CN106031793B - Live vaccine and preparation method and application thereof - Google Patents
Live vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a live vaccine rHVT-HA for preventing and treating avian influenza and Marek's disease, wherein the HVT is recombined with HA gene of avian influenza virus, and the HA gene can express HA protein. The live vaccine of the invention can rapidly generate HI antibody in vivo, has more effective effect of preventing avian influenza than the traditional inactivated vaccine, and can generate good protection effect on Marek's virus infection.
Description
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to a live vaccine for treating avian influenza, a preparation method and application.
Background
The H9 subtype Avian Influenza Virus (AIV) can cause respiratory diseases and egg laying reduction of chickens, has higher morbidity and mortality under the condition of mixed infection or secondary infection of other pathogens, and causes serious economic loss to poultry industry in China. Its genome is composed of 8 segments of single-stranded antisense RNA, and encodes 10 proteins, 8 of which are structural proteins, including HA and NA, and the other two are non-structural proteins. HA is generally considered to be its major antigenic gene, which can elicit protective antibodies in avians. However, the mutation speed of H9 subtype AIV is fast, the existing vaccine strains cannot effectively protect epidemic strains, and most of the existing vaccines are inactivated vaccines, which can stimulate the organism to generate humoral immunity but cannot generate mucosal immunity and cellular immunity, the immune period is short, in addition, the production cost of the inactivated vaccine is high, and the production process may cause the pollution of virus to the environment. Therefore, there is a need to screen epidemic strains to prepare novel vaccines to solve the existing problems.
Marek's Disease (MD) is a lymphoproliferative tumor disease of chicken caused by Marek's virus (MDV). Marek's disease is a neoplastic disease that can be controlled by vaccines, and the disease occurs frequently in many areas of China, even if immune chicken flocks also have morbidity, MDV infection can induce generation of an immunosuppressive phenomenon, and secondary or mixed infection can also cause more serious epidemic situation of MD, cause higher morbidity and mortality, and cause huge economic loss for poultry industry.
Disclosure of Invention
The invention uses the herpesvirus of turkeys (HVT), i.e. Marek's disease virus serotype-3, as the live virus vector to express the avian influenza virus HA gene, so as to overcome the defects of the existing inactivated vaccine for avian influenza.
The invention provides a live vaccine rHVT-HA for preventing and treating avian influenza and Marek's disease, wherein the HVT is recombined with an HA gene of the avian influenza virus, the HA gene can express HA protein, and the HA gene of the avian influenza virus is recombined between 157-167 amino acids of an US2 gene in a non-essential region in an HVT genome.
The avian influenza virus HA gene can be obtained from any subtype or any strain of avian influenza virus.
In one embodiment of the present invention, the HVT is FC126 strain, PB-THV1 strain, H-2 strain, YT-7 strain, WTHHV-1 strain or HPRS-26 strain in the live vaccine rHVT-HA of the present invention.
Turkey herpesviruses are double-stranded linear DNA viruses in the Herpesviridae (Herpesviridae family) and alphaherpesviridae (Alphaherpesvirinae subfamily). HVT is ubiquitous and non-carcinogenic in domestic turkeys and it is classified as marek's disease virus serotype 3. HVT has been widely used to inoculate chickens to prevent marek's disease in chickens. Any HVT can be used in the present invention as long as it is non-pathogenic to chickens. For example, the following HVT strains, FC126, PB-THV1, H-2, YT-7, WTHHV-1 and HPRS-26 are suitable for use in backbone viruses. Of these, preferably, the FC126 strain is advantageously suitable for use in the present invention.
The HVT virus FC126 strain, PB-THV1 strain, H-2 strain, YT-7 strain, WTHHV-1 strain and HPRS-26 strain are commercially available.
In one embodiment of the present invention, the HA gene in the live vaccine rHVT-HA of the present invention includes an HA gene of avian influenza virus subtype H9.
In a preferred embodiment of the present invention, the HA gene of the live vaccine rHVT-HA of the present invention includes HA genes of HL strain, SZ strain, and a155-1-2014 strain of H9 subtype avian influenza virus.
