CN111378017B - Subunit F protein of peste des petits ruminants virus and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of animal vaccines and biological products for animals, and particularly relates to a subunit F protein of peste des petits ruminants virus, a preparation method and an application thereof, wherein the amino acid sequence of the subunit F protein is as follows: an amino acid sequence shown as SEQ ID NO. 1; ② a derived amino acid sequence with immunogenicity obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 1. The subunit F protein is mainly prepared by constructing recombinant plasmids, transfecting cell strains with the recombinant plasmids, screening high-expression cell strains and purifying the subunit F protein of the peste des petits ruminants virus, can be better suitable for the subunit vaccine or diagnostic reagent of the peste des petits ruminants virus, and has the characteristics of high efficiency of secretory expression, high protein purity, easiness in purification, low production cost, high safety performance and the like.

Description

Subunit F protein of peste des petits ruminants virus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of animal vaccines and biological products for animals, and particularly relates to a subunit F protein of peste des petits ruminants virus, and a preparation method and application thereof.

Background

Peste des Petits of ruminates, PPR, is a virulent, contact-infectious viral disease caused by Peste des Petits of ruminates Virus (PPRV). The world animal health Organization (OIE) classifies it as a class A infectious disease, and the world also classifies it as a class epidemic disease. PPRV mainly infects sheep, especially goats are highly susceptible. Once infected, the sheep are characterized by sudden fever, depression, oral ulcer, cough, secretion discharge from eyes and nose and diarrhea, the morbidity and mortality can reach 100 percent, huge economic loss is caused, and the development of sheep raising industry is seriously influenced.

At present, no effective treatment means exists for the disease, and the prevention and control are mainly carried out by means of vaccines. Since PPRV and Rinderpest (RPV) belong to the same genus and have cross-reactivity with antigens, PPRV has historically been prevented by RPV attenuated vaccines, but the vaccines have been stopped because they lead to false positive RPV detection and are not favorable for the implementation of the national rinderpest elimination program. At present, the disease is prevented mainly through an attenuated vaccine, the vaccine is obtained through continuous passage weakening in Vero cells by a conventional means, the duration of the vaccine is long, and the immune animal is safe. However, the vaccine is a conventional attenuated live vaccine, and in the process of large-area use, the vaccine is possibly recombined with homologous virulent strains, the virulence 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.

PPRV belongs to the genus morbillivirus of the family Paramyxoviridae, the virions are oval or round, the outermost layer of the virions is wrapped with a capsule membrane, and the capsule membrane is provided with protruding fibers. The PPRV genome is single-strand negative strand non-segmented RNA, the total length is about 15.6kb, and the genome sequentially encodes capsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), large protein (L), six structural proteins and C, V non-structural proteins. The F protein and the H protein are main proteins of the virus envelope, can stimulate the body to produce neutralizing antibodies, and are the first glycoproteins of subunit vaccines. The subunit vaccine does not contain nucleic acid substances, and does not generate persistent infection or latent infection after inoculation; the generated immune response can be distinguished from wild virus infection, and is beneficial to controlling and eliminating epidemic diseases. However, subunit vaccines also have significant drawbacks: the production cost is high and the application is limited.

The cost of subunit vaccines is mainly in the production of subunit proteins, and the F protein is a membrane glycosylation modified trimer protein. Generally comprises 537-552 amino acids, the F protein does not have complete biological function and is called protein F0, and in order to obtain subunit F protein with biological activity, F0 needs to be hydrolyzed by cell protease into disulfide-linked polypeptides F1 and F2. Therefore, to ensure that the expressed protein can have glycosylation modification and form the complete active F1 and F2, it must be realized in animal cells.

The engineered cells are expression cells widely used in the biopharmaceutical engineering at present. The protein expressed in the system is most similar to natural protein molecules in the aspects of molecular structure, physicochemical properties, post-transcriptional modification and other biological functions. And can achieve high-density culture in a suspension culture mode, the culture volume can reach more than 2,000L, and the method can be used for large-scale production.

However, when the engineered cell is used for expressing the F protein, the encoding gene sequence of the F protein is not optimized, the engineered cell does not substantially express the F protein, and further, the F protein with excellent immunogenicity and stability is difficult to obtain for the control of peste des petits ruminants virus, so that the optimization of the encoding gene sequence of the F protein is a necessary process when the engineered cell is used for expressing the F protein.

