteic purpose fragment of primer amplification coding E2 of Pestivirus suis raq gene, wherein the PCR reaction system is as follows:

Template 1 μ L

10 * PCR damping fluid, 5 μ L

2.5mmol/L?dNTP 5μL

Raq gene upstream primer (50 μ mol/L) 2 μ L

Raq gene downstream primer (50 μ mol/L) 2 μ L

Ex Taq archaeal dna polymerase (5U/ μ L) 1 μ L

Sterilization deionized water 34 μ L

Reaction totally is 50 μ L

PCR reaction conditions: 95 ℃ of pre-sex change 5min; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min 30sec, totally 35 circulations; Last 72 ℃ are extended 10min, 4 ℃ of storages; The PCR product is placed the 8g/L sepharose, behind 60V electrophoresis 20min, cut the fragment of purpose stripe size, reclaim the amplified production of Pestivirus suis raq gene;

B. the structure of adenovirus shuttle vector pAdTrack-E0

(1) the Bgl II of the pcr amplification product of Pestivirus suis E0 gene and Kpn I double digestion

In 1.5mL Eppendorf pipe, add successively:

E0 gene PCR product 15 μ L

10×Buffer 5μL

Bgl?II 3μL

Kpn?I 3μL

H ₂O 24μL

Cumulative volume is 50Ml, and is fully instantaneous centrifugal behind the mixing

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(2) the Bgl II of shuttle vectors pAdTrack-CMV and Kpn I double digestion

In 1.5mL Eppendorf pipe, add successively:

PAdTrack-CMV plasmid 10 μ L

10×Buffer 6μL

Bgl?II 3μL

Kpn?I 3μL

H ₂O 38μL

Cumulative volume is 60 μ L, and is fully behind the mixing, instantaneous centrifugal

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(3) Pestivirus suis E0 gene and shuttle vector pAdTrack-CMV's is connected

The ligation system:

E0 gene fragment 4 μ L

pAdTrack-CMV 2μL

10×Buffer 1μL

T4DNA ligase enzyme 1 μ L

H ₂O 3μL

Cumulative volume is 10 μ L, and is fully instantaneous centrifugal behind the mixing

16 ℃ of water-baths connect spends the night;

(4) Pestivirus suis E0 gene is connected the conversion and the evaluation of product with the pAdTrack-CMV carrier

1. the preparation of DH5 α competent cell

The DH5 α that-70 ℃ of glycerine are protected kind is inoculated in and does not contain on the antibiotic LB nutrient agar, single bacterium colony that 37 ℃ of overnight incubation, picking grow fine is inoculated in new not containing in the antibiotic LB nutrient solution of disposing of 3mL, 37 ℃ of 250r/min shaking culture are spent the night, about 14h; Get 100 μ L cultures, be inoculated in the fresh LB nutrient solution of 100mL in 1: 100 ratio, the 300r/min thermal agitation is cultivated about 3h, to OD ₆₀₀Be 0.6; Aseptic condition shifts down in the 50mL centrifuge tube of bacterial cultures to two precooling, ice bath 10min; 4 ℃ of centrifugal 10min of following 1600r/min abandon supernatant, are inverted centrifuge tube and drain liquid, add the 0.1mol/L CaCl of precooling ₂Solution 10mL, the suspension bacterial sediment is put ice bath and is placed 30min; 4 ℃ of centrifugal 10min of following 1100r/min abandon supernatant, with the CaCl of 2mL 0.1mol/L precooling ₂(containing 150mL/L glycerine) resuspended thalline is sub-packed in the sterilization Eppendorf pipe of precooling then, and every pipe 100 μ L put-70 ℃ of refrigerators and preserve standby;