As a most preferred embodiment of the present invention, in the live vaccine rHVT-HA of the present invention, the HA gene sequence comprises a nucleotide sequence encoding an amino acid sequence of seo.no. 4.
Preferably, the HA gene is obtained from an H9 subtype avian influenza virus.
Preferably, the HA gene is obtained from H9 subtype avian influenza virus HL strain, SZ strain and A155-1-2014 strain.
Preferably, the amino acid sequence of the HA gene of the A155-1-2014 strain is shown in SEQ ID NO. 4.
Preferably, the nucleotide sequence of the HA gene of the H9 subtype avian influenza virus A155-1-2014 strain is shown in SEQ ID NO. 3.
In the present invention, the HA gene is inserted into the HVT genome, preferably, the HA gene is inserted into a region of the HVT genome that is not essential for viral growth, which is referred to herein as a non-essential region. In other words, a non-essential region may be defined as a region in which a modification or insertion of a foreign gene does not prevent successful replication of the virus in vitro or in vivo.
Preferably, the non-essential region in the HVT genome is the region between UL43, US2, UL44 and UL 46.
Preferably, the non-essential region in the HVT genome is US 2.
Preferably, the HA gene is inserted into the non-essential region of the HVT genome at the position of US2 between the amino acids 157-167 of the US2 gene.
The HA gene is a protective antigen gene of avian influenza virus, and the backbone HVT is a marek's disease live vaccine, so the recombinant HVT containing the HA gene in the present invention can be used as a bivalent vaccine against AI and marek's disease, or as a monovalent vaccine against AI.
As an embodiment of the invention, the live vaccine rHVT-HA of the invention also comprises a carrier and an adjuvant.
The vaccine may also include avian cells, media components, buffers such as hydrochloride buffer and HEPES buffer, and/or adjuvants such as cytokines and CpG oligodeoxynucleotides. The vaccine may also contain any ingredient such as a preservative as long as it is not pharmacologically harmful. In addition, the vaccines of the invention may be used in admixture with any recombinant or non-recombinant virus, such as MDV serotype 1 or serotype 2 vaccine strains.
The second aspect of the invention provides an avian influenza HA gene, the nucleotide sequence of which is shown in SEO.No. 3.
The third aspect of the invention provides an avian influenza HA protein, the amino acid sequence of which is shown in SEO.No. 4.
In a fourth aspect of the present invention, there is provided a method for preparing said live vaccine rHVT-HA, said method comprising: 1) providing herpesvirus of turkeys HVT; 2) cloning the HA gene of the avian influenza virus; 3) recombining the HA gene of the avian influenza virus into the genome of the herpesvirus of turkeys HVT; 4) culturing and propagating the recombinant HVT-HA virus; and 5) mixing the propagated recombinant virus with a vector.
Any known method may be suitable for the preparation of the recombinant bivalent vaccine of the present invention. For example, recombinant HVTs can be seeded into permissive culture cells (such as CEF cells) and cultured to appropriate titers. However, cells removed from the spinner flask were trypsinized and collected by centrifugation. The pelleted cells were then suspended in medium containing dimethyl sulfoxide, frozen slowly, and then stored in liquid nitrogen. Alternatively, recombinant HVT can be released from infected cells by disrupting the cells in a diluent containing a stabilizing agent (such as sucrose and bovine serum albumin). These released HVTs become cell-free HVTs. Cell-free HVT can be lyophilized and stored at 4 ℃.
As a preferred embodiment of the present invention, in the method for preparing the live vaccine rHVT-HA according to the present invention, the HA gene sequence of the avian influenza virus includes a nucleotide sequence encoding an seo.no.4 amino acid sequence; recombining the HA gene of the avian influenza virus to 157-167 amino acids of the herpes virus HVT US2 gene of the turkey.
