CN112375126B

CN112375126B - Marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine

Info

Publication number: CN112375126B
Application number: CN202011220361.9A
Authority: CN
Inventors: 贺笋; 李俊辉; 王遵宝; 李延涛; 程兰玲; 刘宏; 郑侃; 王雨朦
Original assignee: Tiankang Biopharmaceutical Co ltd
Current assignee: Tiankang Biopharmaceutical Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2022-07-29
Anticipated expiration: 2040-11-04
Also published as: CN112375126A

Abstract

The invention relates to the field of vaccines, and particularly provides a marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine. The invention provides a marked classical swine fever virus recombinant E2 protein of SEQ ID No.1, the nucleic acid molecule sequence of the protein is shown in SEQ ID No.2, the high-efficiency expression of target protein can be realized through a baculovirus-insect cell expression system, the marked classical swine fever virus recombinant E2 protein can be used for preparing a marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, and the immune efficacy equivalent to subunit vaccines and live vaccines can be realized only by immunizing once. In addition, identification of non-local marker vaccine strains or wild strains infected by pigs can be realized through detection of WH303 antibodies, so that effective purification of swine fever is realized.

Description

Marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine

Technical Field

The invention relates to the field of vaccines, and in particular relates to a marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine.

Background

At present, the conventional hog cholera vaccine has three production methods, namely, inoculating the seed virus to healthy rabbits for propagation in vivo, collecting spleens and lymph nodes of the rabbits, grinding and freeze-drying to obtain the hog cholera vaccine. Secondly, inoculating the seed virus into primary cells or passage cells, collecting supernatant and freeze-drying to prepare the virus vaccine. The first production method requires a large amount of rabbits to collect the antigen, and in addition, the quality of the antigen is not uniform due to the influence of individual differences of the rabbits, and the effect of the vaccine is finally influenced. The second production method needs bovine serum, which not only introduces heterologous animal protein into the product but also has possibility of polluting bovine viral diarrhea, thus affecting the safety of the vaccine. In addition, after the live vaccine is used for immunizing piglets, the influence of maternal antibodies causes poor immune effect or immune failure. The third is a conventional subunit vaccine of hog cholera E2. The swine fever E2 subunit vaccine is immunized twice to generate effective protection under the attack of strong virus; although the differential diagnosis can be carried out by the hog cholera Erns antibody, the application of the Erns antibody can not effectively carry out the differential diagnosis because the Erns antibody has short duration and even some strains can not produce the Erns antibody.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a marked classical swine fever virus recombinant E2 protein.

The second object of the present invention is to provide a nucleic acid molecule.

It is a third object of the present invention to provide a biological material related to a nucleic acid molecule.

The fourth purpose of the invention is to provide a recombinant baculovirus expressing the marked classical swine fever virus recombinant E2 protein.

The fifth object of the present invention is to provide the use of a labeled recombinant baculovirus.

The sixth purpose of the invention is to provide a preparation method of the marked classical swine fever virus recombinant E2 protein.

The seventh purpose of the invention is to provide a recombinant baculovirus inactivated vaccine marked with classical swine fever virus E2 protein.

The eighth purpose of the invention is to provide a preparation method of the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine.

The ninth purpose of the invention is to provide a method for identifying the non-labeled vaccine strain or wild strain infected by the pig.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a classical swine fever virus recombinant E2 protein, wherein the amino acid sequence of the recombinant E2 protein is shown as SEQ ID No. 1.

The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2, and the nucleic acid molecule codes the classical swine fever virus recombinant E2 protein.

Biological materials related to the above nucleic acid molecules include the following:

(a) an expression cassette comprising a nucleic acid molecule of the invention;

(b) a recombinant vector comprising a nucleic acid molecule of the invention or an expression cassette of (a);

(c) a recombinant eukaryotic cell comprising a nucleic acid molecule of the invention, an expression cassette as in (a) or a recombinant vector as in (b).

A recombinant baculovirus expressing the labeled classical swine fever virus recombinant E2 protein of the present invention, the recombinant baculovirus comprising the nucleic acid molecule of the present invention;

preferably, the preservation number of the recombinant baculovirus is CCTCC NO: v202045.

The recombinant baculovirus of the invention is applied to the following (a) or (b):

(a) preparing a hog cholera virus vaccine;

(b) preparing a reagent for identifying the non-labeled vaccine strain or wild strain infected by the pig.

The preparation method of the recombinant E2 protein comprises the steps of transfecting insect cells with the recombinant baculovirus, and culturing and expressing to obtain the recombinant E2 protein;

preferably, the insect cell is an Sf-9 cell;

preferably, the process for culturing expression comprises: inoculating virus with MOI of 2, setting dissolved oxygen of bioreactor at 40-50%, setting temperature at 26-28 deg.C, setting rotation speed at 40-120rpm, culturing for 90-110 hr, and harvesting supernatant;

Preferably, the step of protein purification is also included after the culture expression.

A marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine comprises an adjuvant and the marked recombinant E2 protein;

preferably, adjuvants include ISA201VG, ISA563VG or ISA660VG, further preferably ISA563 VG;

preferably, the mass ratio of the adjuvant to the recombinant E2 protein is 1: 0.5-1.5.

The preparation method of the marked inactivated vaccine is obtained by producing and preparing the marked recombinant baculovirus.

Further, the culture medium is prepared from a culture medium with a preservation number of CCTCC NO: v202045;

preferably, the recombinant baculovirus is transfected into insect cells, and is cultured and expressed to obtain recombinant E2 protein which is emulsified with an adjuvant;

preferably, the insect cell is an Sf-9 cell;

preferably, the mass ratio of the adjuvant to the labeled recombinant E2 protein is 1: 0.5-1.5.

A method for identifying a non-local marker vaccine strain or a wild strain for pig infection is characterized in that whether a WH303 antibody exists in a pig body is detected, and a positive result shows that the pig is infected with the non-local marker vaccine strain or the wild strain;

the non-labeled vaccine strain is the labeled inactivated vaccine of the invention.

Compared with the prior art, the invention has the beneficial effects that:

the inventor optimizes the E2 gene according to codon preference in insect cells on the basis of referring to E2 genes of classical strains and epidemic strains at home and abroad, and simultaneously changes the coded TAVSPTTLR epitope into ATVSPTTRL so as to finally obtain the nucleic acid molecule (SEQ ID NO.2) for coding the marked classical swine fever virus recombinant E2 protein.

The marked classical swine fever virus recombinant E2 protein (SEQ ID NO.1) provided by the invention can be obtained by expressing the nucleic acid molecule through a baculovirus-insect cell expression system, so that the expression level of a target recombinant E2 protein is obviously improved compared with that of the prior art, and the recombinant baculovirus with the expression level of more than 120 microgram/ml is obtained through screening, such as the recombinant baculovirus with the preservation number of CCTCC NO: labeled recombinant baculovirus of V202045. In addition, the preparation method of the recombinant E2 protein provided by the invention can further improve the culture expression quantity of the marked recombinant baculovirus to be more than 140 microgram/ml.

The marked classical swine fever virus recombinant E2 protein can be used for preparing a marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, the marked inactivated vaccine is one of novel subunit marked vaccines, and the marked inactivated vaccine can have the equivalent immune efficacy to a subunit vaccine and a live vaccine only by immunization once. In addition, because the mutation of the linear epitope WH303 of the recombinant E2 protein in the marked inactivated vaccine does not generate an antibody which reacts with the epitope after immunizing pigs, and a wild strain, a live vaccine or a conventional E2 protein can generate a corresponding antibody, the identification of non-marked vaccine strains or wild viruses infected by the pigs can be realized through the detection of the WH303 antibody, so that the effective purification of the swine fever is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following descriptions are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a PCR identification of the classical swine fever virus E2 recombinant baculovirus of example 4, wherein, 1. negative control; 2. a positive control; 3, DNA Marker; 4. classical swine fever virus E2 recombinant baculovirus;

FIG. 2 shows the immunological identification of the classical swine fever virus E2 recombinant baculovirus in example 4, wherein 1-5 are all the proteins E2 expressed by the classical swine fever virus E2 recombinant baculovirus.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as is well understood by those of skill in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention provides a marked classical swine fever virus recombinant E2 protein, the amino acid sequence of which is shown in SEQ ID NO. 1. The nucleotide sequence of the nucleic acid molecule for coding the recombinant E2 protein is preferably the nucleic acid molecule shown in SEQ ID NO. 2.

The inventor optimizes the E2 gene according to codon preference in insect cells on the basis of referring to E2 genes of classical strains and epidemic strains at home and abroad, and simultaneously changes the coded TAVSPTTLR epitope into ATVSPTTRL so as to finally obtain the nucleic acid molecule (SEQ ID NO.2) for encoding the classical swine fever virus recombinant E2 protein.

TTAFLICLVKVLRGQIVQGVIWLLLVTGAQGRLACKEDYRYAISSTD EIGLLGAGGLTTTWKEYTHDLQLNDGTVKATCVAGSFKITALNAVSRRY LASLHKKALPTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTRPVVKGK YNATLVNGSAFYLVCPIGWTGVIECATVSPTTRLTEVVKTFRRDKPFPHR MNCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGLVKQCRWCGFDF NEPDGLPHYPIGKCILANETSYRVVDSTDCNRDGVVISTEGSHECLIGNT TVRVHASDERLGPMPCRPKEIVSSAGPAMKTSCTFNYAKTLKNRYYEPR DSYFQQYMLKGEYQYWFDLDATDHHSDYFAEF(SEQ ID NO.1)。