2. recombinant plasmid transformed competence colibacillus cell

Get a pipe competent cell and place on the mixture of ice and water, after treating to melt fully, the connection product of 10 μ L is added in the pipe, gently mixing; Ice bath 30min; Heat shock 90s in 42 ℃ of water-baths; Ice bath 3min; Add 800 μ L LB substratum, 37 ℃ of following 200～250r/min shaking culture 1h; The centrifugal 1min of 8000r/min abandons the part supernatant, retains the resuspended precipitation of supernatant about 100 μ L; Get 100 μ L mixtures and evenly coat on the LB agar plate that contains kantlex (50 μ g/mL), after treating to dry up fully, 37 ℃ of overnight incubation (12～16h); Picking list bacterium colony contains in the LB liquid nutrient medium of 50 μ g/mL kantlex in 3mL, 37 ℃ of overnight incubation; Carry out behind the alkaline lysis method of extracting plasmid that enzyme is cut, PCR and order-checking identify;

Pestivirus suis E0 gene is inserted into the recombinant plasmid called after that makes up among the shuttle vectors pAdTrack-CMV: adenovirus shuttle vector pAdTrack-E0;

C. the segmental acquisition of the structure of adenovirus shuttle vector pAdTrack-E2 and CMV-E2

(1) the Kpn I of Pestivirus suis raq gene pcr amplification product and Not I double digestion

In 1.5mL Eppendorf pipe, add successively:

Raq gene PCR product 15 μ L

10×Buffer 5μL

Kpn?I 3μL

Not?I 3μL

H2O 24μL

Cumulative volume is 50 μ L, and is fully instantaneous centrifugal behind the mixing

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(2) the Kpn I of shuttle vectors pAdTrack-CMV and Not I double digestion

In 1.5mL Eppendorf pipe, add successively:

PAdTrack-CMV plasmid 10 μ L

10×Buffer 6μL

Kpn?I 3μL

Not?I 3μL

H ₂O 38μL

Cumulative volume is 60 μ L, and is fully instantaneous centrifugal behind the mixing

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(3) Pestivirus suis raq gene and shuttle vector pAdTrack-CMV's is connected

The ligation system:

Raq gene fragment 4 μ L

pAdTrack-CMV 2μL

10×Buffer 1μL

T4DNA ligase enzyme 1 μ L

H ₂O 3μL

Cumulative volume is 10 μ L, and is fully instantaneous centrifugal behind the mixing

16 ℃ of water-baths connect spends the night;

(4) the Pestivirus suis raq gene is connected the conversion and the evaluation of product with the pAdTrack-CMV carrier

1. the preparation of DH5 α competent cell

With step B (4) 1.;

2. recombinant plasmid transformed competence colibacillus cell

With step B (4) 2.;

The Pestivirus suis raq gene is inserted into the recombinant plasmid called after that makes up among the shuttle vectors pAdTrack-CMV is: adenovirus shuttle vector pAdTrack-E2;

(5) acquisition of CMV-E2 gene

Primer according to the sequences Design of CMV promotor in pAdTrack-E2 carrier amplification CMV-E2 gene:

Upstream primer P1:5 '-AAA CTCGAGTAGTTATTAATAGTAATCAAT-3 '

Downstream primer P2:5 '-CAC TCTAGAACCAGCGGCGAGTTGTTCTG-3 '

In the primer of the upstream and downstream of CMV-E2 gene, add Xho I, XbaI and restriction enzyme digestion sites respectively, and in downstream primer, introduce terminator codon;

D. the structure of recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2

(1) the Xho I of CMV-E2 gene PCR product and Xba I double digestion

In 1.5mL Eppendorf pipe, add successively:

CMV-E2 fragment PCR products 15 μ L

10×Buffer 5μL

Xho?I 3μL

Xba?I 3μL

H2O 24μL

Cumulative volume is 50 μ L, and is fully instantaneous centrifugal behind the mixing

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(2) the Xho I of shuttle vectors pAdTrack-E0 and Xba I double digestion

In 1.5mL Eppendorf pipe, add successively:

