CN111705083A - Rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, vaccine, preparation method and application thereof - Google Patents
Rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, vaccine, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, vaccine, a preparation method and application thereof. The invention amplifies rabbit hemorrhagic disease virus type 2 capsid protein VP60 gene by reverse transcription PCR, synthesizes the gene after insect cell codon optimization of the gene sequence and clones the gene into a eukaryotic expression vector, transfects Sf9 cell with recombinant plasmid, and packages to obtain recombinant baculovirus rBAC-RHDV2-VP60 expressing rabbit hemorrhagic disease virus type 2VP60 protein. VP60 protein expressed after recombinant baculovirus inoculated cells is used as antigen, and an aluminum hydroxide gel adjuvant is added to prepare the gene engineering vaccine of rabbit hemorrhagic disease virus type 2VP60 protein. The invention can provide an RHDV2VP60 genetic engineering vaccine with good immune effect and simple and convenient process, which is used for preventing and controlling the new variant rabbit hemorrhagic disease strain RHDV 2.
Description
Technical Field
The invention relates to the technical field of biology, in particular to rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, vaccine, a preparation method and application thereof.
Background
Rabbit Hemorrhagic Disease (RHD) is an acute and high-contact infectious disease caused by Rabbit Hemorrhagic Disease Virus (RHDV), is characterized by respiratory system hemorrhage, hepatic necrosis, parenchymal organ edema and extravasated blood hemorrhage, and has been reported in many countries and regions around the world, the disease is fulminant epidemic, the morbidity and mortality rate is up to 90%, and great economic loss is caused to the Rabbit raising industry. In 1989, the disease was formally classified as a type B infectious disease by the world animal health Organization (OIE), and as a type two infectious disease in our country.
The classical RHDV GI.1 strain is mainly susceptible to rabbits of 2 months age, hares have certain resistance to the strain, and the strain is mainly used for immune prevention by using tissue inactivated vaccines and genetic engineering vaccines at present, and the immune protection efficiency of the vaccine is 100%. In 2010, the first finding in france of RHDV gi.2 was reported, this variant strain was also called RHDV2, which subsequently rapidly spread to many parts of the world. China firstly discovers that the variant strain explodes in a Sichuan rabbit farm in 2020 to cause death of a large number of rabbits, and gene sequencing shows that the virus belongs to RHDV GI.2. The death rate of the RHDV2 infected rabbits reaches 90%, the strain is susceptible to rabbits and hares with age of 7-15 days (non-weaned rabbits), and the typical RHDV vaccine has no cross protection effect on the variant strain. RHDV2 has become popular in China and has great harm to rabbit raising industry in China. Therefore, in order to effectively control the epidemic spread of RHDV2 and reduce the harm of virus to rabbit industry, the rapid development of effective vaccine is urgent.
Disclosure of Invention
In view of the above, the invention aims to provide a rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, a vaccine, a preparation method and an application thereof, wherein the recombinant RHDV2VP60 baculovirus inactivated vaccine has a 100% attack protection rate on RHDV2 and has good immune efficacy.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a recombinant transfer vector of rabbit hemorrhagic disease virus type 2 capsid protein gene, which takes a eukaryotic expression vector as a basic vector, and the basic vector is connected with an RHDV2VP60 gene segment.
Preferably, the eukaryotic expression vector is plasmid pFastBac TM1; the RHDV2VP60 gene fragment is connected to plasmid pFastBac TM1 EcoRI and HindIII sites.
Preferably, the nucleotide sequence of the RHDV2VP60 gene fragment is shown as SEQ ID NO. 4.
The invention provides a construction method of the recombinant transfer vector, which comprises the following steps:
(1) carrying out reverse transcription PCR amplification on RHDV2 virus RNA to obtain cDNA;
(2) using the cDNA as a template, and obtaining an RHDV2VP60 gene fragment through PCR amplification, wherein the nucleotide sequence is shown as SEQID NO. 3;
(3) synthesizing the obtained RHDV2VP60 gene sequence after insect cell codon optimization, wherein the nucleotide sequence is shown as SEQ ID NO.4, cloning the synthesized gene to plasmid pFastBac TM1 between the EcoRI and HindIII restriction sitesGroup transfer vector pFastBacTM1-RHDV2-VP60。
Preferably, the nucleotide sequence of the upstream primer P1 amplified by reverse transcription PCR in the step (1) is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer P2 is shown as SEQ ID NO. 2.
