CN115772231A - Fusion protein for simultaneously expressing proteins H and F of peste des petits ruminants, double-gene subunit vaccine and preparation method - Google Patents

Abstract

The invention provides a fusion protein for simultaneously expressing Peste des petits ruminants H and F proteins, a double-gene subunit vaccine and a preparation method thereof, and relates to the technical field of biological products for livestock. The fusion protein is subjected to enzyme digestion and in vitro refolding to obtain a dimer H protein and a trimer F protein, and the dimer H protein and the trimer F protein are mixed and emulsified with an adjuvant to form the double-gene subunit vaccine. The invention innovatively realizes the simultaneous production of two protective antigens of the Peste des petits ruminants in one expression process, the purification filler and the enzyme for enzyme digestion can be repeatedly used, the purification process is simple, the production cost is low, the antigen purity is high, the immune side reaction is effectively reduced, and the immune protection of the double-gene subunit vaccine is more comprehensive.

Description

Fusion protein for simultaneously expressing proteins H and F of peste des petits ruminants, double-gene subunit vaccine and preparation method

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a fusion protein for simultaneously expressing proteins H and F of peste des petits ruminants, a double-gene subunit vaccine and a preparation method thereof.

Background

Peste des petits ruminants (PPR), also called as plague, is a highly contagious, acute, virulent infectious disease caused by Peste des Peste virus (PPRV), mainly infecting Peste des ruminants such as goats, sheep and the like, wherein the goats are highly susceptible. In susceptible groups, mortality at severe outbreaks is 100%. Although peste des petits ruminants virus does not infect people and does not belong to zoonosis, once epidemic, serious economic loss is caused to local animal husbandry, and public health and safety are harmed.

PPRV belongs to the Paramyxoviridae (Paramyxoviridae) Paramyxovirinae (Paramyxoviridae) measles virus (morblivirus) genus. PPRV is a single-stranded negative-strand RNA virus with an envelope containing two glycoproteins on the envelope that are involved in viral entry into the cell: hemagglutinin protein (H) and fusion protein (F). The two proteins are not only the main virulence proteins of peste des petits ruminants virus, but also the main protective antigens. The H protein of PPRV is a homodimer, is formed by the same monomer through a disulfide bond, and further forms a tetramer through non-covalent bond actions such as a hydrophobic bond on the surface of the protein. The H protein mediates the adsorption of viral particles to sialic acid-containing receptors on the target cell surface, thereby initiating the infection process and promoting cell fusion of the F protein. The F protein is a trimer structure and mediates the fusion of the virus envelope and the host cell through violent conformational change. Before fusion is initiated, the F protein adopts a prefusion conformation which is unstable and has a low energy barrier, and when the fusion peptide is inserted into the host cell membrane at a close distance from the host, the F protein can cross the viral envelope and the host cell membrane. Subsequently, the F protein forms a trimeric hairpin structure, which connects the two membranes together to promote fusion, and the fused conformation is very stable. The F protein trimer in the pre-fusion conformation is in the shape of lollipop, the F protein in the post-fusion conformation is in the shape of crutch, and the two conformations are greatly different in structure and have different antigen epitopes. Ideally, to prevent viral entry, vaccines need to be developed with epitopes in the pre-fusion conformation of the F protein. Due to the stability of the post-fusion conformation, the intact F protein obtained by recombinant expression systems is only able to maintain the post-fusion stable conformation. Therefore, a series of gene mutations are carried out on the gene coding sequence of the F protein, and the Furin protease site and the transmembrane region are replaced by a flexible GSGSGR sequence by removing the transmembrane region and the intracellular region at the C terminal of the F protein and introducing a trimer structure stabilizing motif at the C terminal, so that the prefusion conformation of the F protein is stabilized.

There is no effective treatment for PPR, and the prevention and control of PPR mainly depends on vaccine immunization. A common attenuated vaccine for the peste des petits ruminants is a Nigeria7511 attenuated vaccine which can cross protect the attack infection of each group of strains, but in the process of large-area use, the live vaccine is possibly recombined with homologous virulent strains, the toxicity becomes strong or a novel strain appears, so that the immunity fails, and even a new epidemic situation occurs. In addition, after the vaccine is used for immunizing animals, the immunized animals and naturally infected animals cannot be distinguished through a serological means, so that the vaccine is not beneficial to the detection of PPR epidemic situations and the implementation of PPR elimination plans. In recent years, some novel vaccines are developed and comprise nucleic acid vaccines, recombinant vaccines and the like, for example, CN110684782A provides a method for preparing F gene nucleic acid vaccines of peste des petits ruminants virus, the speed of antibody generation induced by the nucleic acid vaccines is slow, the inoculation dose is large, potential risks of chromosome integration and transformation are caused, and the F protein alone is not high in immunogenicity and cannot provide enough protection of neutralizing antibodies; CN101422607A provides a method for recombining gene of PPRV immune protection antigen H protein and F protein into goat pox virus genome, although PPR and goat pox can be prevented at the same time, the effective immune duration and safety of the vaccine need to be demonstrated; CN107236047A provides a method for expressing Peste des petits ruminants virus H-F fusion protein by a coliform expression system, but the folding property of the coliform expression system protein is poor, an inclusion body is easy to form, and the coliform expression system is modified without glycosylation and cannot be folded to form a protein structure with complete space conformation. CN111732667A provides a fusion protein (PPRV-Fu) linked after the modification of H and F proteins, which is finally presented in the form of heterodimer, and does not present the dimer of H protein and the trimer of F protein, and has insufficient epitope, and cannot provide high level immune protection.