ACTACTGCTTTCCTGATCTGCCTGGTGAAGGTGCTGAGAGGTCAG ATCGTGCAAGGTGTGATCTGGCTGCTGCTGGTGACTGGTGCTCAAGGT AGACTGGCTTGCAAGGAGGACTACAGATACGCTATCTCCTCCACTGAC GAGATCGGTCTGCTGGGTGCTGGTGGTCTGACTACTACTTGGAAGGA GTACACTCACGACCTGCAGCTGAACGACGGTACTGTGAAGGCTACTT GCGTGGCTGGTTCCTTCAAGATCACTGCTCTGAACGCTGTGTCTAGAA GATACCTGGCTTCCCTGCACAAGAAGGCTCTGCCAACTTCCGTGACTT TCGAGCTGCTGTTCGACGGTACTAACCCATCCACTGAGGAGATGGGTG ACGACTTCGGTTTCGGTCTGTGCCCATTCGACACTAGACCTGTGGTGA AGGGTAAGTACAACGCTACTCTGGTGAACGGTTCCGCTTTCTACCTGG TGTGCCCAATCGGTTGGACTGGTGTGATCGAGTGCGCTACTGTGTCCC CAACTACTAGACTGACTGAGGTGGTGAAGACTTTCAGAAGAGACAAG CCATTCCCACACAGAATGAACTGCGTGACTACTACTGTGGAGAACGA GGACCTGTTCTACTGCAAGCTGGGTGGTAACTGGACTTGCGTGAAGG GTGAGCCTGTGGTGTACACTGGTGGTCTGGTGAAGCAGTGCAGATGG TGCGGTTTCGACTTCAACGAGCCTGACGGTCTGCCACACTACCCAATC GGTAAGTGCATCCTGGCTAACGAGACTTCCTACAGAGTGGTGGACTC CACTGACTGCAACAGAGACGGTGTGGTGATCTCCACTGAGGGTTCCC ACGAGTGCCTGATCGGTAACACTACTGTGAGAGTGCACGCTTCCGAC GAGAGACTGGGTCCAATGCCATGCAGACCAAAGGAGATCGTGTCCTC CGCTGGTCCTGCTATGAAGACTTCCTGCACTTTCAACTACGCTAAGAC TCTGAAGAACAGATACTACGAGCCAAGAGACTCCTACTTTCAGCAGT ACATGCTGAAGGGTGAGTATCAGTACTGGTTCGACCTGGACGCTACTG ACCACCACTCCGACTACTTCGCTGAGTTC(SEQ ID NO.2)。

The invention also protects biological materials related to the nucleic acid molecules, which comprise the following components:

(a) an expression cassette comprising the nucleic acid molecule of claim 2;

(b) a recombinant vector comprising the nucleic acid molecule of claim 2 or the expression cassette of (a);

(c) a recombinant eukaryotic cell comprising the nucleic acid molecule of claim 2, the expression cassette of (a), or the recombinant vector of (b).

The "expression cassette" in the present invention comprises a polynucleotide sequence encoding the classical swine fever virus recombinant E2 protein to be expressed and sequences controlling its expression, such as a promoter and optionally an enhancer sequence, including any combination of cis-acting transcriptional control elements. Sequences that control the expression of a gene (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory units are located upstream of and operably linked to the coding sequence of the gene. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.

The vector of the "recombinant vector" of the present invention may be a plasmid, a phage, or a virus.

The host cell of the "recombinant eukaryotic cell" of the present invention may be an insect cell or the like.

The invention also provides a recombinant baculovirus expressing the marked classical swine fever virus recombinant E2 protein, which contains the nucleic acid molecule and is used for expressing the classical swine fever recombinant E2 protein. Compared with the prior art, the protein expression quantity of the recombinant baculovirus is obviously improved, the recombinant baculovirus with the expression quantity of more than 120 micrograms/ml can be obtained through experiments by the inventor, and the preservation number CCTCC NO: the recombinant baculovirus of V202045. The recombinant baculovirus marked with classical swine fever virus E2 protein provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 13 days of 2020, and the preservation number is as follows: CCTCC NO: v202045, specifically classified as a recombinant baculovirus tagged with hog cholera virus E2 protein, accession number: the preservation center of Wuhan university in Wuhan City of Hubei province in China.

The recombinant baculovirus provided by the invention can be used for preparing a classical swine fever virus vaccine and also can be used for preparing a reagent for identifying a non-labeled vaccine strain or a wild strain infected by a pig.

In addition, the invention also provides a method for preparing the recombinant E2 protein by using the marked recombinant baculovirus, which comprises the following steps: transfecting the recombinant baculovirus into insect cells, and culturing and expressing to obtain the recombinant baculovirus.

In a preferred embodiment, the insect cell is an Sf-9 cell.

In a more preferred embodiment, the process of culturing expression is specifically: inoculating virus with MOI of 2, setting dissolved oxygen of bioreactor at 40-50%, temperature at 26-28 deg.C, rotation speed at 40-120rpm, culturing for 90-110 hr, and harvesting supernatant. The process condition can further improve the culture expression quantity of the recombinant baculovirus to be more than 140 micrograms/ml.