The invention also provides a recombinant baculovirus rBAC-RHDV2-VP60 strain of rabbit hemorrhagic disease virus type 2 capsid protein gene, wherein the recombinant baculovirus comprises the recombinant transfer vector pFastBacTM1-RHDV2-VP60。
The invention provides a construction method of the recombinant baculovirus, which comprises the following steps:
transfer of the recombinant vector pFastBacTMThe 1-RHDV2-VP60 is transformed into E.coli DH10Bac competent cells containing a shuttle vector Bacmid, Sf9 cells are transfected, and the rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus rBAC-RHDV2-VP60 is obtained.
The invention also provides virus-like particles formed by the recombinant RHDV2VP60 protein and the recombinant RHDV2VP60 protein expressed by the recombinant baculovirus.
The invention also provides application of virus-like particles formed by the recombinant transfer vector, the recombinant baculovirus or the recombinant RHDV2VP60 protein and the recombinant RHDV2VP60 protein in preparation of medicines for preventing, diagnosing and treating RHDV2 infection.
The invention also provides a vaccine for preventing and treating rabbit hemorrhagic disease type 2, which comprises the recombinant RHDV2VP60 protein expressed by the recombinant baculovirus rBAC-RHDV2-VP60 and an adjuvant.
Preferably, the adjuvant is aluminum hydroxide gel, and the mass ratio of the RHDV2VP60 protein antigen to the aluminum hydroxide gel is (8.2-9.5): (1.8-0.5).
The invention provides a rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus and a vector inactivated vaccine, wherein an RHDV2VP60 gene sequence is synthesized into a gene after being optimized by insect cell codons and cloned into a baculovirus vector, an Sf9 cell is transfected to obtain a recombinant RHDV2VP60 baculovirus, the recombinant virus is inoculated into an Sf9 cell to induce and express an RHDV2VP60 protein, and the recombinant RHDV2VP60 baculovirus inactivated vaccine is prepared by combining with an aluminum hydroxide gel adjuvant. The vaccine is used for carrying out immune efficacy evaluation tests, the attack protection rate of the recombinant RHDV2VP60 baculovirus inactivated vaccine to RHDV2 is 100%, and the vaccine has good immune efficacy and important application value in the aspects of preventing and treating RHDV 2.
Biological preservation Instructions
The rabbit hemorrhagic disease virus type 2 capsid protein gene recombination baculovirus rBAC-RHDV2-VP60 is classified and named as follows: the autographa californica nuclear polyhedrosis virus rBAC-RHDV2-VP60 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v202035, preservation date 2020, 6/21, preservation address: wuhan university in Wuhan, China.
The 3D7 cell strain is classified and named as follows: the hybridoma cell strain 3D7 is preserved in China center for type culture Collection with a preservation number of CCTCC NO: c2020111, date of deposit 2020, 6/21, deposit address: wuhan university in Wuhan, China.
Drawings
FIG. 1 shows the result of PCR amplification of VP60 gene fragment; wherein M1 is DNA molecular mass standard (DL 15000); m2 is DNA molecular mass standard (DL 2000); 1 is VP60 amplification product;
FIG. 2 shows the restriction enzyme digestion identification result of the recombinant vector Bacmid-RHDV2-VP 60; wherein M is DNA molecular mass standard (DL 15000); 1 is Bacmid-RHDV2-VP60 double enzyme digestion product;
FIG. 3 shows the Western blot detection result of recombinant RHDV2VP60 protein; wherein, 1, recombinant rBAC-RHDV2-VP60 is inoculated with Sf9 cell culture; 2 inoculating Sf9 cell culture for wild baculovirus; sf9 cell culture control 3;
FIG. 4 shows the results of detecting the wild RHDV2 virus particles and the recombinant RHDV2 protein virus-like particles by an electron microscope; wherein A is wild type RHDV2 virus; b is recombinant RHDV2 protein virus-like particle.
Detailed Description
The invention provides a recombinant transfer vector of rabbit hemorrhagic disease virus type 2 capsid protein gene, which takes a eukaryotic expression vector as a basic vector, and the basic vector is connected with an RHDV2VP60 gene segment.
In the invention, the eukaryotic expression vector for loading the RHDV2VP60 gene fragment preferably comprises pFastBac TM1. The RHDV2VP60 gene fragment is preferably connected to plasmid pFastBac TM1 EcoRI and HindIII sites. The invention relates to the plasmid pFastBacTM1 is not particularly limited, and the plasmid pFastBac used in the embodiment of the present invention TM1 is preferably purchased from Invitrogen. In the invention, the nucleotide sequence of the RHDV2VP60 gene fragment is shown as SEQ ID NO. 4.