Specifically, the recombinant live vector HVT vaccine prepared by the invention is realized by the following modes:
1. amplifying HA gene of H9N2 avian influenza virus, and cloning the HA gene into eukaryotic expression vector pcDNA;
2. cloning a gene expression cassette containing lacZ and HA controlled by a CMV promoter on the eukaryotic expression vector into a Marek's disease virus US2 gene to construct a transfer vector of the Marek's disease virus;
3. transfecting Chicken Embryo Fibroblast (CEF) infected with Marek's disease virus by a liposome method, and screening blue spots to obtain a recombinant virus;
and 4, identifying whether the genome contains complete HA genes by a PCR (polymerase chain reaction) method, verifying whether the recombinant virus can express the avian influenza HA protein by an immunoenzyme reaction and Western-blot, and identifying whether the recombinant virus HAs serum activity.
The fifth aspect of the invention provides the application of the vaccine in preparing medicines for preventing and treating avian influenza and Marek's disease.
The use may be by administering a bivalent recombinant HVT vaccine to chickens by any known method of vaccination with marek's disease vaccine. For example, the vaccine of the present invention is diluted to 10-10 per dose with a diluent containing a buffer component, a saccharide and a dye5Or, more advantageously, 102-104Each plaque forming unit (pfu), the diluted vaccine may be inoculated subcutaneously, behind the neck of a day-old chick.
The sixth aspect of the invention is to provide the application of the avian influenza HA gene shown in the nucleotide sequence SEO.No.3 in the preparation of drugs for preventing and treating avian influenza.
The avian influenza HA gene can be applied to the development of expression vectors, live vectors, nucleic acid vaccines and diagnostic reagents.
The invention inserts the protective antigen HA gene of avian influenza H9N2 into the nonessential region US2 of cell-associated HVT virus, so that HA protein is continuously expressed along with the reproduction and replication of recombinant virus on CEF cells. The vaccine prepared by the strain can effectively resist the attack of MDV virulent virus and can also induce an organism to generate humoral immunity and cellular immune response for resisting the attack of H9 subtype avian influenza. And the MDV is a strict cell-bound virus, can break through the interference of maternal antibodies, is suitable for early immunity, and is more convenient to operate through chick embryo immunity.
In the above preferred embodiment of the present invention, the HA gene of the recombinant avian influenza virus is inserted between 157-167 amino acids of the HVT US2 gene, and the insertion site of 157-167 amino acids of the HVTUS2 gene is a new insertion site, and the live vaccine prepared by the present invention can functionally express HA protein for effective immunization.
Drawings
FIG. 1 shows the results of the HI antibodies generated in chickens immunized with rHVT-HA live vaccine and H9 inactivated vaccine of the present invention.
In the sequence table:
the sequence 1 is a nucleotide sequence of an avian influenza virus HL strain HA gene;
the sequence 2 is an amino acid sequence of HA protein of an avian influenza virus HL strain;
the sequence 3 is the nucleotide sequence of HA gene of avian influenza virus A155-1-2014 strain;
the sequence 4 is an amino acid sequence of HA protein of avian influenza virus A155-1-2014 strain;
the sequence 5 is a nucleotide sequence of HVT virus FC126 strain US2 gene;
the sequence 6 is an amino acid sequence of HVT virus FC126 strain US2 protein.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 construction of transfer vectors
1. Extracting H9 subtype avian influenza virus genome RNA: inoculating H9 subtype AIV A155-1-2014 strain to SPF chick embryos of 10 days old, collecting allantoic fluid of dead chick embryos within 24-72 hours, and extracting total RNA of the allantoic fluid by using Trizol reagent. The specific operation is carried out according to the instruction of the reagent.
2. RT-PCR: designing a primer pair, wherein the sequence of the primer pair is as follows: an upstream primer: 5'-GAAGCGGCCGCAAAATGGAGACAATATCACTAATAAC-3', downstream primer: 5'-GGTGGGCCCTTATATACAAATGTTGCATCTGC-3' are provided. And respectively introducing Not I and Apa I at the 5' ends of the upstream primer and the downstream primer, wherein the expected length of the amplified fragment is 1700bp, cloning the amplified fragment into a T vector, and identifying the positive recombinant plasmid containing the HA gene through enzyme digestion analysis and PCR.