In one embodiment, after the insect cells are transfected by the recombinant baculovirus, the recombinant baculovirus is cultured and expressed, and the supernatant is the target recombinant E2 protein (containing the recombinant baculovirus); on the basis, purified recombinant E2 protein can also be obtained by further protein purification steps. Both can be used for later stage vaccine preparation, in the invention, the antigen in the later stage marker inactivated vaccine preparation can be culture expressed supernatant (virus inactivation is needed), and can also be purified recombinant E2 protein.

The marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine provided by the invention is one of novel subunit marked vaccines, and can have the equivalent immune efficacy with subunit vaccines and live vaccines only by immunization once. The adjuvant in the vaccine can be ISA201VG, ISA563VG or ISA660VG, and more preferably ISA563 VG. In addition, because the linear epitope WH303 mutation of the recombinant E2 protein in the marked inactivated vaccine provided by the invention does not generate an antibody which reacts with the epitope after immunizing a pig, and a wild strain, a live vaccine or a conventional E2 protein can generate a corresponding antibody, the identification method of the pig infected non-marked vaccine strain or the wild strain can realize the identification of the pig infected non-marked vaccine strain or the wild strain through the detection of the WH303 antibody, thereby realizing the effective purification of the swine fever.

The invention finally provides a preparation method of the marked inactivated vaccine, wherein the marked recombinant baculovirus is inoculated to insect cells, cultured and expressed, and the supernatant is collected to obtain classical swine fever virus E2 protein solution, and the classical swine fever virus E2 protein solution is emulsified with an adjuvant after being inactivated to obtain the marked inactivated vaccine. The mass ratio of the adjuvant to the recombinant E2 protein is preferably 1:0.5-1.5, and more preferably 1: 1.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Used in the present invention

The vector was manufactured by Invitrogen corporation, cat no: k2400-20; TOP10 competent cells were purchased from Tianzhu Biochemical (Beijing) science and technology Limited, cat #: CB 104-01; SOC media was purchased from Beijing Solebao Biotechnology Ltd, L1025-1; preparing LB culture solution according to literature reports; QIAprepTM Spin Plasmid Kit is a product of Qiagen, cat #: 12123, respectively; SF900II serum-free insect fineThe cell culture solution is product of Invitrogen company, and the product number is: 10902-088; the BaculoDirectTM Baculoviral Expression System kit is produced by Invitrogen company, and all reagents used in transfection are provided in the kit, and the cargo number: 12562013, respectively; BEI manufactured by Sigma, cat # I: b65705; swine fever positive serum purchased from the institute of veterinary medicine of China; the horseradish peroxidase-labeled rabbit anti-pig secondary antibody is a product of Sigma company, and the product number is: a5760; DAB color was obtained from bio-engineering (shanghai) gmbh, cat #: PW023-5 membranes. Sodium thiosulfate is chemical reagent research institute in Tianjin, CAS No.: 7772-98-7; adjuvant 563VG is manufactured by sibira corporation, cat No.: 36025H. Sf-9 cells, purchased from Invitrogen, cat #: b825-01; hog cholera lapinized attenuated vaccine (passage cell source) purchased from guangdong Yongshun biopharmaceutical, Inc., lot number: 2018002 batches; the subunit vaccine of hog cholera E2 is produced by Tiankang biological products Co., Ltd, batch No. 2018003, and the hog cholera Shimen-based hematotoxin is purchased from the inspection institute of Chinese veterinary drugs. The WH303 polypeptide was synthesized by gill chemical limited. WH303 mAb, a premium from Taiwan university of Zhongxing laboratory.

Example 1 hog cholera virus E2 Gene optimization, mutation and Synthesis

A published classical swine fever virus E2 sequence and a recently epidemic new strain E2 sequence are downloaded from Genebank for reference, optimization is carried out on the basis of a C strain E2 gene, the optimized E2 gene is compared with reference strains of various subgroups, and the result shows that the optimized E2 gene is determined to be a 1.1 group. Meanwhile, the optimized E2 gene sequence epitope 'TAVSPTTLR' is mutated into 'ATVSPTTRL, TASVPTTLR, TAVSTPTLR, TAVSPTTRL, TAPVSTTRL or TASPVTTRL', and the obtained 6 optimized mutated sequences are handed to a gene company for synthesis.

EXAMPLE 2 construction of transfer vectors

The experimental procedures were described in the Invitrogen operating manual, as follows.

Preparation of

A clone reaction mixture comprising the following components: 0.5-4 μ l of synthesized E2 nucleosideAcid, 1. mu.l salt solution (1.2mol/LNaCl, 0.06mol/LMgCl2)), 1. mu.l

The carrier was added with double distilled water to make the total volume 6. mu.l.