Disclosure of Invention

In view of the above, the invention aims to provide a fusion protein and a double-gene subunit vaccine for simultaneously expressing H and F proteins of Peste des petits ruminants and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a fusion protein for simultaneously expressing proteins H and F of Peste des petits ruminants, which has a structure from an N end to a C end and comprises: signal peptide, protein H of Peste des petits ruminants with the N-terminal intracellular region and the transmembrane region removed, fc, TEV enzyme cutting motif, protein F of Peste des petits ruminants and trimerization sequence of Fibritin.

Preferably, the source of the signal peptide comprises a mouse IgG kappa chain signal peptide and/or a bombyx mori immunoglobulin signal peptide;

sources of the Fc include ovine IgG Fc.

The invention provides the fusion protein expressed by CHO cells, and the structure of the fusion protein from N end to C end sequentially comprises: a mouse IgG kappa chain signal peptide, a Peste des petits ruminants H protein, a sheep IgG Fc, a TEV enzyme cutting motif, a Peste des petits ruminants F protein and a Fibritin trimerization sequence with an N-terminal intracellular region and a transmembrane region removed;

the nucleotide sequence of the mouse IgG kappa chain signal peptide is shown as SEQ ID No.1, the nucleotide sequence of the Peste des petits ruminants H protein with the N-terminal removed intracellular region and the transmembrane region removed is shown as SEQ ID No.2, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.3, the nucleotide sequence of the TEV enzyme cutting motif is shown as SEQ ID No.4, the nucleotide sequence of the Peste des petits ruminants F protein is shown as SEQ ID No.5, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 6.

Preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 8.

The invention provides the fusion protein expressed by insect cells, and the structure of the fusion protein from N end to C end sequentially comprises: silkworm immunoglobulin signal peptide, small ruminants H protein, sheep IgG Fc, TEV enzyme cutting motif, small ruminants F protein and Fibritin trimerization sequence with an N-terminal intracellular region and a transmembrane region removed;

the nucleotide sequence of the silkworm immunoglobulin signal peptide is shown as SEQ ID No.9, the nucleotide sequence of the peste des petits ruminants H protein with the N-terminal intracellular region and the transmembrane region removed is shown as SEQ ID No.10, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.11, the nucleotide sequence of the TEV enzyme digestion motif is shown as SEQ ID No.12, the nucleotide sequence of the peste des petits ruminants F protein is shown as SEQ ID No.13, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 14.

Preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 16.

Preferably, the fusion protein is secreted in culture supernatant after signal peptide is cut off, and the amino acid sequence after the signal peptide is cut off is shown in SEQ ID NO. 17.

The invention provides a recombinant vector containing the fusion protein.

The invention provides a recombinant cell line for expressing the fusion protein.

The invention also provides the application of the fusion protein, the recombinant vector or the recombinant cell line in preparing the Peste des petits ruminants H and F protein double-gene subunit vaccine.

The invention also provides a Peste des petits ruminants H and F double-gene subunit vaccine which takes the fusion protein expressed by the recombinant cell line as an antigen.

Has the advantages that: the invention provides a fusion protein for simultaneously expressing proteins H and F of peste des petits ruminants, wherein an N-terminal intracellular region and a transmembrane region of the protein H are deleted, and an FC (fiber channel) sequence is added at a C terminal to promote the protein H to form a dimer. The full-length F protein has no biological function and is called as proprotein F0, and after F0 is hydrolyzed into F1 and F2 proteins by Furin protease, the proprotein F0 is aggregated into tripolymer F protein with biological activity through hydrophobic effect; the Furin protease site and the transmembrane region from 105 th to 131 th of the F protein are deleted, a flexible 'GSGSGSGR' sequence is changed, and meanwhile, the transmembrane region from 488 th to 546 th at the C end and the intracellular region are removed for enhancing the trimerization of the monomer, and a Fibritin trimerization sequence is replaced. Finally, the modified H and F proteins are connected by using a TEV enzyme digestion motif to form a fusion protein, and the H and F double-gene subunit vaccine with complete structure function is obtained through enzyme digestion and in-vitro refolding in one production process.

The FC label is added into the fusion protein, so that the expression of the H protein in a dimer form is promoted, the half-life period of the protein is prolonged, and the antigen presentation is promoted; the fusion protein is subjected to a one-time expression process to simultaneously produce subunit vaccines of two genes of Peste des petits ruminants H and F, and finally the H protein forms a dimer and the F protein forms a vaccine in a trimer form. The fusion protein can purify protein by using commercial ProteinG filler, has simple purification process and high purity of vaccine antigen, and effectively reduces immune side reaction.