3. Construction of eukaryotic expression vectors containing the HA gene: the HA gene was cloned into a pcDNA vector via Not I and Apa I sites, and named pcDNA-HA.
4. Construction of pUS2 vector, primers were designed based on the HVT Fc126 strain full-length genome sequence (accession No. AF291866) published in GenBank, Sal I and Hind III cleavage sites were introduced into the 5' ends of the upstream and downstream genes, and the HVT genomic DNA was used as a template to amplify the US2 region of HVT and its homologous sequences at both ends. The amplified target fragment was then cloned into pUC18 vector and named pUS 2.
5. Construction of HVT transfer vector containing HA Gene: after the pcDNA-HA was digested with endonucleases Bgl II and Ava II, a 6.8kb expression cassette fragment containing a CMV promoter, LacZ gene, HA gene and SV40polyA tail was obtained, and this fragment was subcloned into pUS2 to obtain a transfer plasmid vector, which was named pUS 2-HA. The specific insertion site is between 157-167 amino acids of the US2 gene.
As a result: cloning the HA gene of 1.7kb into pcDNA vector to obtain pcDNA-HA, then cutting the pcDNA-HA with endonuclease, subcloning the obtained fragment into pUS2 to obtain transfer plasmid vector, namely pUS 2-HA. The transfer recombinant plasmid contains lacZ reporter gene and homologous arms for in vivo homologous recombination, and the sizes of the upstream and downstream homologous arms are 3.5 kb and 0.85kb respectively.
Example 2 construction, screening and characterization of recombinant Marek's virus
Preparing Chicken Embryo Fibroblast (CEF) by conventional method, growing into 80% monolayer, inoculating HVT at a dose of 50PFU per well of 24-well plate, acting at 37 deg.C for 4 hr, and treating with LipofectamineTM2000 instructions, transfer vector plasmid transfection of cells, after 2h, pour out transfection solution, add 5% fetal bovine serum in MEM medium at 37 ℃ CO2After 4-5 days in the incubator, to show typical cytopathic effects, a nutrient solution containing X-Gal (200ug/ml) was added, blue plaques were observed, infected CEF cells in 24 wells were digested with 0.25% trypsin and seeded onto 96-well plates with a fresh CEF cell monolayer. The blue spots were observed, cells with blue wells were digested and plated onto new cells, and this was repeated for several cycles until all plaques were deemed blue, and purified recombinant virus was obtained by blue spot screening.
As a result: the transfer plasmid pUS2-HA is wrapped by liposome to transfer CEF cells infected by HVT, 3-4 days after transfection, substrate X-Gal is added, occurrence of blue spots is observed, and the recombinant virus is proved to be stable through 6 generations of experiments.
Identification of recombinant viruses: the total DNA of the recombinant virus was extracted by a conventional method, and the HA gene fragment was amplified using the previously designed HA gene primers.
As a result: extracting and purifying the obtained stable recombinant virus DNA, and amplifying a PCR product with the size of about 1.7kb by using a specific primer of the avian influenza HA gene.
Example 3 identification of recombinant Virus expressed HA proteins
1. The indirect immune enzyme reaction is to infect the recombinant virus (rHVT-HA) to CEF cells in 96 holes for 4-5 days. Pouring off the culture solution and fixing the cells with a pre-cooled 95% ethanol and acetone (4:6) mixture; the immune enzyme reaction was performed with SPF chicken anti-H9N 2 serum and HRP-labeled rabbit anti-chicken IgY.
As a result: the CEF cells containing the recombinant virus showed blue plaques after X-Gal staining. The result of the immune enzyme reaction shows that the blue spot formed by the recombinant virus can specifically react with the H9N2 serum.
2. Western-blot detection CEF cells are infected by recombinant viruses (rHVT-HA), the collected cells are cracked after 4-5 days, supernatant is taken, 12% SDS-PAGE electrophoresis is carried out, the supernatant is transferred to a membrane, and Western-blot analysis is carried out by SPF chicken anti-H9N 2 serum and HRP marked rabbit anti-chicken IgY.