The cloning reaction was performed as follows: the tube wall of the centrifuge tube was tapped to mix the reaction mixture uniformly, and the reaction tube was placed on ice after 15 minutes at room temperature (22-23 ℃). Taking 2 μ l

Adding clone reaction liquid into the sensitive cells, lightly beating the tube wall to uniformly mix the cells, placing the cells on ice for 30 minutes, placing the cells in a 42 ℃ water bath for 30 seconds to perform heat shock action, immediately placing the reaction tube back to the ice bath, then adding 250 mu l of SOC culture solution with returned temperature into the reaction tube, and placing the reaction tube into a 37 ℃ constant temperature shaking incubator to perform shaking culture at the rotating speed of 200r/min for 60 minutes. 50 μ l of the bacterial liquid was applied to an LB-kana plate (containing 50 μ g/ml kanamycin), and the remaining bacterial liquid was taken out and applied to another LB-kana plate, and cultured overnight in a 37 ℃ incubator.

Single colonies were picked and subjected to primary screening of classical swine fever virus E2 recombinant transfer vectors by PCR. First, PCR reaction solution was prepared and divided into 24 tubes, each of which contained 0.5. mu.l each of BacE2-F1 (5'-CACCATGGCATTTCTCATCTGCTTG GTA-3', SEQ ID NO.3) and BacE2-R1 (5'-AAATTCTGCGAAGTAATCTGA GTG-3', SEQ ID NO.4) primers, 2.5. mu.l dNTPs (2.5mM), 2.5. mu.l 10 XPCR buffer, 0.5. mu.l Pfu DNA polymerase, and 18.5. mu.l double distilled water, and was heated at 95 ℃ for 3 minutes, then subjected to 35 cycles under the procedures of 94 ℃ for 30 seconds, 57 ℃ for 30 seconds, and 72 ℃ for 1 minute, and finally extended at 72 ℃ for 10 minutes. After the reaction, the reaction was analyzed by electrophoresis on a 1% agar gel at 100 volts. If the expected product appears, 4-5 corresponding colonies are selected and inoculated into 3ml LB-kana, and the mixture is subjected to shaking culture in a constant temperature shaking culture box at 37 ℃ and at the rotating speed of 200r/min overnight. Extraction of the recombinant vector was performed using QIAprepTM Spin Plasmid Kit. The recombinant vector was cleaved with the restriction endonuclease NotI, and then analyzed by electrophoresis on a 1% agarose gel at 100 volts. If the size of the recombinant vector is consistent with the expected product size, 2 extracted recombinant vectors are selected to be recombined with the linear baculovirus.

Example 3 recombination of transfer vectors with Linear baculoviruses

The extracted recombinant vector was subjected to spectrophotometric measurement at a wavelength of 260nm for the concentration of nucleic acid in the recombinant vector. The LR recombination reaction was then performed according to the operating manual BaculoDirectTM. mu.s Expression System, available from Invitrogen. The specific operation is as follows: sterile-manipulating 1. mu.l of recombinant transfer vector (100-300 ng), 4. mu.l of 5 xlr

The reaction buffer and 1. mu.l of double distilled water were added to 10. mu.l of BaculoDirectTM linear DNA (300ng), respectively. Taking LR out of refrigerator at-70 deg.C

After the enzyme mix was warmed on ice for 2 minutes, it was shaken 2 times for 2 seconds, and 4. mu. lLR were collected

The enzyme mix is added to the mixture and the tube wall is tapped several times to mix it evenly without vigorous shaking to avoid the baculovirus DNA breaking. After 18 hours at 25 ℃ 2. mu.l proteinase K was added and left to act at 37 ℃ for 10 minutes, the LR recombination reaction was interrupted.

Then 6 wells of cell culture plate were inoculated with 1.5X 10 cells per well ⁶ 2 wells were repeated with good (greater than 95% cell viability) Sf-9 cells. And culturing the cells at 26-28 ℃ for 1 hour to ensure that the cells are completely attached to the bottom of the culture plate. Before transfection, the following mixed solution is prepared, namely a transfection mixed solution A: 20 mul LR recombination reaction solution (containing recombined baculovirus DNA) and 200 mul lSF900II serum-free insect cell culture solution; transfection mixture B: 12 μ l

reagent and 200. mu.l SF900II serum-free insect cellsAnd (4) a culture solution. The transfection mixture B was added to the transfection mixture A, and the tube wall was gently tapped and mixed, and left to stand at room temperature for 45 minutes. The adhered Sf-9 cells were carefully washed with SF900II serum-free insect cell culture medium, and then A, B mixture was divided into two tubes, each tube was added with 800. mu.l of SF900II serum-free insect cell culture medium, and the tubes were inverted several times. Finally, the culture solution in the 6-well plate is pumped to the greatest extent, and A, B mixed solution is slowly added into the 2-well plate along the tube wall. The cell culture plate was sealed and cultured at 26-28 ℃ for 5 hours. The mixed solution is pumped out, 3ml of Sf-900 II SFM containing antibiotics and 100 mu M ganciclovir is added into each hole, finally, the holes are sealed by transparent adhesive tapes, and the holes are placed in a constant temperature incubator at 26-28 ℃ for 5 days.