The invention provides a construction method of the recombinant transfer vector, which comprises the following steps:
(1) carrying out reverse transcription PCR amplification on RHDV2 virus RNA to obtain cDNA;
(2) using the cDNA as a template, and obtaining an RHDV2VP60 gene fragment through PCR amplification, wherein the nucleotide sequence is shown as SEQID NO. 3;
(3) synthesizing the obtained RHDV2VP60 gene sequence after insect cell codon optimization, wherein the nucleotide sequence is shown as SEQ ID NO.4, cloning the synthesized gene to plasmid pFastBac TM1, obtaining a recombinant transfer vector pFastBac between EcoRI and HindIII enzyme cutting sitesTM1-RHDV2-VP60。
In the invention, the RHDV2 virus RNA is preferably derived from RHDV2SC2020/04 strain; the RHDV2SC2020/04 strain is preferably selected from organ tissues such as liver, spleen and the like of acute death rabbit inspected by a certain rabbit farm in Sichuan China, and the gene serial number is MT 383749. The method for extracting RNA from RHDV2 virus is not particularly limited, and the method can be any conventional method for extracting viral RNA in the field. In the specific implementation process of the invention, the Trizol method is preferably adopted for extracting the RHDV2 virus RNA. The invention takes the cDNA as a template, after an RHDV2VP60 gene segment is obtained by PCR amplification, the RHDV2VP60 gene segment is preferably subjected to nucleic acid electrophoresis identification, and whether the amplified RHDV2VP60 gene nucleotide sequence is correct or not is verified.
In the invention, the obtained RHDV2VP60 gene sequence is synthesized after codon optimization. The optimization specifically comprises the following steps: on the premise of not changing the amino acid sequence of the wild type RHDV2, all codons are replaced by codons with the highest frequency of use by insect cells, and on the basis, the following four aspects are adjusted: eliminating more than 12 repetitive sequences; eliminating potential secondary structures; avoid too high local GC and AT content; eliminating potential splice sites. The optimized gene has the total length of 1740bp and the GC content of 52 percent, the EcoRI enzyme cutting site is introduced into the upstream of the optimized gene of the RHDV2, and the HindIII enzyme cutting site is introduced into the downstream of the optimized gene. In the invention, the whole gene synthesis and the sequence determination are both completed in the Kingsler Biotechnology GmbH, and the sequencing result confirms that the synthesis is correct. In the invention, the codon optimization of the amino acid sequence of the wild RHDV2 can guide the regulation and control of protein expression and optimize the expression yield and quality.
The invention also provides a rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus rBAC-RHDV2-VP60, which comprises the recombinant transfer vector pFastBacTM1-RHDV2-VP60。
In the present invention, the recombinant transfer vector pFastBacTM1-RHDV2-VP60 preferably transforms E.coli DH10Bac competent cells containing shuttle vector Bacmid to obtain recombinant baculovirus shuttle plasmid Bacmid-RHDV2-VP 60. The invention preferably transfers the successfully constructed recombinant baculovirus shuttle plasmid into escherichia coli for screening and identification. In the practice of the invention, the screening is preferably a blue-white screening, specifically the recombinant transfer vector pFastBacTMTransferring the 1-RHDV2-VP60 into escherichia coli competent cells, inoculating the escherichia coli competent cells into an LB culture medium containing kanamycin, gentamicin and tetracycline for culture, and screening a white single colony; after further propagation, the recombinant baculovirus shuttle plasmid identified as positive was extracted. In the present invention, the methods and reagents used for extracting the recombinant baculovirus shuttle plasmid are all the methods and reagents which are conventional in the field for extracting baculovirus plasmids. In this embodiment, the extraction is preferably carried out by using the methods and reagents described in the handbook of baculovirus expression systems.
The invention provides a construction method of the recombinant baculovirus, which comprises the following steps:
will weigh heavilyGroup transfer vector pFastBacTMThe 1-RHDV2-VP60 is transformed into E.coli DH10Bac competent cells containing a shuttle vector Bacmid, Sf9 cells are transfected, and the rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus rBAC-RHDV2-VP60 is obtained.
In the present invention, the transfection reagent used for transfecting Sf9 cells is preferably LipofectaminTM3000. The source of the transfection reagent is not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used. In the invention, after the recombinant shuttle plasmid Bacmid-RHDV2-VP60 transfects Sf9 cells, whether the transfection is successful or not is preferably verified in a cell morphology, and the method specifically comprises the following steps: after 5 days of transfection, the cells were observed to swell and become round, the division was stopped, the intercellular tight junctions disappeared, the refractive index of the cells was enhanced, and the normal cells did not have the change, indicating that the transfection was successful. The method of culturing transfected cells in the present invention is not particularly limited, and a conventional method of culturing transfected cells in the art may be used.
The invention also provides virus-like particles formed by the recombinant RHDV2VP60 protein and the recombinant RHDV2VP60 protein expressed by the recombinant baculovirus.