As a result: western-blot analysis of cell lysates containing recombinant viruses showed that there was a specific reaction band at a molecular weight of about 70KD, further indicating that the HA protein was expressed in the recombinant viruses.
Example 4 identification of hemagglutinating Activity of recombinant viruses
Infecting CEF cells with recombinant virus (rHVT-HA), collecting 24-hole cells after 4-5 days, dissolving in 0.2ml physiological saline, breaking by ultrasonic wave, passing through microcentrifuge 8000r/min for 2min, collecting supernatant, diluting with physiological saline at multiple ratio, adding equal volume of 0.5% chicken red blood cells, acting at room temperature for 30min, observing results, and using the CEF cells infected with HVT as negative control.
As a result: HA expressed by the recombinant virus had hemagglutination activity with a hemagglutination titer of 1:64, whereas lysates of control HVT-infected CEF cells had no hemagglutination activity.
Example 5 determination of growth curves of recombinant viruses
rHVT-HA and HVT were inoculated separately into confluent monolayers of CEF (3 PFU/well) cultured in 24-well plates. Respectively collecting virus liquid 1, 2, 3, 4, 5, 6 and 7 days after inoculation, and connecting to a fresh cell monolayer, wherein the virus liquid collected every day is respectively repeated for 4 times; after 6d, the number of plaques in each well was calculated, and the number of plaques on each day was calculated.
As a result: according to the number of rHVT-HA and HVT plaques on CEF, the rHVT-HA HAs a similar growth curve with the parental virus, which shows that the insertion of HA gene does not influence the normal growth and proliferation process of HVT, and the recombinant virus HAs stable structure.
Example 6 preparation of inactivated vaccine against H9 subtype avian influenza
1. Preparation of virus liquid
Taking H9 subtype avian influenza virus A155-1-2014 strain seed, diluting with sterile normal saline to 10%-3(0.1 ml of virus liquid is added into 0.9ml of sterile physiological saline, and diluted for 2 times after shaking and mixing uniformly) to pass through allantoic cavityInoculating 10 day old susceptible chick embryo (self-hatching with SPF hatching egg purchased from Beijing Meiliya Winton laboratory animals technology Co., Ltd.) 0.1ml per embryo (containing 10 days)5EID50). After inoculation, the pinholes are sealed, and incubation is continued at 36-37 ℃ without turning eggs. And taking out the mixture for 96 hours, standing the mixture in an upward air chamber, and cooling the mixture for 12 to 24 hours at the temperature of between 2 and 8 ℃. And (5) harvesting the embryo liquid of the cooled chick embryo. The harvested virus liquid was sampled and the virus content was measured to be 10 according to the method in appendix of pharmacopoeia of the people's republic of China (2010 version)8.5EID50/0.1ml。
2. Vaccine preparation
Inactivating the prepared virus solution with formaldehyde solution, mixing with injectable white oil, and emulsifying at 2800r/min for 40 min. The specific ratio of the vaccine is shown in table 1.
TABLE 1H9 subtype avian influenza A155-1-2014 strain inactivated vaccine component ratio
| Components | Vaccine 1 |
| A155-1-2014 strain (EID)50/0.1ml) | 108.0 |
| White oil (V/V) | 60％ |
Example 7 immune protection experiment of rHVT-HA against H9 subtype avian influenza
Randomly dividing 40 SPF (specific pathogen free) chickens of 1 day old into 2 groups of 20 chickens, wherein the first group immunizes recombinant virus rHVT-HA with the dose of 5000 PFU; the second group immunized the H9 subtype avian influenza inactivated vaccine prepared in example 6 (vaccine 1) at a dose of 0.3 ml; all are administered by subcutaneous injection at the neckAnd (5) immunizing chicks. HI antibody was assayed by collecting blood weekly in the wing vein after immunization and isolating serum, and then administered intravenously at 10 weeks after immunization7.0EID50The H9N2 subtype avian influenza virus A155-1-2014 strain at the dose infects 10 chickens in each of the two immunization groups, and the 10 chickens in each of the two immunization groups are attacked at the same dose 3 weeks after the immunization. After the challenge, the observation was continued for two weeks, and cotton swabs of larynx and cloaca of all groups of chickens were collected on the 5 th day after the challenge, and after treatment, 10-day-old SPF chick embryos were inoculated for virus isolation.