Disclosure of Invention

Aiming at the defects in the prior art, the first purpose of the invention is to provide the subunit F protein of the peste des petits ruminants virus, and the subunit F protein has excellent immunogenicity and stability of the peste des petits ruminants virus F protein, and is convenient for stable and efficient secretion expression in an engineered cell strain.

The second purpose of the invention is to provide a preparation method of the subunit F protein of peste des petits ruminants virus, which is convenient for large-scale industrial production of the subunit F protein and reduces the production cost of the F protein.

The third purpose of the invention is to provide the application of the subunit F protein of the peste des petits ruminants virus, which can be better applied to the subunit vaccine and the diagnostic reagent of the peste des petits ruminants virus, thereby facilitating the prevention and control of people on the peste des petits ruminants virus.

In order to achieve the first object, the invention provides the following technical scheme:

a subunit F protein of Peste des petits ruminants virus, the amino acid sequence of said subunit F protein:

an amino acid sequence shown as SEQ ID NO. 1;

② a derived amino acid sequence with immunogenicity obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO. 1.

According to the technical solution of the present invention, preferably, one of poly-His, FLAG, c-myc, HA and poly-Arg is linked to the amino terminus or the carboxy terminus of the amino acid sequence shown in SEQ ID NO. 1.

According to the technical scheme of the invention, preferably, the coding gene sequence of the subunit F protein is shown as SEQ ID NO.2, or is obtained by codon optimization of SEQ ID NO. 2.

According to the technical scheme of the invention, preferably, the coding gene sequence of the subunit F protein is shown as SEQ ID NO. 3.

In order to achieve the second object, the invention provides the following technical scheme:

a preparation method of subunit F protein of peste des petits ruminants virus comprises the following steps:

firstly, constructing an encoding gene sequence of subunit F protein of peste des petits ruminants virus;

secondly, cloning the coding gene sequence of the subunit F protein into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit F protein;

transfecting recombinant plasmids containing subunit F protein coding gene sequences into engineering cells of animals to obtain cell strains;

fourthly, screening out cell strains with high expression from the cell strains obtained in the third step;

fifthly, purifying the highly expressed cell strain obtained in the fermentation culture step to obtain subunit F protein of the peste des petits ruminants virus.

In the technical scheme of the invention, preferably, in the step (II), the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.1.

In the technical scheme of the invention, preferably, in the step two, the eukaryotic expression vector is pEE12.4.

In the technical solution of the present invention, preferably, in the step (c), the cell line is one of a CHO cell line, a HEK293 cell line, and a 293T/17 cell line.

In the technical solution of the present invention, preferably, in the step (c), the CHO cell line is one of a DG44 cell line, a DXB11 cell line, a CHO-K1 cell line and a CHO-S cell line.

In order to achieve the second object, the invention provides the following technical scheme:

use of a subunit F protein of peste des petits ruminants virus, said subunit F protein being suitable for use in a subunit vaccine or diagnostic reagent for peste des petits ruminants virus.

The invention provides a first subunit F protein, which has excellent immunogenicity and stability of the peste des petits ruminants virus F protein, is convenient for stable and efficient secretion expression in an engineered cell strain, has high yield and easy purification, ensures that the purity of target protein in cell culture supernatant can reach more than 70 percent, can ensure that the purity of the target protein reaches more than 90 percent by only one step of affinity chromatography, far meets the requirements of subunit vaccines and diagnostic reagents, and is convenient for large-scale production, thereby solving the technical problem of high production cost of the peste des petits ruminants virus subunit F protein. In addition, engineering cell strains such as CHO cell strains, HEK293 cell strains, 293T/17 cell strains and the like for production are high in controllability during culture, easy to control quality and stable among production protein batches, so that the protein of the peste des petits ruminants produced by the invention has small amount of other viruses in subunit F protein, effectively reduces the risk of virus dispersion and has excellent biological safety.

Drawings

FIG. 1 is a schematic diagram showing the three-dimensional structure of a predicted subunit F protein;

FIG. 2 shows the results of alignment before and after optimization of subunit F gene sequences;

FIG. 3 shows a map of the pEE12.4-OPTI-F plasmid;

FIG. 4 shows the results of the double restriction enzyme identification of pEE12.4-OPTI-F: m is a DNA Marker: DL10000 Marker; 1 is the result of pEE12.4-OPTI-F double-enzyme digestion electrophoresis;

FIG. 5 shows SDS-PAGE of subunit F protein after purification, wherein 1 is subunit F protein and M is Marker;

FIG. 6 shows the results of a Werstern blot assay after purification of subunit F protein, wherein 1 is subunit F protein and M is Marker;

FIG. 7 shows the results of stability measurements after purification of subunit F protein, wherein 1 in A is Marker and 2 is subunit F protein after 4 ℃ treatment; in B, 1 is Marker, and 2 is subunit F protein treated at-20 ℃.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.