Detailed Description

The invention provides a fusion protein for simultaneously expressing proteins H and F of Peste des petits ruminants, the structure of the fusion protein from an N end to a C end comprises: signal peptide, protein H of Peste des petits ruminants with the N-terminal intracellular region and the transmembrane region removed, fc, TEV enzyme cutting motif, protein F of Peste des petits ruminants and trimerization sequence of Fibritin.

The source of the signal peptide of the invention preferably comprises a mouse IgG kappa chain signal peptide and/or a silkworm immunoglobulin signal peptide; the source of the Fc preferably comprises sheep IgG Fc. In the present invention, before the fusion, the fusion protein preferably further comprises the step of modifying the F protein and the H protein, which specifically comprises: the H protein mediates receptor attachment and the F protein causes fusion of the viral envelope and cell membrane. Through the research of protein structure prediction, the first 58 amino acids of the N end of the H protein are an intracellular region and a transmembrane region, the rest C end part is an extracellular region, and the natural H protein is attached to the surface of the virus in a homodimer mode. The invention deletes the N-terminal intracellular region and transmembrane region of the H protein, and adds a sheep IgG FC sequence at the C terminal to promote the H protein to form a dimer. The full-length F protein has no biological function and is called as a proprotein F0, and after the F0 is hydrolyzed into F1 and F2 proteins by Furin protease, the proteins are aggregated into a trimeric F protein with biological activity through hydrophobic interaction. Through the research of protein structure prediction, the F protein is a triple transmembrane protein, and is respectively a transmembrane region from positions 4 to 26, 109 to 131 and 488 to 510, a signal peptide from positions 1 to 18, and a Furin proteolytic cleavage site from positions 108 and 109, namely 19C-108R is the F2 protein, and 109F-487P is the F1 protein. In order to realize the complete function of the F protein and not to break the fusion protein before secreting the cell due to the hydrolysis of Furin protease, the invention deletes the Furin protease site and transmembrane region from 105 th to 131 th of the F protein, changes the F protein into a flexible GSGSGR sequence, and simultaneously removes the transmembrane region and intracellular region from 488 th to 546 th of the C end for enhancing the trimerization of monomers and replaces the C end with a Fibritin trimerization sequence. Through the transformation of the invention, a transmembrane region and a Furin enzyme hydrolysis site which do not participate in trimerization folding in the F protein are deleted, a flexible sequence GSGSGR is replaced, the folding complexity of the protein is reduced, the structural stability is increased, and a Fibritin trimerization sequence is added at the C end of the F protein, so that the F protein is trimerized.

When the fusion protein is expressed by using different cell lines, codon optimization is required according to the codon preference of an expression cell, so that the nucleotide sequences of the fusion protein are slightly different although the structures of the fusion protein are the same based on different cell expression systems.

The invention provides the fusion protein expressed by CHO cells, and the structure of the fusion protein from N end to C end sequentially comprises: a mouse IgG kappa chain signal peptide, a Peste des petits ruminants H protein, a sheep IgG Fc, a TEV enzyme cutting motif, a Peste des petits ruminants F protein and a Fibritin trimerization sequence with an N-terminal intracellular region and a transmembrane region removed;

the nucleotide sequence of the mouse IgG kappa chain signal peptide is shown as SEQ ID No.1, the nucleotide sequence of the Peste des petits ruminants H protein with the N-terminal removed intracellular region and the transmembrane region removed is shown as SEQ ID No.2, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.3, the nucleotide sequence of the TEV enzyme cutting motif is shown as SEQ ID No.4, the nucleotide sequence of the Peste des petits ruminants F protein is shown as SEQ ID No.5, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 6.

The invention synthesizes CHO-H-F sequence by using the fusion protein expressed by CHO cell, such as PPRV-FY strain complete gene sequence (KX 354359.1) in the embodiment, but it can not be only identified as the full protection scope of the invention. The CHO-H-F sequence of the invention sequentially comprises: a mouse IgG kappa chain signal peptide with the size of 63 basic groups and the nucleotide sequence shown in SEQ ID NO. 1; the protein sequence of Peste des petits ruminants H with the N-terminal intracellular region and the transmembrane region removed has 1653 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 2; the goat IgG FC sequence has the size of 696 nucleotides, and the nucleotide sequence is shown as SEQ ID No. 3; TEV enzyme cutting motif with the size of 21 basic groups and the nucleotide sequence shown in SEQ ID NO. 4; the Peste des petits ruminants F protein sequence has the size of 1344 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 5; the Fibritin trimerization sequence has the size of 75 bases, and the nucleotide sequence is shown as SEQ ID NO. 6; the two ends of the synthesized CHO-H-F sequence respectively contain HindIII and EcoRI restriction sites, and the total length is 3873 basic groups, which is shown in SEQ ID NO. 7. The amino acid sequence coded by the CHO-H-F sequence is shown in SEQ ID NO. 8.