The invention also provides application of virus-like particles formed by the recombinant transfer vector, the recombinant baculovirus or the recombinant RHDV2VP60 protein and the recombinant RHDV2VP60 protein in preparation of medicines for preventing, diagnosing and treating RHDV2 infection. In the invention, the recombinant transfer vector and the recombinant baculovirus take the expressed RHDV2VP60 protein as immunogen to prepare the RHDV2VP60 baculovirus inactivated vaccine for preventing, diagnosing and treating RHDV 2. In the invention, the virus-like particles formed by the recombinant RHDV2VP60 protein are virus-like particles with the diameter of about 32-36 nm, are similar to the RHDV2 particles, and can be directly used as immunogen to prepare the RHDV2VP60 baculovirus inactivated vaccine for preventing, diagnosing and treating RHDV 2.
The invention also provides a vaccine for preventing and treating rabbit hemorrhagic disease type 2, which comprises the recombinant RHDV2VP60 protein and an adjuvant.
The invention preferably uses the recombinant RHDV2VP60 protein as immunogen to prepare the RHDV2VP60 baculovirus inactivated vaccine. In the invention, the adjuvant is preferably aluminum hydroxide gel; the mass ratio of the RHDV2VP60 protein antigen to the aluminum hydroxide gel is preferably (8.2-9.5): (1.8-0.5), and more preferably 9: 1. The injection dose of the vaccine of the invention is preferably 1 ml/tube.
The RHDV2VP60 gene sequence is obtained through RT-PCR amplification, the gene is synthesized after codon optimization and cloned into a baculovirus expression vector to construct recombinant RHDV2VP60 baculovirus as vaccine virus seeds, RHDV2VP60 protein is obtained through inoculation insect cell expression and is used as immunogen, the recombinant RHDV2VP60 baculovirus vector inactivated vaccine is prepared after mixing with adjuvant, the attack virus immune protection determination shows that the rabbit immune vaccine is attacked, the mortality of a control group is 100 percent, the immune group is totally survived, the protection rate is 100 percent, the RHDV2 can be effectively prevented and controlled through research and development of the vaccine, and the healthy development of the rabbit breeding industry in China is maintained.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Separation and identification of novel mutant rabbit hemorrhagic disease virus RHDV2 strain from Sichuan China
In 4 months of 2020, a novel variant rabbit hemorrhagic disease virus is separated from liver, spleen and other organ tissues of acute death rabbit collected from a certain rabbit farm in Sichuan by the veterinary institute of the academy of agricultural sciences in Jiangsu province, and is named as RHDV2SC2020/04 strain, and the gene sequence number is: MT 383749.
Example 2
Construction and identification of recombinant RHDV2VP60 baculovirus
(1) Amplification of RHDV2VP60 Gene
Mixing water treated by diethyl pyrocarbonate DEPC with liver tissue infected and killed by RHDV2 according to the mass ratio of 9:1, grinding into suspension, placing into a DEPC-treated 1.5ml EP tube, and repeatedly freezing and thawing at-20 deg.C for 3 times; centrifuging for 20 minutes at 7200r/min at the temperature of 2-8 ℃; taking 200 mu l of supernatant, adding 700 mu l of Trizol, shaking and mixing uniformly, and standing for 10 minutes at room temperature; adding 300 mu l of chloroform, oscillating vigorously, and standing at room temperature for 30 minutes; centrifuging for 15 minutes at 2-8 ℃ and 10000 r/min; taking 750 mu l of upper-layer water phase, adding 750 mu l of isopropanol, uniformly mixing, standing for 15 minutes at the temperature of below-20 ℃; centrifuging for 10 minutes at the temperature of 2-8 ℃ and the speed of 10000 r/min; removing supernatant, adding 1ml of 75% ethanol, and washing; centrifuging for 5 minutes at 2-8 ℃ at 8000r/min, and discarding the supernatant; the mixture was dried at room temperature for 20 minutes to obtain total RNA of RHDV2, and 10. mu.l of DEPC-treated water was added thereto to dissolve it sufficiently.
Design of synthetic upstream primer P1:
5′-ATAGAATTCATGGAGGGCAAAGCCCGC-3′(SEQ ID NO.1)；
the downstream primer P2:
5′-GCAAGCTTTCAGACATAAGAAAAAC-3′(SEQ ID NO.2)。
EcoRI restriction enzyme cutting site is added to the 5 'end of the upstream primer, and HindIII restriction enzyme cutting site is introduced to the 5' end of the downstream primer.