The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

The sources of the reagent and the medicine of the invention are listed as follows:

the engineered cells are derived from a cell bank of the China academy of sciences (CCTCC) and a cell bank of the Shanghai institute of Life sciences of China academy of sciences; cell culture medium and serum were purchased from gibco, usa; the eukaryotic expression vector pEE12.4 is purchased from Shanghai Linyuan Biotech, Inc.; lipofectamine LTX was purchased from Thermo Fisher, USA; methionine sulfoxide iminium (L-methionine sulfoximine, MSX) was purchased from Sigma company; BCA protein quantification kit was purchased from Thermo Fisher, usa; PLUS^TMreagent is available from thermo corporation as LAn addition of ipofectamine LTX transfection reagent; CB5 was purchased from thermo and fed to the fermentation medium.

Example 1

1 a: the structure analysis and optimization are carried out on the peste des petits ruminants virus F protein, so as to construct the coding gene sequence of the subunit F protein of the peste des petits ruminants virus.

By analyzing the F protein sequence (GenBank: KM091959.1) of the peste des petits ruminants virus, the F protein sequence of the peste des petits ruminants coded by the genome sequence 5532-. Further analysis 1M-19C may be a secretion signal peptide of the F protein, 20Q-491N may be an extracellular domain of the F protein, 492M-510I may be a transmembrane domain of the F protein, and 511C-546L may be an intracellular domain protein of the F protein. In addition, the enzyme cutting sites of the F protein are found to be between 109R and 110F through analysis, namely 20Q to 109R are F2 protein, and 110F to 491N are F1 protein.

In combination with the experience of the studies on the expression of the viral envelope protein in the previous period, the extracellular region of the subunit F protein expressed by CHO-K1 cells was selected as the immunogenic protein, i.e., the amino acid sequence of 1M-491N. Through the protein structure prediction and the research of the amino acid structure, the amino acid sequence of 1M-491N is shown in SEQ ID NO.1, the predicted three-dimensional structure of the amino acid sequence is similar to that of other measles virus F proteins, and the three-dimensional structure pattern is shown in figure 1.

Wherein, the person skilled in the art can easily prepare a derivative protein by substituting, deleting or adding an amino acid or several amino acids on the basis of the amino acid sequence SEQ ID No.1 by conventional technical means, and the homology of the derivative protein and the amino acid sequence (shown as SEQ ID No. 1) of the subunit F protein in the embodiment is as high as 80% -100%, so as to ensure that the two proteins have the same immunogenicity, and therefore, the derivative protein also falls into the protection scope of the present invention.

In order to facilitate the purification of the subunit F protein, a tag as shown in Table I can be attached to the amino terminus or the carboxy terminus of the amino acid sequence shown in SEQ ID NO.1, specifically Poly-His in this example, which is attached to the amino terminus of the amino acid sequence shown in SEQ ID NO. 1.

TABLE-TAG AND ITS AMINO ACID SEQUENCE

The coding gene sequence of the amino acid sequence SEQ ID NO.1 can be shown as SEQ ID NO.2, or can be obtained by codon optimization of SEQ ID NO. 2. In the embodiment, the coding gene sequence of the subunit F protein is subjected to codon optimization on the basis of SEQ ID No.2 to obtain an OPTI-F sequence, which is shown as SEQ ID No.3 and is the subunit F protein coding gene sequence of the peste des petits ruminants virus, and the artificial synthesis work of the gene sequence is finished by Nanjing Kingsry Biotech Co.

The codon-optimized sequence (shown as SEQ ID No. 3) was aligned with the sequence before codon optimization (shown as SEQ ID No. 2), and the result is shown in fig. 2, where 358/1473 ═ 24.3% difference.

1 b: construction of pEE12.4-OPTI-F recombinant plasmid

1b.1 PCR amplification of the target fragment OPTI-F

1b.1.1 PCR reaction

(1) Primer design and Synthesis

An upstream primer: 5'-CGGAAGCTTATGACCAGGGTGGCTATCCTG-3'

A downstream primer: 5'-GGCGAATTCTCAATGGTGATGGTGATGGTGATTG-3'

(2) The sample addition system of 50. mu.L is shown in Table II below.