The invention also provides the fusion protein expressed by insect cells, and the structure of the fusion protein from the N end to the C end sequentially comprises: silkworm immunoglobulin signal peptide, small ruminants H protein, sheep IgG Fc, TEV enzyme cutting motif, small ruminants F protein and Fibritin trimerization sequence with an N-terminal intracellular region and a transmembrane region removed;

the nucleotide sequence of the silkworm immunoglobulin signal peptide is shown as SEQ ID No.9, the nucleotide sequence of the peste des petits ruminants H protein with the N-terminal intracellular region and the transmembrane region removed is shown as SEQ ID No.10, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.11, the nucleotide sequence of the TEV enzyme digestion motif is shown as SEQ ID No.12, the nucleotide sequence of the peste des petits ruminants F protein is shown as SEQ ID No.13, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 14.

The present invention is not limited to the types of insect cells, and in the examples, insect cell SF9 is used as an example, but the present invention is not to be construed as being limited to the full scope of the present invention. In the embodiment of the invention, an SF9-H-F sequence is synthesized by codon optimization with reference to a PPRV-FY strain complete gene sequence (KX 354359.1). The SF9-H-F sequence is as follows: the silkworm immunoglobulin signal peptide sequence has the size of 60 bases, and the nucleotide sequence is shown as SEQ ID NO. 9; the protein sequence of Peste des petits ruminants H with the N-terminal intracellular region and the transmembrane region removed has the size of 1653 basic groups, and the nucleotide sequence of the protein sequence is shown as SEQ ID NO. 10; the goat IgG FC sequence has the size of 696 nucleotides, and the nucleotide sequence is shown as SEQ ID No. 11; TEV enzyme cutting motif with the size of 21 basic groups and the nucleotide sequence shown in SEQ ID NO. 12; the Peste des petits ruminants F protein sequence has the size of 1344 bases, and the nucleotide sequence of the Peste des petits ruminants F protein sequence is shown as SEQ ID NO. 13; the size of the Fibritin trimerization sequence is 75 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 14; the synthesized SF9-H-F sequence contains BamHI and HindIII restriction sites at two ends respectively, and has a total length of 3867 basic groups as shown in SEQ ID NO. 15. The amino acid sequence coded by the SF9-H-F sequence is shown in SEQ ID NO. 16.

The CHO-H-F sequence or the SF9-H-F sequence is secreted in culture supernatant after signal peptide is cut off, and the amino acid sequence after the signal peptide is cut off is shown in SEQ ID NO. 17: <xnotran> YPDCKPEFSYAIARNEKIGPLGAEGLTTIWHEYSAKMRLKDTMVEVWCKDGQFMHLKKCAREARYLAVLHTRALPTSVVFKQLFSGQKSVGMVDMNDDFEFGLCPCDAKPVIRGKFNTTLLNGPAFQMVCPIGWTGTVSCALVNEDTLDTTTVHIYRRTRPFPHRQGCITHKLVGEDLYNCTLGGNWTCIPGERLTYKGGPIETCKWCGYNFKKDEGLPHYPIGKCKLKNETGYRLVDDTSCNRDGVAIVPTGTLKCKIGDTVIQVIAMDTNLGPMPCKPYEIIQSEGPVEKTACTFNYTKTLRNKYFEPRDRYFQQYMLKGEYQYWFDLDAVDHHKDYVDPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGKENLYFQGQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNSTDSKVKLIKQELERYNNAVVELQSLMQNEPASFSRAKGSGSGRFLGFLLGIGSAIASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISKIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVGYPEAPPRDGQAYVRKWWVLLSTFL. </xnotran>

The invention provides a recombinant vector containing the fusion protein.

In the invention, aiming at different expression cells, the recombinant vectors constructed in the middle are different, if the fusion protein is a CHO-H-F sequence, hindIII and EcoRI are preferably used for enzyme digestion of the CHO-H-F sequence and the pEE12.4 vector, the enzyme digestion fragments are respectively recovered and then are connected, transformed and cloned, the sequencing is correct, and the CHO-H-F-pEE12.4 recombinant plasmid is constructed. When the fusion protein is an SF9-H-F sequence, preferably, bamHI and HindIII are respectively used for enzyme digestion of the SF9-H-F sequence and the pFastBac1 vector sequence, enzyme digestion fragments are recovered and then connected with a transformation clone, and after sequencing verification, the SF9-H-F-pFastBac1 transfer vector is constructed.

The invention provides a recombinant cell line for expressing the fusion protein.

The construction method of the recombinant cell line is not particularly limited, and the conventional construction method in the field can be used, for example, after a CHO-H-F-pEE12.4 recombinant plasmid is obtained through construction, a CHO-K1 cell is used for transfection and high expression cell strain screening, so that a stable transgenic cell strain (high expression cell strain) is screened out, and the high expression cell strain is subjected to serum-free suspension domestication to obtain the recombinant cell line. The invention preferably performs cell shake flask fermentation on the recombinant cell line, and after the fermentation is finished, cell supernatant is harvested and protein is purified by using commercial ProteinG filler. After the purified protein is obtained, the method preferably further comprises the steps of enzyme digestion according to the ratio of adding 1U TEV protease per 8 mu g of protein, loading the enzyme-digested protein into a dialysis bag for VLPs self-assembly, taking out liquid in the dialysis bag, loading a nickel column, removing the TEV enzyme containing a His label, and obtaining a flow-through liquid, namely an antigen liquid containing a Peste des petits ruminants H protein dimer and an F protein trimer, wherein the flow-through liquid can be applied to preparation of a double-gene subunit vaccine of PPRV.