Performing reverse transcription PCR to amplify capsid protein VP60 gene, wherein the reverse transcription system comprises 10 × Buffer 2 μ L, 10mmol/L dNTPs 2 μ L, downstream primer 1 μ L, RNase inhibitor 0.5 μ L, RNA template 12 μ L, AMV reverse transcriptase 0.5 μ L, DEPC treated H2O2 mul, the total volume is 20 mul, the mixture is instantly centrifuged and mixed, reacted for 15 minutes at 65 ℃, incubated for 60 minutes at 42 ℃, reacted for 5 minutes at 95 ℃ and stored below minus 20 ℃ for standby.
The PCR Reaction system was 10 × Reaction buffer 2.5. mu.l, 25mmol/L MgCl21.5. mu.l, 2.5mmol/L dNTPs 0.5. mu.l, 50mmol/L P10.5.5. mu.l, 50mmol/L P20.5.5. mu.l, template cDNA 4.0. mu.l, ddH2O 15μl、EX TaqTM0.5 mul of polymerase, the total volume of 25 mul, mixing by instant centrifugation, and performing PCR amplification reaction by a hot cover: denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 1 min, annealing at 57 ℃ for 1 min, extension at 72 ℃ for 2 min, 30 cycles; extension at 72 ℃ for 10 min. The PCR product is identified by agarose gel electrophoresis, the result is shown in figure 1, the size of the RHDV2VP60 gene fragment obtained by amplification is 1740bp, and the sequencing shows that the nucleotide sequence is SEQ ID NO.3。
(2) Recombinant transfer vector pFastBacTMConstruction and identification of 1-RHDV2-VP60
Carrying out insect cell codon optimization on the obtained RHDV2VP60 gene sequence to synthesize a gene, wherein the nucleotide sequence is shown as SEQ ID NO.4, and digesting donor plasmid pFastBac by using restriction enzymes EcoRI and HindIII respectivelyTM1, synthesizing RHDV2VP60 gene fragment of the gene after codon optimization, connecting for 12 hours by T4 DNA Ligase at 2-8 ℃, transforming escherichia coli DH5 α competent cells, screening positive clone to obtain a recombinant transfer vector pFastBacTM1-RHDV2-VP60。
(3) Construction and identification of recombinant baculovirus shuttle plasmid
Will pFastBacTME.coli DH10Bac competent cells containing shuttle vector Bacmid are transformed by 1-RHDV2-VP60 plasmid, after culturing for 4 hours at 37 ℃ in LB liquid culture medium, 100 mul of transformation product is taken and smeared on an LB plate containing 50 mu g/ml kanamycin, 7 mu g/ml gentamicin, 10 mu g/ml tetracycline, 100 mu g/ml X-gal and 20 mu g/ml IPTG, after culturing for 48 hours at 37 ℃, white colonies are selected, inoculation and passage once again are carried out on the same LB plate containing three antibiotics to obtain pure culture, the pure culture is inoculated in LB liquid culture medium containing three antibiotics, shaking (180r/min) at 37 ℃ for 24 hours, and Bacmid plasmid is extracted by a BAC/PAC large-scale plasmid extraction kit to obtain recombinant baculovirus shuttle plasmid Bacmid-RHDV2-VP 60. After the recombinant baculovirus shuttle plasmid Bacmid-RHDV2-VP60 is subjected to double enzyme digestion by EcoR I/HindIII, the result of agarose gel electrophoresis verification is shown in FIG. 2, and two fragments with the sizes of 4800bp and 1740bp respectively can be seen.
(4) Acquisition of recombinant RHDV2VP60 baculovirus
With LipofectaminTM3000 is a transfection reagent, according to LipofectaminTM3000 reagent transfection method Bacmid-RHDV2-VP60 was transfected into Sf9 monolayers. After transfection, observation was carried out every 12 hours until cytopathic effect became evident, and cells and supernatant were collected and stored as a recombinant baculovirus stock solution at below-20 ℃. The recombinant baculovirus of RHDV2VP60 gene is named as autographa californica nuclear polyhedrosis virus rBAC-RHDV2-VP60 strain, and the recombinant baculovirus is named as autographa californica nuclear polyhedrosis virus rBAC-RHDV2-VP60 strainThe recombinant virus is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university with the preservation number of CCTCC NO: v202035.
(5) Passage and preliminary identification of recombinant viruses
Collecting Sf9 cells of the 2 nd generation inoculated recombinant virus, extracting cell DNA, and carrying out PCR identification on the recombinant virus by using an upstream primer P3 with a target gene sequence shown as SEQ ID NO.5 and a downstream primer P4 with a sequence shown as SEQ ID NO. 6. The agarose gel electrophoresis identification shows that the electrophoresis band is 1740bp, which indicates that the target gene is expressed in Sf9 cells and the recombinant baculovirus is successfully constructed.