Example 4 molecular biological and immunological identification of recombinant baculovirus

Molecular biological identification: extracting DNA in the supernatant of the 6-pore plate, and performing PCR identification by using identification primers BacE2-F1 and BacE2-R1, wherein the PCR reaction conditions are the same as those in the construction of the transfer carrier. After the reaction, electrophoresis analysis was performed on a 1% agar gel at 100 volts. The successfully recombined baculovirus can obtain a specific band at the 1119bp position, and the result is shown in figure 1(1. negative control; 2. positive control; 3.DNA Marker; 4. classical swine fever virus E2 recombined baculovirus).

Immunological identification: and determining the expression of the E2 protein, and verifying the reactogenicity of the E2 protein with polyclonal antibody and WH303 monoclonal antibody by an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western-blot method. The swine fever E2 protein which can be effectively expressed by the mutant strain ATVSPTTRL, TAVSPTTRL and does not react with the WH303 monoclonal antibody is detected; the mutant "TASVPTTLR, TAVSTPTLR, TAPVSTTRL" expresses classical swine fever E2 protein which weakly reacts with WH303 monoclonal antibody, while the mutant "TASPVTTRL" fails to express classical swine fever E2 protein. And finally screening the mutant strain ATVSPTTRL as a mutant recombinant virus strain for preparing the vaccine by verifying the expression yield of the swine fever E2 protein. The nucleotide sequence of the strain is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO. 1. The recombinant baculovirus can generate a specific band around 48KDa, and the verification result is shown in figure 2 (1-5 are E2 proteins expressed by classical swine fever virus E2 recombinant baculovirus).

Example 5 Strain Collection

Identifying the E2 gene of the hog cholera virus with correct expression to recombine in baculovirus for 10-fold gradient dilution, inoculating in Sf-9 cells and covering low-melting point glue for single virus purification, selecting a plurality of monoclonovirus to culture and then verifying protein expression, finally obtaining 3 strains with the expression quantity of more than 120 microgram/ml and establishing a virus seed bank. The expression amount is more than 2 times of that of the conventional baculovirus, and the advantages of obviously improving the yield per unit volume after sequence optimization and screening of monocrotovirus clone are revealed.

EXAMPLE 6 vaccine preparation

(1) Expression of classical swine fever virus recombinant E2 protein: the Sf-9 cells (preservation number CCTCC NO: V202045 recombinant baculovirus inoculation) are cultured by using a bioreactor, after condition optimization, the dissolved oxygen of the bioreactor is set to be between 40 and 50 percent, the temperature is set to be 27 ℃, the rotating speed is set to be 40 to 120rpm, the cell density is 200 ten thousand/ml during expression, the MOI is 2, and when the cells are harvested after 100 hours of culture, the stable expression of more than 140 micrograms/ml can be obtained, which is 20 micrograms higher than that of the cells cultured in a conventional shake flask. Removing cell debris, and collecting supernatant, namely marked classical swine fever virus E2 protein liquid (2) marked classical swine fever virus E2 recombinant baculovirus marked inactivation: preparing 0.1mol/L BEI inactivation solution, weighing 0.2mol NaOH solution, dissolving in 1L water for injection, adding 0.1mol 2-Bromoethylla Ehydrobromide (BEA), and reacting in water bath at 37 deg.C for 1 hr to obtain 0.1mol/L BEI solution. Adding 0.1mol/L BEI solution into CSFV E2 protein solution in (1) to a final concentration of 1mmol/L, inactivating at 37 ℃ for 48 hours, and stirring evenly for 10 minutes every 2 hours. After inactivation, 1mol/L sodium thiosulfate with the final concentration of 10mmol/L is added, and the reaction is carried out for 1 hour at 37 ℃ for subsequent vaccine preparation.

(2) The hog cholera virus E2 protein and 563VG are emulsified to prepare the vaccine: preparation of adjuvant, 563VG oily adjuvant (121 ℃, 15pa, 20 minutes) sterilization, protein antigen and adjuvant 1:1 (mass ratio) mixing, in 5000rpm emulsification for 10 minutes, stop 10 minutes, in 5000rpm emulsification for 10 minutes. Thus obtaining the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine.

Example 7 vaccine validation

Safety test of the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine: the method is characterized in that 10 healthy susceptible piglets (taking classical swine fever virus antibodies as negative) with 4-5 weeks of age are injected with 4ml (2 times of dosage) of classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine at the neck part of the back of 5 ears, and 5 pigs are not injected as negative control. Continuously observing for 14 days, measuring temperature every day, wherein the body temperature should not exceed 40 ℃, and all piglets should not have any adverse reaction and death.