After the SF9-H-F-pFastBac1 transfer vector is obtained, preferably, the method also comprises the steps of transforming the vector into a competent cell DH10Bac, culturing at 37 ℃, screening by using a blue-white spot screening method and carrying out PCR identification to obtain an SF9-H-F-Bacmid recombinant Bacmid; and transfecting the SF9-H-F-Bacmid recombinant Bacmid into SF9 cells in logarithmic growth phase, and harvesting P after culturing ₁ Generations of recombinant baculovirus and continued transmission to P ₃ Instead, P is ₃ Centrifuging for virus generation, and collecting supernatant as virus solution; by P ₃ Infecting High Five cells with the substitute virus, culturing, and collecting supernatant containing target protein. The purification, enzyme digestion and antigen solution preparation methods of the target protein are the same, and are not described herein again.

The invention also provides the application of the fusion protein, the recombinant vector or the recombinant cell line in preparing the Peste des petits ruminants H and F protein double-gene subunit vaccine.

The application of the present invention is preferably the same as described above and will not be described further herein.

The invention also provides a peste des petits ruminants H and F protein double-gene subunit vaccine, which takes the fusion protein expressed by the recombinant cell line as an antigen.

After the antigen solution is obtained, the invention preferably further comprises diluting the antigen solution to a final concentration of 60 μ g/mL by using a PBS buffer solution, and mixing and emulsifying the antigen solution and ISA201 VG adjuvant according to a volume ratio of 46.

The present invention provides a fusion protein and a two-gene subunit vaccine for simultaneously expressing proteins H and F of peste des petits ruminants and a preparation method thereof, which are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Sequence Synthesis

The full gene sequence (KX 354359.1) of PPRV-FY strain of Peste des petits ruminants is referred, a CHO-H-F sequence is synthesized through codon optimization, the two ends of the synthesized CHO-H-F sequence respectively contain HindIII and EcoRI enzyme cutting sites, the total length is 3873 bases, the nucleotide sequence is shown as SEQ ID NO.7, and the coded amino acid sequence is shown as SEQ ID NO. 8.

Example 2

Construction of CHO-H-F-pEE12.4 recombinant plasmid

The CHO-H-F sequence and pEE12.4 vector (purchased from Chimana organisms) synthesized in example 1 are digested by HindIII and EcoRI, the fragments of the digested CHO-H-F sequence and pEE12.4 sequence are recovered, and are connected, transformed and cloned, and the recombinant plasmid of CHO-H-F-pEE12.4 is constructed after the sequencing verification.

Example 3

CHO-K1 cell transfection and high expression cell strain screening

1. Preparing cells:

selecting CHO-K1 cells with confluency of 80% -90% in one dish (10 cm cell culture dish) within 24h of subculture, discarding the culture medium, adding 10mL PBS to wash the cells once, discarding the PBS, adding 15mL serum-free antibiotic-free DMEM/F12 culture medium, placing at 37 deg.C for 5% ₂ Culturing in a cell culture box.

2. Transfection plasmids

According to

2000, 24. Mu.g of the CHO-H-FpEE12.4 recombinant plasmid and 60. Mu.L of the recombinant plasmid were diluted with 1.5mL of serum-free, antibiotic-free DMEM/F12 medium, respectively

2000, mixing, standing at room temperature for 5min. The two tubes of liquid were mixed and left at room temperature for 20min. Taking prepared CHO-K1 cells, adding the above mixing reagent dropwise, and adding into the mixture at 37 deg.C for 5% CO ₂ A cell culture box. Culturing for 4-6 h, discarding the medium, adding 10mL DMEM/F12 medium (10% serum, 1% penicillin-streptomycin double antibody (100X), no glutamine), and 5% CO at 37 ℃% ₂ Culturing in a cell culture box.

3. Pressure screening

24h after transfection, each dish of transfected CHO-K1 cells and untransfected CHO-K1 cells (negative control) was removed, the supernatant medium was discarded, 10mL of DMEM/F12 medium (10% serum + 25. Mu.M MSX,1% double antibody, no glutamine) was added, pressurized screening was performed for 7d, cells were observed in the middle, and when many dead cells were present, the medium was changed.

4. Selection of stably transformed cell lines

When 25 μ M MSX was pressure-screened until the negative control cells were essentially dead, about 7 days, cell line screening was initiated. Taking transfected CHO-K1 cells, discarding the culture medium, washing with PBS once, adding 500 mu L of 0.25% pancreatin, digesting at room temperature for 2-5 min until the cells are single cells, adding 10mL of DMEM/F12 culture medium (10% serum +25uM MSX,1% double antibody, no glutamine) to terminate the digestion reaction, blowing off the cells with a pipette, and counting the cells. Cells were diluted to 10 with DMEM/F12 medium (10% serum + 25. Mu.M MSX,1% double antibody, glutamine free) ⁴ cells/mL, transferred to 96-well plates at 200. Mu.L per well, charged at 37 ℃ 5% CO ₂ Culturing in a cell culture box. And when the 96-well plate is full, taking the supernatant, carrying out ELISA detection, and continuously carrying out amplification culture and cryopreservation on the high-expression positive cell strain.