PAdTrack-E0 plasmid 10 μ L

10×Buffer 6μL

Xho?I 3μL

Xba?I 3μL

H ₂O 38μL

Cumulative volume is 60 μ L, and is fully instantaneous centrifugal behind the mixing

Behind 37 ℃ of water-bath 6h, use the 8g/L agarose gel electrophoresis, downcut the gel that contains the purpose band and reclaim;

(3) CMV-E2 gene and shuttle vector pAdTrack-E0's is connected

The ligation system:

CMV-E2 gene fragment 4 μ L

pAdTrack-E0 2μL

10×Buffer 1μL

T4DNA ligase enzyme 1 μ L

H ₂O 3μL

Cumulative volume is 10 μ L, and is fully instantaneous centrifugal behind the mixing

16 ℃ of water-baths connect spends the night;

(4) the CMV-E2 fragment is connected the conversion and the evaluation of product with the pAdTrack-E0 carrier

1. the preparation of DH5 α competent cell

With step B (4) 1.;

2. recombinant plasmid transformed competence colibacillus cell

With step B (4) 2.;

The CMV-E2 fragment is inserted into the recombinant plasmid called after that makes up among the shuttle vectors pAdTrack-E0: recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2;

E. the structure of recombinant adenovirus skeleton carrier pAdEasy-E0-CMV-E2

(1) linearizing of recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2

With PmeI to shuttle vectors pAdTrack-E0-CMV-E2 linearization for enzyme restriction, by each component that adds respectively as follows:

pAdTrack-E0-CMV-E2?25μL

PmeI 4μL

0.1g/L?BSA 10μL

10×Buffer?4 10μL

H ₂O 51μL

Cumulative volume is 100 μ L

Put 6h in 37 ℃ of water-baths after agarose gel electrophoresis reclaims;

(2) linearizing recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2 is transformed into the intestinal bacteria BJ5183 competent cell that contains adenovirus skeleton carrier pAdEasy-1

With step B (4) 2.;

With the reorganization after adenovirus skeleton carrier called after: recombinant adenovirus skeleton carrier pAdEasy-E0-CMV-E2;

F. the acquisition of recombinant adenovirus rAd-E0-CMV-E2

(1) linearizing of recombinant adenovirus skeleton carrier pAdEasy-E0-CMV-E2, recovery and purifying

Cut with the PacI enzyme and to make recombinant adenovirus skeleton carrier pAdEasy-E0-CMV-E2 linearizing, the endonuclease reaction system is:

pAdeasy-E0-CMV-E2 25μL

Pac?I 5μL

0.1g/L?BSA 10μL

10×Buffer?4 10μL

H ₂O 50μL

Cumulative volume is 100 μ L, puts 8h in 37 ℃ of water-baths.

In the product after enzyme is cut, add the dehydrated alcohol of 2.5 volumes and the 3mol/L NaAc of 1/10 volume ,-20 ℃ the precipitation 1h, 4 ℃ with the centrifugal 15min of 12000r/min, supernatant discarded adds 70% ethanol and washes once, after the seasoning, adds 20 μ L H ₂The O dissolving DNA;

(2) acquisition of recombinant adenovirus rAd-E0-CMV-E2

To human embryonic kidney cell HEK 293 cells, Lipofectamine is pressed in concrete operations with linearizing recombinant adenovirus skeleton carrier pAdEasy-E0-CMV-E2 transfection ^TM2000 product descriptions carry out; The viral liquid called after recombinant adenovirus rAd-E0-CMV-E2 of results;

(3) amplification of recombinant adenovirus rAd-E0-CMV-E2 and titer determination

Virus titer when in 293 cells, reaching for 15 generations according to plaque ethods mensuration rAd-E0-CMV-E2.