Efficacy test of the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine: taking 25 healthy pigs with swine fever antibodies as negatives, wherein the first group of 10 immune labeled swine fever virus E2 protein recombinant baculovirus inactivated vaccines, the second group of 5 immune swine fever lapinized attenuated viruses (passage cell source), the third group of 5 immune swine fever E2 subunit vaccines and the fourth group of 5 swine fever virus inactivated vaccines are control groups, separately feeding under the same condition, and injecting 1ml (containing 10) of swine fever phylum haemoviruses together with the 5 control pigs respectively 21 days after the first immunization ⁵ MLD) was performed for challenge and continuously observed 16. 16 days after virus attack, 3 immunity groups, namely, a swine autopsy test group inoculated with a marked swine fever virus E2 protein recombinant baculovirus inactivated vaccine, a swine fever lapinized attenuated vaccine (a passage cell source), a swine fever E2 subunit vaccine group and a control group, have no bleeding point or other abnormal pathological conditions in tonsil, larynx, lymph node, kidney, spleen and ileum. After the pigs in the control group are attacked by toxin, bleeding spots or bleeding spots exist in tonsils, throats, lymph nodes, kidneys, spleens and ileums, marginal peduncles of the spleens die, sections of the lymph nodes are marbled, a large number of bleeding spots exist on the surfaces of the kidneys, the pathological changes of typical sparrow-spot kidneys appear, and statistical results are shown in attached table 1.

TABLE 1

National standard determination standard: the control pigs should be full of disease and at least 3 dead, and the immunized pigs should be fully healthy or slightly temperature responsive, but without clinical symptoms of swine fever. The results show that: the test pigs of the immune labeled classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine group, the immune classical swine fever lapinized attenuated vaccine (passage cell source) group and the classical swine fever E2 subunit vaccine group do not show clinical symptoms of classical swine fever, while the control pigs are all attacked and die within 6-10 days. Thus, the protective capability of the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, the classical swine fever lapinized attenuated vaccine and the classical swine fever E2 subunit vaccine to pigs is proved to be equivalent.

The synthetic WH303 epitope coated Elisa antibody detection kit is used for detecting serum of each immune group, all pig WH303 antibodies of 0 day and 7 days of immunization are negative, 14 days after immunization are marked with classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, classical swine fever E2 subunit vaccine, classical swine fever lapinized attenuated vaccine and negative control group WH303 antibody positive conversion is 0/10, 3/5, 2/5 and 0/5 respectively, and the WH303 antibodies of the classical swine fever E2 subunit vaccine and the classical swine fever lapinized attenuated vaccine are all positive conversion 21 days after immunization. The marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine and the negative control are negative after immunization before virus challenge, the classical swine fever E2 subunit vaccine and the classical swine fever lapinized attenuated vaccine WH303 antibody are still positive after virus challenge, the marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine is 2/10 positive after virus challenge, and other pigs are negative all the time.

Thus proving that the marked inactivated vaccine can effectively identify the antibody generated by mutation and non-mutation and wild virus infection. The marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine provides a substance guarantee for the purification of classical swine fever viruses in the future, and shows technical progress.

The marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine is a novel subunit marked vaccine, not only has good safety, but also has equivalent immunization efficacy to that of classical swine fever virus attenuated vaccine. In addition, the vaccine does not generate an antibody aiming at WH303 when used for immunizing animals, and the Elisa antibody diagnostic kit prepared by taking the WH303 polypeptide as an antigen can distinguish the antibody of the immune-labeled classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine from the antibody generated by the infected wild virus, so that the vaccine has wide prospect in future clinical application.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Tiankang biological products Ltd

<120> marked classical swine fever virus E2 protein recombinant baculovirus marked inactivated vaccine