(6) Amplification and sequencing of VP60 Gene
Extracting recombinant virus DNA from the recombinant baculovirus according to a BAC/PAC large-scale plasmid extraction kit, amplifying the VP60 gene by using an upstream primer P3 with a target gene sequence shown as SEQ ID NO.5 and a downstream primer P4 with a sequence shown as SEQ ID NO.6, and cloning and then carrying out sequencing verification. The VP60 gene amplification band in the recombinant baculovirus is positive, the size is 1740bp, and the sequencing result is completely coincided with the optimized VP60 sequence in the virus RHDV 2.
Example 3
Expression and identification of recombinant RHDV2VP60 protein
(1) Western blot identification of expression products
The experimental group was inoculated with recombinant RHDV2VP60 baculovirus cell culture, while the two control groups were inoculated with Sf9 cell lysate and Sf9 cell lysate of wild Bacmid, respectively. Respectively subpackaging the products of the experimental group and the control group in 1.5ml centrifuge tubes, centrifuging for 5 minutes at 3000r/min, discarding the supernatant, resuspending the cells by using 0.01mol/L PBS solution with the pH value of 7.0-7.2, washing for 2 times, dissolving in 500 mul PBS, repeatedly freezing and thawing for 3 times, ultrasonically cracking, centrifuging for 5 minutes at 3000r/min, taking the supernatant, adding a loading buffer solution, boiling for 5 minutes, centrifuging for 1 minute at 11000r/min, and taking 20 mul of the supernatant for SDS-PAGE. The immunity transfer adopts a semi-dry transfer method. Western blot identification was performed using anti-rabbit hemorrhagic disease virus type 2 monoclonal antibody 3D7 (secreted from 3D7 cell line) as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the VP60 gene expressed a specific band with a size of about 60kDa in insect cells, whereas both control samples inoculated with the Sf9 cell lysate and the Sf9 cell lysate of wild Bacmid did not show the corresponding bands, and immunoblotting was matched with the position of the electrophoresed band of interest, indicating that the target protein was expressed.
(2) Blood coagulation potency
And (3) repeatedly freezing and thawing the recombinant RHDV2VP60 baculovirus cell culture for 3 times, and centrifuging for 5 minutes at 1000g to obtain a supernatant, namely the recombinant VP60 protein. According to the 50-well plate method in the erythrocyte agglutination assay. Taking 50 mu L of recombinant VP60 protein to a reaction plate, diluting by 2 times using PBS (phosphate buffer solution) with 0.01mol/L and pH value of 7.0-7.2, adding 1% human O-shaped erythrocyte suspension with the same volume, vibrating uniformly, and reacting for 45 minutes at 2-8 ℃ to observe the result. The result is judged after the reaction plate is inclined, and the result shows that the agglutination titer of the recombinant RHDV2VP60 protein to human O-type red blood cells is more than or equal to 1: 64.
(3) Observation by electron microscope
Inoculation of recombinant RHDV2VP60 baculovirus with serum-free Medium Sf-900TMII SFM cultured Sf9 insect cells are centrifuged at 3000r/min for 5 minutes, supernatant culture solution is discarded, bottom cells are resuspended by PBS and washed twice, centrifuged at 3000r/min for 5 minutes, the PBS is resuspended, frozen and thawed at-20 ℃ for 3 times, then centrifuged at 11000r/min for 3 minutes, supernatant is taken and dropped on a copper mesh for adsorption for 2 minutes, redundant samples are removed by filter paper, then 2% phosphotungstic acid staining solution is dropped on the copper mesh for fixation for 2 minutes, finally redundant phosphotungstic acid staining solution is removed, the cells are dried at room temperature for 5 minutes and then observed on a transmission electron microscope, and the result is shown in figure 4.
As shown in the electron microscope result of FIG. 4, when Sf9 cells are inoculated with the recombinant virus, the target gene is expressed, and the expressed protein can form virus-like particles with the diameter of about 32-36 nm, which is similar to RHDV2 particles.
Example 4
1. Preparation of recombinant RHDV2VP60 baculovirus vector inactivated vaccine
Culturing the cells in suspension in SF-SFM medium until the cell concentration is about 2 × 10 after 24 hours of culture6One/ml, 1% of the total volume inoculatedThe recombinant RHDV2VP60 baculovirus is cultured continuously under the same conditions, and the cell culture is harvested when the cytopathic effect reaches more than about 85 percent after continuous observation for 5 days. And (3) measuring the hemagglutination titer of a cell culture, namely the expressed recombinant 2-type VP60 protein antigen, wherein the hemagglutination titer of the antigen to human O-type erythrocytes is not lower than 1:64, inactivating the antigen by using formaldehyde solution, and mixing the antigen and aluminum hydroxide gel according to the mass ratio of 9:1 to prepare the recombinant RHDV2VP60 baculovirus inactivated vaccine.