Example 4

Serum-free suspension domestication of high-expression cell strain

Recovering the high-expression cell strain in DMEM/F12 medium (10% serum, 1% double antibody and no glutamine), and continuously using the medium for passage for 2 to 3 times until the cell growth is stable and when the cell density reaches 2 x 10 ⁶ Passage is carried out after cells/mL, and the density of passage cells is controlled to be 0.3-0.5 multiplied by 10 ⁵ cells/mL, medium 10%DMEM/F12 medium for serum and

basal medium (purchased from kindred) was mixed at a ratio of 75 ₂ Culturing in a constant temperature shaking table with the rotation speed of 110 rpm. When the cell density reaches 2 multiplied by 10 after 3 to 4 days ⁶ cells/mL and cell viability>DMEM/F12 medium with 90%, 10% serum at passage and

the basal medium is mixed according to the proportion of 50 ⁶ cells/mL. If the cells grow slowly, the supernatant can be centrifuged, 20% of the original culture medium can be retained, and 80% of 10% serum DMEM/F12 culture medium can be added to the supernatant

Continuously culturing in a mixed culture medium with a basic culture medium ratio of 75; repeating the above steps to increase gradually

The proportion of the basic culture medium is up to 100%

A basal medium.

Example 5

Cell shake flask fermentation

Taking out the shake flask cell from the constant temperature shaking table, diluting the cell to 0.3-0.5X 10 ⁵ cells/mL inoculated 30mL

Basic medium to a 125mL shake flask, placed at 37 ℃,5% CO ₂ Culturing in a constant temperature shaking table with the rotation speed of 110 rpm. The glucose concentration was measured every day, and when the glucose concentration was less than 4g/L, a glucose solution was added to the culture solution to give a glucose concentration of 4g/L. Adding 3% of the extract on day 3, day 5, day 7 and day 9

And (3) feeding a culture medium. On day 12, cell supernatants were harvested.

Example 6

Insect cell expression sequence Synthesis

By referring to the whole gene sequence (KX 354359.1) of PPRV-FY strain of peste des petits ruminants, an SF9-H-F sequence is synthesized through codon optimization, the two ends of the synthesized SF9-H-F sequence respectively contain BamHI and HindIII enzyme cutting sites, the total length is 3867 basic groups, and is shown as SEQ ID NO.15, and the coded amino acid sequence is shown as SEQ ID NO. 16.

Example 7

Baculovirus transfer vector construction

The SF9-H-F sequence and the pFastBac1 vector sequence synthesized in the example 6 are digested by BamHI and HindIII, the digested SF9-H-F and pFastBac1 sequence fragments are recovered, and are connected and transformed to clone, and after the sequencing verification is correct, the SF9-H-F-pFastBac1 transfer vector is constructed.

Example 8

Preparation of bacon

The SF9-H-F-pFastBac1 transfer vector prepared in example 7 was transformed into competent cell DH10Bac, cultured at 37 ℃, and then screened by blue-white spot screening and identified by PCR to obtain SF9-H-F-Bacmid recombinant Bacmid. Bacmid transformation and screening methods Invitrogen company Bac-to-Bac is referenced ^TM Baculovirus expression system user guide for operation.

Example 9

Recombinant baculovirus harvest

The SF9-H-F-Bacmid recombinant Bacmid obtained in example 8 was used with a transfection reagent

SF9 cells in logarithmic growth phase were transfected, cultured for 72h, and P was harvested ₁ Generations of recombinant baculovirus SF9-H-F-rBV. The harvested P1 generation recombinant baculovirus is continuously subcultured on SF9 cells until P ₃ Instead, P is ₃ Centrifuging the virus, collecting the supernatant as virus solution, and determining P by plaque assay ₃ Titer of the virus. According to an inoculum size of 1 MOI, using P ₃ And infecting High Five cells by the substitute virus, and culturing for 96h to obtain culture supernatant containing the target protein.

Example 10

Protein purification

The cell supernatants of examples 5 and 9 were collected, centrifuged at 8000g for 30min at 4 ℃ and the supernatants were filtered through 0.8 μm filters. 5-10 times column volume PBS balance Protein G column material, processed cell supernatant is repeatedly loaded on the column for 3 times, 10 times PBST washes column material, 2 times PBS washes column material, 2 times column material volume 0.1M glycine (pH3.0) elution Protein, eluent is collected, 1M Tris (pH9.0) is added to neutralize to pH7.5, and the purified Protein is obtained.