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 373

<212> PRT

<213> Artificial sequence

<400> 1

Thr Thr Ala Phe Leu Ile Cys Leu Val Lys Val Leu Arg Gly Gln Ile

1 5 10 15

Val Gln Gly Val Ile Trp Leu Leu Leu Val Thr Gly Ala Gln Gly Arg

20 25 30

Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asp Glu

35 40 45

Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu Tyr

50 55 60

Thr His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Thr Cys Val

65 70 75 80

Ala Gly Ser Phe Lys Ile Thr Ala Leu Asn Ala Val Ser Arg Arg Tyr

85 90 95

Leu Ala Ser Leu His Lys Lys Ala Leu Pro Thr Ser Val Thr Phe Glu

100 105 110

Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp Asp

115 120 125

Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Arg Pro Val Val Lys Gly

130 135 140

Lys Tyr Asn Ala Thr Leu Val Asn Gly Ser Ala Phe Tyr Leu Val Cys

145 150 155 160

Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Ala Thr Val Ser Pro Thr

165 170 175

Thr Arg Leu Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro Phe

180 185 190

Pro His Arg Met Asn Cys Val Thr Thr Thr Val Glu Asn Glu Asp Leu

195 200 205

Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu Pro

210 215 220

Val Val Tyr Thr Gly Gly Leu Val Lys Gln Cys Arg Trp Cys Gly Phe

225 230 235 240

Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys Cys

245 250 255

Ile Leu Ala Asn Glu Thr Ser Tyr Arg Val Val Asp Ser Thr Asp Cys

260 265 270

Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu Cys Leu

275 280 285

Ile Gly Asn Thr Thr Val Arg Val His Ala Ser Asp Glu Arg Leu Gly

290 295 300

Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly Pro Ala

305 310 315 320

Met Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys Asn Arg

325 330 335

Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys Gly

340 345 350

Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Ala Thr Asp His His Ser Asp

355 360 365

Tyr Phe Ala Glu Phe

370

<210> 2

<211> 1119

<212> DNA

<213> Artificial sequence

<400> 2

actactgctt tcctgatctg cctggtgaag gtgctgagag gtcagatcgt gcaaggtgtg 60

atctggctgc tgctggtgac tggtgctcaa ggtagactgg cttgcaagga ggactacaga 120

tacgctatct cctccactga cgagatcggt ctgctgggtg ctggtggtct gactactact 180

tggaaggagt acactcacga cctgcagctg aacgacggta ctgtgaaggc tacttgcgtg 240

gctggttcct tcaagatcac tgctctgaac gctgtgtcta gaagatacct ggcttccctg 300

cacaagaagg ctctgccaac ttccgtgact ttcgagctgc tgttcgacgg tactaaccca 360

tccactgagg agatgggtga cgacttcggt ttcggtctgt gcccattcga cactagacct 420

gtggtgaagg gtaagtacaa cgctactctg gtgaacggtt ccgctttcta cctggtgtgc 480

ccaatcggtt ggactggtgt gatcgagtgc gctactgtgt ccccaactac tagactgact 540

gaggtggtga agactttcag aagagacaag ccattcccac acagaatgaa ctgcgtgact 600

actactgtgg agaacgagga cctgttctac tgcaagctgg gtggtaactg gacttgcgtg 660

aagggtgagc ctgtggtgta cactggtggt ctggtgaagc agtgcagatg gtgcggtttc 720

gacttcaacg agcctgacgg tctgccacac tacccaatcg gtaagtgcat cctggctaac 780

gagacttcct acagagtggt ggactccact gactgcaaca gagacggtgt ggtgatctcc 840

actgagggtt cccacgagtg cctgatcggt aacactactg tgagagtgca cgcttccgac 900

gagagactgg gtccaatgcc atgcagacca aaggagatcg tgtcctccgc tggtcctgct 960

atgaagactt cctgcacttt caactacgct aagactctga agaacagata ctacgagcca 1020

agagactcct actttcagca gtacatgctg aagggtgagt atcagtactg gttcgacctg 1080

gacgctactg accaccactc cgactacttc gctgagttc 1119

<210> 3

<211> 28

<212> DNA

<213> Artificial sequence

<400> 3

caccatggca tttctcatct gcttggta 28

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<400> 4

aaattctgcg aagtaatctg agtg 24

Claims

1. The marked classical swine fever virus recombinant E2 protein is characterized in that the amino acid sequence of the marked recombinant E2 protein is shown as SEQ ID No. 1.

2. The nucleic acid molecule encoding the marked classical swine fever virus recombinant E2 protein of claim 1, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No. 2.

3. Biological material associated with the nucleic acid molecule of claim 2, comprising any one of:

(a) an expression cassette comprising the nucleic acid molecule of claim 2;

4. A recombinant baculovirus expressing the labeled classical swine fever virus recombinant E2 protein of claim 1, comprising the nucleic acid molecule of claim 2;

the preservation number of the recombinant baculovirus is CCTCC NO: v202045.

5. Use of a recombinant baculovirus as defined in claim 4 for the preparation of a classical swine fever virus vaccine.

6. The method for preparing the labeled classical swine fever virus recombinant E2 protein according to claim 1, wherein the labeled classical swine fever virus recombinant E2 protein is obtained by culturing and expressing insect cells transfected with the recombinant baculovirus according to claim 4;

the insect cell is an Sf-9 cell;

the culture expression process comprises the following steps: inoculating virus with MOI of 2, setting dissolved oxygen of bioreactor at 40-50%, temperature at 26-28 deg.C, rotation speed at 40-120rpm, culturing for 90-110 hr, and harvesting supernatant.

7. The method according to claim 6, further comprising a step of protein purification after the culture expression.

8. A marked classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, which is characterized by comprising an adjuvant and the marked classical swine fever virus recombinant E2 protein as claimed in claim 1;

The adjuvant comprises ISA201VG, ISA563VG or ISA660VG, and the mass ratio of the adjuvant to the marked classical swine fever virus recombinant E2 protein is 1: 0.5-1.5.

9. The method for preparing the inactivated vaccine against the classical swine fever virus E2 protein recombinant baculovirus of claim 8, which is produced from the recombinant baculovirus of claim 4.

10. The preparation method according to claim 9, wherein the insect cell Sf-9 cell is transfected by the recombinant baculovirus, and the labeled classical swine fever virus recombinant E2 protein is obtained by culture expression and is emulsified with an adjuvant;

the adjuvant comprises ISA201VG, ISA563VG or ISA660 VG;

the mass ratio of the adjuvant to the marked classical swine fever virus recombinant E2 protein is 1: 0.5-1.5.

11. The method according to claim 10, wherein the culturing expression process comprises: inoculating virus with MOI of 2, setting dissolved oxygen of bioreactor at 40-50%, temperature at 26-28 deg.C, rotation speed at 40-120rpm, culturing for 90-110 hr, and harvesting supernatant.