The following test of recombinant adenovirus rAd-E0-CMV-E2 process that the present invention obtains:

1.rAd-E0-CMV-E2 immune protective test

(1) laboratory animal grouping

25 pigs are divided into 5 groups at random, and first group is the rAd-E0-CMV-E2 immune group, 5.Second group is swine fever spleen pouring seedling immune group, 5.The 3rd group is the recombinant adenovirus rAd-E0-E2 immune group of amalgamation and expression E0-E2 gene, 5.The 4th group is non-recombinant adenovirus immune group, 5.The 5th group is the blank group, 5.

(2) immunity and attack poison

The immunizing dose of recombinant adenovirus group and non-recombinant adenovirus group is 2.4 * 10 ⁹Pfu (quantitatively to 2mL) is in musculi colli and subcutaneous abdomen multi-point injection; The immunization method of hog cholera lapinised virus vaccine and dosage carry out immunity according to the actual using dosage of vaccine; The blank DMEM nutrient solution immunity of cultivating recombinant adenovirus, the 2mL/ head.During the 20d of all test pig after immunity with 100 times of TCID ₅₀The strong malicious intramuscular injection of swine fever, eye droppings, collunarium and oral attack.

(3) temperature check and clinical observation

All test pig are from attacking the 1st day beginning take temperature of poison, and later every day, take temperature was observed clinical symptom.

(4) detection of serum antibody

Each week after immunity beginning and immunity is gathered blood, collects serum, is used for the detection of hog cholera antibody, and another part adds antithrombotics, is used for the detection of blood Pestivirus suis.

(5) attack the detection that virus exists in the blood of poison back

Gather anticoagulated whole blood every other day after attacking poison, with the Pestivirus suis in the RT-PCR method detection blood.

(6) pathological observation

Inspection is all put to death and cutd open in after attacking poison 14 days after the clinical symptom of all survival pigs disappears substantially,, and whether observe lymphoodi mandibulares, mesenteric lymph nodes, superficial inguinal lymph nodes, kidney, spleen, bladder and heart has pathological change.

2. result

(1) body temperature and clinical symptom

Any clinical symptom does not appear in first group (rAd-E0-CMV-E2 immune group) after immunity, attacking poison back the 4th day, and 1 temperature of pig body reaches more than 40 ℃ in 5 immune swines, continue about 48h, in the time of the 7th day about temperature recovery normal.Deadly protection ratio is 100%.

Second group (swine fever spleen drench seedling immune group) after attacking poison the 4th day, 1 temperature of pig body reaches more than 40 ℃ in 5 immune swines, continues about 72h, and appetite descends a little, recovers normal the 8th day gentle appetite of body.Deadly protection ratio is 100%.

Any clinical symptom does not appear in the 3rd group (the recombinant adenovirus rAd-E0-E2 immune group of amalgamation and expression E0-E2 gene) after immunity, attacking the poison back the 4th day, 2 temperature of pig body reach more than 40 ℃ in 5 immune swines, continue about 72h, in the time of the 8th day about temperature recovery normal.Deadly protection ratio is 100%.

All test pig of the 4th group (non-recombinant adenovirus immune group) and the 5th group (blank group) are being attacked more than the poison back body temperature rise to 40 in the 1st～2 day ℃ sick pig constipation; The 3rd day appetite stimulator, spirit is depressed, and some sick pig vomits, and seldom searches for food, and only drinks water on a small quantity, spirit is highly depressed, and tetraparesis is unable, and is slow in action, waves shakiness, generally have loose bowels, two are not had refreshingly, and volume mucus purulent secretion is arranged, under the abdomen, there is the blutpunkte ear and four limbs inboard; Sick pig can not lie prostrate in the 4th day, general tremor, and four limbs are swimming shape paddling, and body temperature is delaied about 41 ℃ always.Began death successively on the 5th day, all dead to the 7th day, dead preceding body temperature decline.Mortality ratio is 100%.

Body temperature and death condition see Table 1.