Example 11

Protease cleavage and refolding

The concentration of the purified protein prepared in example 10 was determined according to the BCA protein quantitative determination kit (purchased from Shanghai Processori), the total mass of the protein was calculated, and 1U of TEV protease (His-tag, purchased from Byunnan Bio) was added to 8. Mu.g of protein, and 10 Xdigestion buffer (500 mM NaH) was added by volume ₂ PO ₄ 150mM NaCl,10mM EDTA,10mM DTT,1% Tween-20, pH8.0), and digested at 4 ℃ for 12 to 16 hours. After the enzyme digestion is finished, loading the enzyme-digested protein into a dialysis bag (3500D), wherein the dialysate is 50mM NaH ₂ PO ₄ 500mM NaCl, pH8.0, dialyzing for 12-16 h, and changing the solution 2-3 times. After the first dialysis, standing for 8-12 h at 4 ℃ to complete protein refolding. Changing the dialyzate into PBS buffer solution, dialyzing for 12-16 h, and changing the dialyzate 2-3 times. And after the second dialysis is finished, taking out the liquid in the dialysis bag, loading the sample on a nickel column, removing the TEV enzyme containing the His label, and obtaining the flow-through liquid which is the antigen liquid containing H protein dimer and F protein trimer of the peste des petits ruminants. Eluting the nickel column with PBS buffer solution containing 200mM imidazole, collecting eluent, namely recovered TEV protease, and directly using the recovered TEV protease in the enzyme digestion process of the next production after enzyme activity determination.

Example 12

Vaccine preparation

The antigen solution containing the H protein dimer and the F protein trimer of the peste des petits ruminants described in the example 11 is diluted to the final concentration of 60 mu g/mL by using PBS buffer solution, and is mixed and emulsified with ISA201 VG adjuvant according to the volume ratio of 46.

Example 13

Detection of PPRV antibody by H and F protein coated ELISA plate

The antigen solution containing H protein dimer and F protein trimer of Peste des petits ruminants described in example 11 was diluted with PBS buffer to 0.2. Mu.g/ml, coated on an ELISA plate at 100. Mu.l/well, and coated at 4 ℃ for 16 hours. After coating, the wells were discarded, and 300. Mu.l of PBST wash solution was added to each well and rinsed 1 time. Freshly prepared blocking solution (5% skim milk, PBS) was added to each well in 200. Mu.l and blocked at 37 ℃ for 2h. After the blocking was complete, the wells were discarded, 300. Mu.l of PBST wash solution was added to each well, rinsed 1 time, and patted dry on absorbent filter paper. Diluting the BRSV serum to be detected by 100 times by using PBS buffer solution, adding an antigen coated plate, and incubating for 1h at 37 ℃. The wells were discarded and 300. Mu.l of wash solution was added to each well and rinsed 3 times. The HRP-labeled rabbit anti-goat IgG Fab secondary antibody was diluted 10000-fold with 5% skim milk-containing PBS, 100. Mu.l was added to each well, and incubated at 37 ℃ for 1h. The wells were discarded, and 300. Mu.l of PBST wash solution was added to each well, followed by 3 rinses. Adding 100 μ l of TMB color developing solution into each well, and developing for 10min at room temperature in dark. Add 50. Mu.l of stop solution into each well, and read the absorbance at 450nm on the microplate reader. Judging the positive standard of the antibody, wherein P/N is more than or equal to 2.1 ₄₅₀ ≥0.4。

Example 14

Immunization experiment

Screening 7 goats (PPRV antibody negative before immunization) with the age of more than 1 year, randomly dividing the goats into 2 groups, a vaccine immunization group with 5 goats, a blank control group with 2 goats, a vaccine immunization group for immunizing the Peste des petits ruminants H and F double-gene subunit vaccine, and a blank control group for immunizing PBS. The neck was injected intramuscularly at 1ml each time, three weeks after the priming immunization was performed once, and serum was collected before, 21 days after the first immunization, and 21 days after the second immunization. Referring to the method for detecting PPRV antibody by using the protein-coated ELISA plates H and F in example 13, the serum to be detected is diluted to 1. The maximum dilution times of the detection result P/N is more than or equal to 2.1 and the OD450 is more than or equal to 0.4 are the antibody titer of the serum sample. The results are shown in table 1, with numbers 1 to 5 being vaccine immunization groups and numbers 6 and 7 being blank groups. The results show that the PPRV antibodies of the pre-immunization group and the blank group are negative, the PPRV antibody titer 21 days after the first immunization of the vaccine immunization group is not less than 1. PPRV antibody detection results show that the Peste des petits ruminants H and F double-gene subunit vaccine has good immunogenicity.