The detection result of serum HI antibody is shown in figure 1, the generation of rHVT-HA immune group HI antibody is earlier than that of H9 inactivated vaccine immune group HI antibody by 1 week, and 8log can be reached after 2 weeks of immunization2(ii) a The challenge protection results are shown in tables 2 and 3, the recombinant virus rHVT-HA immunization group chickens can obtain 100% protection only 2 weeks after immunization, and viruses are not separated from all chickens after challenge; 6/10 toxin expelling is carried out on the H9 subtype avian influenza inactivated vaccine immunized chicken 2 weeks after immunization. The results show that the HI antibody is generated faster after the recombinant virus rHVT-HA is used for immunizing the chicken, and can provide complete protection against H9N2 subtype avian influenza 2 weeks after the immunization.
TABLE 2 challenge protection test results 2 weeks after immunization
TABLE 3 protective test results of challenge 3 weeks after immunization
Example 8 Cross-immune protection assay of rHVT-HA against H9 subtype avian influenza
In order to study the cross protection of rHVT-HA on other strains, according to the preparation method of the vaccine in example 6, the research on the antigen correlation and the cross immunity of avian influenza (H9 subtype) inactivated vaccine (HL strain, see the literature: avian influenza virus (H9N2 subtype, HL strain) and domestic part provincial avian influenza virus (H9 subtype) epidemic strain is prepared, the annual meeting of the animal husbandry and veterinary medical society in 2007 is collected, 2007, 35-38, the applicant holds the strain and is willing to release to the public within 20 years from the application date according to the relevant provisions of patent law, and the specific vaccine proportion is shown in Table 4.
TABLE 4 inactivated vaccine component ratio for H9 subtype avian influenza HL strain
| | Vaccine | 2 |
| HL strain (EID)50/0.1ml) | 108.0 | |
| White oil (V/V) | 60％ |
The recombinant virus rHVT-HA, the vaccine 1 prepared in the example 6 and the vaccine 2 prepared in the example are selected to immunize SPF chickens (purchased from Beijing Meiliya Winton laboratory animal technology Co., Ltd.), HL strain and A155-1-2014 strain are selected to attack respectively 3 weeks after immunization, and virus separation is carried out. The results show that the detoxification rates of the HL strain inactivated vaccine immune group are 0/10 and 4/10 respectively, the detoxification rates of the A155-1-2014 strain inactivated vaccine immune group are 0/10 and 0/10 respectively, the detoxification rates of the recombinant virus rHVT-HA immune group are 0/10 and 0/10 respectively, and the detoxification rates of the challenge control group are 5/5 and 5/5 respectively. The specific results are shown in Table 5.
TABLE 5 results of 3 weeks after Cross-Immunoprotective assay of rHVT-HA against H9 subtype avian influenza
The A155-1-2014 strain inactivated vaccine and the recombinant virus rHVT-HA are proved to generate effective protection on the attack of the two strains of viruses, and the cross immunogenicity is good.
However, the above-described test was subjected to a two-week challenge test after immunization, and virus isolation was performed. The results show that the detoxification rates of the HL strain inactivated vaccine immune group are 6/10 and 10/10 respectively, the detoxification rates of the A155-1-2014 strain inactivated vaccine immune group are 6/10 and 6/10 respectively, the detoxification rates of the recombinant virus rHVT-HA immune group are 0/10 and 0/10 respectively, and the detoxification rates of the challenge control group are 5/5 and 5/5 respectively. The specific results are shown in Table 6.