(2) swine fever virus resistant and swine fever virus resistant detection of antibodies

The swine fever ELISA antibody assay kit that provides with China Veterinary Drugs Supervisory Inst. detects the antibody horizontal of all test pig.The antibody of rAd-E0-CMV-E2 immune group is in rising trend, antibody OD before attacking poison ₆₃₀Value reaches 0.696 respectively, and the antibody horizontal of attacking all test group of poison back all is tangible ascendant trend.The antibody horizontal of rAd-E0-E2 immune group is also in rising trend, but attack the poison before antibody OD ₆₃₀Value reaches 0.595 respectively.The OD of the antibody horizontal of hog cholera lapinised virus seedling immune group before attacking poison ₆₃₀Maximum is 0.713.Non-recombinant adenovirus control group and blank group never detect antibody horizontal before attacking poison, but can detect the hog cholera antibody level before on one's deathbed after attacking poison.Fig. 1.

Body temperature situation behind the different vaccine immunity group of the table 1 CSFV strong virus attack

Table 2 is attacked the detection of Pestivirus suis in the blood of poison back

(3) attack the detection of Pestivirus suis and Pestivirus suis in the blood of poison back

Attack the anticoagulation of gathering every other day behind the poison, detect Pestivirus suis, found that having 1/5 in the rAd-E0-CMV-E2 immune group can detect Pestivirus suis with the RT-PCR method.There is 2/5 in the rAd-E0-E2 immune group and can detects Pestivirus suis.The swine fever spleen drenches the seedling immune group also has 1/5 can detect Pestivirus suis, but not recombinant adenovirus contrasts and blank all 100% detects, and the results are shown in Table 2.

(4) attack the pathological observation of organizing internal organs behind the poison

The inspection result that cuts open of the dead pig of non-recombinant adenovirus and blank group shows, pig lymphoodi mandibulares, mesenteric lymph nodes, inguinal lymph nodes, and kidney, spleen, bladder and heart all have tangible bleeding.

The inspection result that cuts open of all survival pigs of rAd-E0-CMV-E2 immune group, rAd-E0-E2 immune group and conventional vaccine immune group shows that although clinical symptom disappears, also there are some pathological changes in some internal organs.In 5 of the rAd-E0-CMV-E2 immune group survival pig, 1/5 lymphoodi mandibulares, mesenteric lymph nodes are hemorrhage.In 5 survivals of rAd-E0-E2 immune group pig, 2/5 cephalogaster mesentery lymphoglandula and kidney are hemorrhage, and 1/5 lymphoodi mandibulares is hemorrhage.The swine fever spleen drenches in 5 survivals of seedling immune group pig, 1/5 lymphoodi mandibulares, and 1/5 head-kidney is dirty hemorrhage.

(5) discuss

The present invention adopts the ELISA method to detect the humoral immunity level of immune swine.Can both induce body to produce specific humoral immune response behind recombinant adenovirus rAd-E0-CMV-E2, rAd-E0-E2 and the conventional vaccine immune swine, after this showed recombinant adenovirus inoculation pig body, albumen expressed in host cell had better immunogenicity.Inductive antibody horizontal and recombinant adenovirus rAd-E0-CMV-E2 inductive antibody horizontal indifference behind the conventional vaccine inoculation pig body, but be higher than recombinant adenovirus rAd-E0-E2 inductive antibody horizontal, although this may be because recombinant adenovirus rAd-E0-CMV-E2 and rAd-E0-E2 express E0 and E2 albumen, but E0 that recombinant adenovirus rAd-E0-CMV-E2 expresses and E2 albumen are the promotors that has separately to be started, E0 and E2 albumen are two independently albumen, the approach that this and Pestivirus suis itself duplicates when breeding in animal body is the same, and E0 that recombinant adenovirus rAd-E0-E2 expresses and E2 albumen are not albumen independently separately, but a kind of fusion rotein, the space that both form in vivo is folding may to cause some antigen site by embedding, can not produce antibody response by the effective stimulus body.