Table 1 detection of PPRV antibodies by protein-coated ELISA plates of H and F

Example 15

Neutralizing antibody assay

Day before the experiment, fetal goat kidney cells were digested with pancreatin, resuspended using 10% FBS-containing DMEM, and plated in 96-well cell culture plates at a cell seeding density of 2X 10 ⁵ cells/mL, 0.1mL per well inoculated, placed at 37 ℃ C. 5% ₂ Culturing under the condition. The day of the experiment, serum samples 21 days after the immunization were diluted with 2% FBS-containing DMEM cell culture solution at a dilution of 2 ¹ ，2 ² ，2 ³ ，2 ⁴ ，2 ⁵ ，2 ⁶ ，2 ⁷ ，2 ⁸ And (4) uniformly mixing. Diluting Peste des petits ruminants virus (obtained from Clone9 strain, institute of veterinary medicine, china) to 200 TCIDs in DMEM cell culture medium containing 2% FBS according to the virus titer ₅₀ 0.1ml. 100 μ L of serum dilution was mixed with an equal volume of PPRV virus (200 TCID) ₅₀ ) Mixing uniformly, and the final toxicity is 100TCID ₅₀ 0.1ml, standing at 37 ℃,5% CO ₂ Culturing for 90min in the incubator; taking a 96-well culture plate fetal goat kidney cell cultured for 24 hours, discarding a growth medium, transferring a mixture of virus and serum neutralized for 90min into a corresponding hole of the 96-well cell culture plate, wherein the volume of the mixture is 0.1 mL/hole, the pipette tip is replaced in time when serum/virus mixed liquid with different dilutions is transferred to the cell plate, each dilution is repeated for 4 times, and meanwhile, a normal negative control cell without the neutralized virus is arranged. serum-PPRV virus mixed liquor with different dilutions is inoculated to fetal goat kidney cells, cultured for 72h in a cell culture box, and cytopathic effect is observed. If it occursCells become round, cells gather and are in a grape string shape to form cytopathic effects such as syncytial and the like, which indicates that PPRV is not neutralized by neutralizing antibodies generated in serum; if the growth state of the cells is good, it indicates that PPRV is neutralized by neutralizing antibodies generated in serum and the cells do not show pathological conditions, as in the control group results. The maximum dilution at which no CPE was present in fetal goat kidney cells was the neutralizing antibody titer of this serum sample. The PPRV neutralizing antibody experimental results are shown in table 2, and the PPRV neutralizing antibody titers in the immunised group 21 days after diabrosis are not less than 1, which complies with OIE regulations for PPR vaccine serum neutralizing antibody levels of 100% not less than 1. The small ruminant animal plague H and F double gene subunit vaccine can provide protection for the immune goat.

TABLE 2 PPRV neutralizing antibody test results table

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. A fusion protein for simultaneously expressing proteins H and F of Peste des petits ruminants, wherein the structure of the fusion protein from N-terminal to C-terminal comprises: signal peptide, protein H of Peste des petits ruminants with the removal of the intracellular region and the transmembrane region at the N end, fc, TEV enzyme cutting motif, protein F of Peste des petits ruminants and Fibritin trimerization sequence.

2. The fusion protein of claim 1, wherein the source of the signal peptide comprises a mouse IgG kappa chain signal peptide and/or a bombyx mori immunoglobulin signal peptide;

sources of the Fc include sheep IgG Fc.

3. The fusion protein of claim 1 or 2 expressed by CHO cells, wherein the structure from N-terminus to C-terminus of the fusion protein comprises, in order: a signal peptide of a mouse IgG kappa chain, a Peste des petits ruminants H protein with an N-terminal intracellular region and a transmembrane region removed, a sheep IgG Fc, a TEV enzyme digestion motif, a Peste des petits ruminants F protein and a Fibritin trimerization sequence;

the nucleotide sequence of the mouse IgG kappa chain signal peptide is shown as SEQ ID No.1, the nucleotide sequence of the Peste des petits ruminants H protein with the N-terminal removed intracellular region and the transmembrane region removed is shown as SEQ ID No.2, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.3, the nucleotide sequence of the TEV enzyme cutting motif is shown as SEQ ID No.4, the nucleotide sequence of the Peste des petits ruminants F protein is shown as SEQ ID No.5, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 6.

4. The fusion protein according to claim 1 or 2, which is expressed by an insect cell, and comprises the following structures from the N-terminus to the C-terminus: silkworm immunoglobulin signal peptide, small ruminant H protein with N-terminal intracellular region and transmembrane region removed, sheep IgG Fc, TEV enzyme digestion motif, small ruminant F protein and Fibritin trimerization sequence;

the nucleotide sequence of the silkworm immunoglobulin signal peptide is shown as SEQ ID No.9, the nucleotide sequence of the Peste des petits ruminants H protein with the N-terminal intracellular region and the transmembrane region removed is shown as SEQ ID No.10, the nucleotide sequence of the sheep IgG Fc is shown as SEQ ID No.11, the nucleotide sequence of the TEV enzyme cutting motif is shown as SEQ ID No.12, the nucleotide sequence of the Peste des petits ruminants F protein is shown as SEQ ID No.13, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID No. 14.

5. The fusion protein of claim 3 or 4, wherein the fusion protein is secreted in the culture supernatant after cleavage of the signal peptide, and the amino acid sequence after cleavage of the signal peptide is shown in SEQ ID No. 17.

6. A recombinant vector comprising the fusion protein of claim 1 or 2, the fusion protein of claim 3, or the fusion protein of claim 4 or the fusion protein of claim 5.

7. A recombinant cell line expressing the fusion protein of claim 1 or 2, the fusion protein of claim 3 or the fusion protein of claim 4 or the fusion protein of claim 5.

8. Use of the fusion protein of claim 1 or 2, the fusion protein of claim 3 or the fusion protein of claim 4 or the fusion protein of claim 5, the recombinant vector of claim 6 or the recombinant cell line of claim 7 for the preparation of a double-gene subunit H and F peste des petits ruminants vaccine.

9. A Peste des petits ruminants H and F double-gene subunit vaccine, which is characterized in that the fusion protein expressed by the recombinant cell line of claim 8 is used as an antigen.