TABLE 6 results of 2 weeks post immunization of the cross-immune protection assay for H9 subtype avian influenza with rHVT-HA
Further shows that the recombinant virus rHVT-HA can effectively protect against the attack of two strains of viruses, HAs good cross immunogenicity, can generate antibodies more quickly, and can provide complete protection against H9N2 subtype avian influenza two weeks after immunization.
Example 9rHVT-HA vaccine MDV immune challenge protection assay
201 day old SPF white leghorn chickens were randomized into 2 groups, the first group immunized with 10 rHVT-HA vaccines injected subcutaneously into each neck at 0.2ml (containing 3000PFU), and the second group not immunized as a control. Two groups of chickens were raised in isolators separately and fed with sterilized feed and drinking water.
After 21d of immunization, 10 chickens in each group attacked MDV Jing-1 strain virulent virus, and were inoculated with virus in the abdominal cavity at a virus-attacking dose of 1000 PFU/chicken. The morbidity and the mortality are observed and recorded every day, and the dead chickens are dissected one by one and observed for macroscopic lesions. And (5) killing all live chickens 60 days after the virus attack, carrying out dissection, observing pathological change characteristics, and calculating a protection index.
Protection index (positive rate of virus attacking control group MD-positive rate of immunity group MD) × 100%/positive rate of virus attacking control group MD
The results show that the rHVT-HA vaccine immunization group can effectively resist the attack of MD virulent virus, the difference is very obvious (P is less than 0.01) compared with the attack control group, the protection index is 100%, and the specific results are shown in Table 7.
TABLE 7rHVT-HA vaccine immunization challenge results
Example 10 efficacy test of HA Gene recombinant live vectors of avian influenza viruses of different strains
The HA gene of avian influenza HL strain (SEQ ID NO: 1) was inserted into the expected HVT site according to the method of examples 1 to 5 to obtain recombinant virus rHVT-HA-HL.
Randomly dividing 60 SPF (specific pathogen free) chickens of 1 day into 6 groups of 10 chickens, and immunizing recombinant virus rHVT-HA by the first group and the second group at the dose of 5000 PFU; immunizing recombinant virus rHVT-HA-HL by the third group and the fourth group at the dose of 5000 PFU; chicks are immunized in a neck subcutaneous injection mode; the fifth and sixth groups are non-immune controls. HI antibody was assayed by collecting blood weekly in the wing vein after immunization and isolating serum, and then administered intravenously at 10 weeks after immunization7.0EID50The H9N2 subtype avian influenza virus A155-1-2014 strain at the dose infects 10 chickens in the first group, the third group and the fifth group, and 10 chickens7.0EID50The second group, the fourth group and the sixth group are infected by 10 chickens with the H9N2 subtype avian influenza virus HL strain. After the challenge, the observation was continued for two weeks, and cotton swabs of larynx and cloaca of all groups of chickens were collected on the 5 th day after the challenge, and after treatment, 10-day-old SPF chick embryos were inoculated for virus isolation.
The detection result of the serum HI antibody shows that the rHVT-HA immune group HI antibody and the rHVT-HA-HL immune group HI antibody are generated earlier, and can reach 6 logs 2 weeks after immunization2Higher up to 8log in rHVT-HA immunization group2(ii) a The challenge protection results are shown in table 8, 100% protection can be obtained only 2 weeks after immunization of recombinant virus rHVT-HA immunization group chickens, and viruses are not separated from all chickens after challenge of the two virus strains; the recombinant virus rHVT-HA-HL immune chicken is 100 percent protected after being attacked by the HL strain 2 weeks after being immunized, and is 4/10 detoxified after being attacked by the A155-1-2014 strain.
The results show that after the recombinant virus rHVT-HA and the rHVT-HA-HL are used for immunizing chickens, HI antibodies can be quickly generated, complete protection against the attack of the H9N2 subtype avian influenza self-strain can be provided 2 weeks after immunization, and particularly complete protection against the latest epidemic strain can be provided by the recombinant virus rHVT-HA. Both controls were all affected.