2. Application of recombinant RHDV2VP60 baculovirus vector inactivated vaccine
The HI titer of rabbit hemorrhagic disease virus type 2 antibody is determined before immunization by 40 healthy and susceptible rabbits with the age of 2 months, and the obtained titer is not higher than 1: 2. Dividing 40 healthy and susceptible rabbits with the age of 2 months into an immunization group and a control group, wherein the immunization group comprises 4 groups, each group comprises 5 rabbits, the control group comprises 4 groups, each group comprises 5 rabbits, the immunization group is inoculated with 1 ml/of the recombinant RHDV2VP60 baculovirus inactivated vaccine, the control group comprises physiological saline with the same volume, the two groups are injected with 1ml of RHDV2 hepatotoxin subcutaneously at the neck of the rabbit after 3 days, 7 days, 14 days and 21 days of immunization respectively to carry out a virus challenge test, observing for 21 days, and recording the result to obtain the table 1.
TABLE 1 detection of immune protection effect of inactivated vaccine of rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus vector
As can be seen from the above table 1, when the vaccine of the present invention is used for combating poison 3 days after immunization, the control group died completely, and the immune group died 3 mice; after 7 days, 14 days and 21 days of vaccine immunization, the control group rabbits all die, and the immunized rabbits all survive healthily. Wherein, the dead rabbits in the control group have the typical pathological changes of rabbit hemorrhagic disease, and the human O-type erythrocyte agglutination tests of dead rabbit liver suspensions are all positive.
The embodiment shows that the recombinant RHDV2VP60 baculovirus is constructed by the molecular biology technology, the rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus vector inactivated vaccine is prepared for the first time by taking the virus as a vaccine virus seed, and an immune efficacy evaluation test is carried out by utilizing the vaccine, so that the attack protection rate of the recombinant RHDV2VP60 baculovirus inactivated vaccine to RHDV2 is 100 percent, the vaccine has good immune efficacy, the immune production period is 7 days after immunization, and the vaccine has important application value in the aspect of preventing and treating RHDV 2.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> rabbit hemorrhagic disease virus type 2 capsid protein gene recombination baculovirus, vaccine, preparation method and application thereof
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ccgttcacag ctgttctaag tcaaatgtac gctggctggg caggtggcat gcagttccgg 360
ttcatagttg ctgggtcagg tgtcttcggt gggcgtctgg tcgcagcggt tataccaccg 420
ggcattgaga ttgggccagg tttggaagtc agacaattcc ctcatgttgt cattgacgct 480
cgttcactcg aaccagtcac catcaccatg ccggacttgc gccccaacat gtaccaccca 540
acaggcaacc ctggcctcgt tcccacgttg gtcctgagcg tttacaacaa cctcatcaac 600
ccatttggtg gatccacgag cgcaatccag gtcacggtgg aaacaaggcc cagtgaggac 660
tttgagtttg tgatgatccg tgccccctcc agtaagaccg ttgactcgat ctcgcccgca 720
gatctcctca caaccccagt tctcactggg gttggcaccg ataacagatg gaatggtgag 780
atagttgggc tgcaaccagt ccccggtggg ttttctacgt gcaacagaca ctggaactta 840
aacggtagca catatggatg gtcaagcccg cggtttgccg ccattgacca cgacagaggc 900
aacgcaagtt tccctggaag cagttcgtca aacgtgcttg agctttggta tgctagtgcc 960
gggtctgcag ctgacaaccc catctcccaa attgctccag atggtttccc tgacatgtca 1020
tttgtaccct tcagcggtat caccatccct accgcagggt gggtcgggtt cggtgggatc 1080
tggaacagca gtaatggtgc cccctacgtc acgaccatgc aggcttatga gttgggtttt 1140
gccactggag taccgagcaa cccccaaccc accaccacca cttcaggggc tcagattgtt 1200
gccaagtcca tctatggcgt tgcaaatggc ataaaccaga caacagccgg gttgtttgtg 1260
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atcatgttcg cgtctgttgt taggcgcacc ggcgacatca acgctgaggc cggttcaact 1440
aacggaaccc agtacggcgc gggatcacaa ccgctgccgg tgacaattgg actttcactg 1500
aacaattatt catcggcact tatgcctggg cagttcttcg tttggcagct aaactttgct 1560
tccggcttca tggaacttgg cttgagtgtt gatggatact tctacgcggg aacaggggct 1620
tcagccaccc tcattgacct gtcagacctt gttgacatcc gccctgtggg gcccagaccg 1680
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atcaatccgt tcggagggag tacctcggct atccaagtga ctgttgaaac gcgtccgtcc 660