When using the strong virus attack of lethal quantity; although recombinant adenovirus rAd-E0-CMV-E2, recombinant adenovirus rAd-E0-E2 immune group and conventional vaccine immune group all provide 100% deadly protection ratio; but to be lighter than the animal of recombinant adenovirus rAd-E0-E2 immunity in the clinical symptom of attacking poison back recombinant adenovirus rAd-E0-CMV-E2 and conventional vaccine immune animal; recombinant adenovirus rAd-E0-CMV-E2 and conventional vaccine have 1/5 clinical symptom to occur, and recombinant adenovirus rAd-E0-E2 has 2/5 clinical symptom to occur.Non-recombinant adenovirus immune group and blank group are all dead.

Detect from the blood poison of attacking behind the poison, do not have difference between rAd-E0-CMV-E2 immune group and the conventional vaccine immune group, 1/5 recall rate is all arranged, recombinant adenovirus rAd-E0-E2 has 2/5 recall rate.

From above-mentioned result as can be seen, the present invention can effectively prevent the generation of swine fever, provides identical protection ratio with conventional vaccine, and rAd-E0-E2 provides the better protection rate than the reorganization adenovirus.The present invention compares the security that has improved vaccine with conventional vaccine, and has saved artificial and cost in a large number.

Claims (4)

1. recombinant adenovirus of expressing Pestivirus suis E0, raq gene, it is characterized in that in the Bgl II of the CMV of adenovirus promotor downstream multiple clone site and Kpn I restriction enzyme site, inserting Pestivirus suis E0 gene, in Xho I and XbaI enzyme cutting site, insert the CMV-E2 fragment of adenovirus CMV promotor and Pestivirus suis raq gene.

2. the recombinant adenovirus of expression Pestivirus suis E0 as claimed in claim 1, raq gene, it is characterized in that described Pestivirus suis is conventional commercial virus, the accession number of E0 gene in GenBank of Pestivirus suis is: AF058715.1, the accession number of the raq gene of Pestivirus suis in GenBank is: AY775178.2

3. the recombinant adenovirus of expression Pestivirus suis E0 as claimed in claim 1, raq gene is characterized in that described adenovirus is conventional commercial people-5 type non-replicating adenovirus.

4. the construction process of the recombinant adenovirus of an expression Pestivirus suis E0 as claimed in claim 1, raq gene is characterized in that comprising following construction step:

A. from commercial Pestivirus suis and Pestivirus suis, amplify E0 and raq gene respectively;

B. the Pestivirus suis E0 gene that steps A is obtained is inserted among the adenovirus shuttle vector pAdTrack by Bgl II and Kpn I restriction enzyme site, forms adenovirus shuttle vector pAdTrack-E0;

C. the Pestivirus suis raq gene that steps A is obtained is inserted among the adenovirus shuttle vector pAdTrack by Kpn I and Not I restriction enzyme site, forms adenovirus shuttle vector pAdTrack-E2;

D. the adenovirus shuttle vector pAdTrack-E2 PCR method routinely with step C amplifies the CMV-E2 gene that contains the CMV promotor, be inserted into by Xhol I and Xba I restriction enzyme site among the Xhol I and Xba I restriction enzyme site among the adenovirus shuttle vector pAdTrack-E0 of step B, form recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2;

E. with the recombinant adenovirus shuttle vectors pAdTrack-E0-CMV-E2 of step D behind Pme I linearization for enzyme restriction, be transformed in the intestinal bacteria BJ5183 competent cell that contains adenovirus skeleton carrier pAd-easy-1, form recombinant adenovirus skeleton carrier pAd-easy-E0-CMV-E2;

F. with recombinant adenovirus skeleton carrier pAd-easy-E0-CMV-E2 after Pac I linearizing, among the transfection human embryonic kidney cell HEK293, pack out recombinant adenovirus rAd-E0-CMV-E2.

Images