TABLE 8 protection test results against challenge 2 weeks after immunization
Example 11 comparison of antibody production by HA Gene recombinant live vector of HL Strain of avian influenza
Randomly dividing 60 SPF (specific pathogen free) chickens of 1 day old into 6 groups of 10 chickens, and immunizing the recombinant virus rHVT-HA-HL prepared in the example 10 by the first group and the second group at the dose of 5000 PFU; a third and fourth group immunizes vaccine 2 prepared in example 8; chicks are immunized in a neck subcutaneous injection mode; the fifth and sixth groups are non-immune controls. HI antibody was assayed by collecting blood weekly in the wing vein after immunization and isolating serum, and then administered intravenously at 10 weeks after immunization7.0EID50The first group, the third group and the fifth group are infected by 10 chickens with the dosage of the HL strain of the H9N2 subtype avian influenza virus, and 10 chickens are added after 3 weeks of immunization7.0EID50The second group, the fourth group and the sixth group are infected by 10 chickens with the H9N2 subtype avian influenza virus HL strain. After the challenge, the observation was continued for two weeks, and cotton swabs of larynx and cloaca of all groups of chickens were collected on the 5 th day after the challenge, and after treatment, 10-day-old SPF chick embryos were inoculated for virus isolation.
Serum HI antibody detection result rHVT-HA-HL immune group antibody generation is earlier, and can reach 6 logs 2 weeks after immunization26log was achieved 3 weeks after immunization in vaccine 2 immunization group2(ii) a The challenge protection results are shown in table 9, 100% protection can be obtained only 2 weeks after immunization of recombinant virus rHVT-HA-HL immune group chickens, and no virus is separated from all chickens after challenge; the HL strain immune group immunized chickens are 100% protected after 3 weeks after the immunization and challenge of the HL strain, and no virus is separated from all chickens after the challenge.
TABLE 9 comparison of antibody production by HA gene recombinant live vector of HL strain of avian influenza
The results show that after the recombinant virus rHVT-HA-HL is used for immunizing chickens, HI antibodies can be quickly generated, complete protection against the attack of the H9N2 subtype avian influenza self strain can be provided 2 weeks after immunization, and the inactivated vaccine can be provided with complete protection only 3 weeks after immunization. Both controls were all affected. The antibody rising speed of the live vector vaccine is far higher than that of the inactivated vaccine.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. A live vaccine rHVT-HA for preventing and treating avian influenza and Marek's disease, wherein, HVT recombines HA gene of avian influenza virus, the HA gene can express HA protein, the HA gene of avian influenza virus recombines between non-essential region US2 gene 157-167 amino acids in HVT genome; the HA gene is the HA gene of H9 subtype avian influenza virus A155-1-2014 strain, and the HA gene sequence of the A155-1-2014 strain encodes the protein shown by SEO. number 4.
2. The live vaccine rHVT-HA according to claim 1, wherein the HVT is FC126 strain, PB-THV1 strain, H-2 strain, YT-7 strain, WTHHV-1 strain or HPRS-26 strain.
3. The live vaccine rHVT-HA according to claim 1, further comprising a carrier, an adjuvant.
4. An avian influenza HA gene, the nucleotide sequence of which is shown as SEO. number 3.
5. An avian influenza HA protein, the amino acid sequence of which is shown as SEO. number 4.
6. A method of preparing the live vaccine rHVT-HA of claim 1, the method comprising:
1) providing herpesvirus of turkeys HVT;
2) cloning the HA gene of the avian influenza virus;
3) recombining the HA gene of the avian influenza virus into the genome of the herpesvirus of turkeys HVT;
4) culturing and propagating the recombinant HVT-HA virus; and
5) mixing the propagated recombinant HVT-HA virus with a vector.
7. The production method according to claim 6, wherein the HA gene sequence of the avian influenza virus comprises a nucleotide sequence encoding an SEO. number 4 amino acid sequence; recombining the HA gene of the avian influenza virus to 157-167 amino acids of the herpes virus HVTUS2 gene of the turkey.
8. Use of the live vaccine rHVT-HA according to any one of claims 1-3 in the preparation of a medicament for the prevention and treatment of avian influenza and Marek's disease.
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