gaggattttg aatttgtgat gataagggcg ccttcatcga aaacagtcga tagtatatcc 720
ccggctgacc tgttaacgac accagtcttg acgggtgtcg gcacagacaa ccgatggaat 780
ggggagatcg taggacttca gcccgtcccg ggaggatttt ctacatgtaa tagacattgg 840
aacctgaacg gctcaacata cggatggagt tctccccgtt ttgcagccat cgatcatgac 900
cggggaaatg cgtcgttccc ggggagctcg tcctcgaacg tattggaact ttggtatgct 960
tctgccggat cagccgcgga taatcctata agtcagatag caccggacgg ttttcctgat 1020
atgtcctttg tgcctttctc agggatcacg attccaactg ccggttgggt aggcttcggc 1080
ggtatctgga acagcagcaa tggtgctccc tacgtcacga ccatgcaagc ctatgaattg 1140
ggtttcgcaa cgggggtgcc tagcaacccg caacccacaa cgaccacctc cggcgcccag 1200
attgtggcaa agtcgatata tggagtcgct aatggcatta atcagactac cgctggatta 1260
tttgttatgg cttcaggtgt aatttctaca ccgaactcct cggcaaccac atatactcct 1320
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accccaatta tgttcgccag cgtggtgagg cgcactggtg atattaatgc agaagccggg 1440
agcaccaatg ggactcaata cggtgcgggt tctcagcctc tgcctgttac aattggcctc 1500
tcactaaaca actattcgtc tgctctcatg ccaggacagt tctttgtatg gcaattaaat 1560
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ggagcctcgg cgactctgat agatctgtca gatctcgtgg acatcaggcc cgtgggcccg 1680
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Claims (11)
1. The recombinant transfer vector of the rabbit hemorrhagic disease virus type 2 capsid protein gene is characterized in that the recombinant transfer vector takes a eukaryotic expression vector as a basic vector, and the basic vector is connected with an RHDV2VP60 gene segment.
2. The recombinant transfer vector of claim 1, wherein the eukaryotic expression vector is the plasmid pFastBacTM1; the RHDV2VP60 gene fragment is connected to the plasmid pFastBacTM1, EcoR I and HindIII sites.
3. The recombinant transfer vector of claim 1, wherein the nucleotide sequence of the RHDV2VP60 gene fragment is shown as SEQ ID No. 4.
4. A method of constructing the recombinant transfer vector of any one of claims 1-3, comprising the steps of:
(1) carrying out reverse transcription PCR amplification on RHDV2 virus RNA to obtain cDNA;
(2) using the cDNA as a template, and obtaining an RHDV2VP60 gene fragment through PCR amplification, wherein the nucleotide sequence is shown as SEQ ID NO. 3;
(3) synthesizing the obtained RHDV2VP60 gene sequence after insect cell codon optimization, wherein the nucleotide sequence is shown as SEQ ID NO.4, cloning the synthesized gene to plasmid pFastBacTM1, obtaining a recombinant transfer vector pFastBac between the EcoR I and Hind III enzyme cutting sitesTM1-RHDV2-VP60。
5. The construction method according to claim 4, wherein the nucleotide sequence of the upstream primer P1 amplified by reverse transcription PCR in the step (1) is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer P2 is shown as SEQ ID NO. 2.
6. A rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus rBAC-RHDV2-VP60, wherein said recombinant baculovirus comprises the recombinant transfer vector of claim 1.
7. A method of constructing a recombinant baculovirus as defined in claim 6, comprising the steps of:
transforming the recombinant transfer vector of claim 1 into E.coli DH10Bac competent cells containing a shuttle vector Bacmid, and transfecting Sf9 cells to obtain rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus rBAC-RHDV2-VP 60.
8. The virus-like particle formed by recombinant RHDV2VP60 protein and recombinant RHDV2VP60 protein expressed by the recombinant baculovirus of claim 6.
9. Use of the recombinant transfer vector of claim 1, the recombinant baculovirus of claim 6 or the virus-like particles formed by the recombinant RHDV2VP60 protein and the recombinant RHDV2VP60 protein of claim 8 in the preparation of medicaments for preventing, diagnosing and treating RHDV2 infection.
10. A vaccine for preventing rabbit hemorrhagic disease type 2, which is characterized in that the vaccine comprises the recombinant RHDV2VP60 protein of claim 8 and an adjuvant.
11. The vaccine for preventing and treating rabbit hemorrhagic disease type 2 according to claim 10, wherein the adjuvant is aluminum hydroxide gel, and the mass ratio of the RHDV2VP60 protein antigen to the aluminum hydroxide gel is (8.2-9.5): (1.8-0.5).
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