Sample adding system with 50 mu L of sample

PCR amplification procedure:

1b.1.2 PCR product gel recovery

(1) Marking a sample collection EP tube, an adsorption column CB2 and a collection tube;

(2) weighing the weight of the marked empty EP pipe, and recording the numerical value;

(3) a single DNA band of interest was carefully excised from the agarose gel on a gel cutter with a scalpel and placed into a clean 1.5mL centrifuge tube;

(4) adding 600 mu L of PC buffer into the 1.5mL of centrifuge tube in the step (3), placing in a water bath at 50 ℃ for about 5min, and turning the centrifuge tube up and down continuously and gently to ensure that the gel block is fully dissolved;

(5) column balancing: adding 500 μ L of equilibrium liquid BL into adsorption column CB2 (adsorption column CB2 is put into a collection tube in advance), centrifuging at 12000rpm/min for 1min, pouring off waste liquid in the collection tube, and putting adsorption column CB2 back into the collection tube;

(6) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min, centrifuging at 10000rpm/min for 30s, pouring waste liquid in a collecting pipe, and then putting the adsorption column CB2 into the collecting pipe;

(7) adding 600 mu L of rinsing liquid PW buffer into an adsorption column CB2, standing for 3min, centrifuging at 10000rpm/min for 30s, pouring waste liquid in a collecting pipe, and putting the adsorption column CB2 into the collecting pipe;

(8) repeating the step (7);

(9) centrifuging with an empty adsorption column at 12000rpm/min for 2min, removing rinsing liquid as much as possible, standing adsorption column CB2 at room temperature for 10min, and air drying completely;

(10) placing adsorption column CB2 in a collecting tube, suspending and dropwise adding 50 μ L of precipitation buffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, and centrifuging at 12000rpm/min for 2 min;

(11) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube with a cover, and keeping the DNA sample in the centrifuge tube;

(12) and (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.

1b.2 double digestion reaction of PCR product and vector

(1) Marking a 1.5mL EP tube which needs to be used, and loading and uniformly mixing the sample in the EP tube according to the third table, wherein the DNA sample in the third table is the DNA fragment finally recovered in the step 1b.1.2 (12);

TABLE 50. mu.L reaction System

(2) Putting the EP pipe in the step (1) into a constant-temperature water bath kettle at 37 ℃ to carry out water bath for 2-3 h;

recovering the double enzyme digestion product gel: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system, wherein the method is the same as that of the PCR product gel recovery in the step 1 b.1.2.

1b.3 ligation reaction

(1) Preparing a plurality of clean 1.5mL EP pipes, marking the EP pipes, and placing the marked EP pipes on an EP pipe frame for later use;

(2) loading and uniformly mixing the sample in the EP tube in the step (1) according to the fourth table, wherein the target fragment in the fourth table is the DNA fragment finally recovered in the step 1b.2 (2);

TABLE four 10. mu.L reaction System

(3) After sample adding is finished according to the table in the step (2), placing each 10 mu L reaction system in a low-temperature cooling liquid circulator at the temperature of 16 ℃ for water bath for 10-16 h;

(4) taking out the EP tube in the step (3), placing the EP tube in a water bath kettle at 65 ℃, and carrying out water bath for 15 min;

(5) and (4) taking out the EP tube in the step (4), and storing at 4 ℃ to obtain a ligation reaction solution.

1b.4 conversion reaction

(1) Quickly adding 10 mu L of the ligation reaction solution prepared in the step 1b.3(5) into 100 mu L of competent cells, uniformly mixing by blowing, and carrying out ice bath for 30 min;

(2) taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min;

(3) taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in a super-clean workbench, then placing the sample tube in a constant temperature shaking table at 37 ℃, and culturing for 1h at 220 rpm/min;

(4) coating a plate: taking out the sample tube in the step (3), centrifuging for 2min at room temperature and 8000rpm/min, removing 600 mu L of supernatant liquid, suspending the thalli at the bottom of the tube by the residual supernatant liquid, putting the resuspended bacterial liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterial liquid in the center of the transformation plate by a bacterial coating rod;

(5) and (3) placing the plate in the transformation step (4) in a biochemical constant-temperature incubator, culturing for 1h at 37 ℃, inverting the transformation plate, culturing for 15h, and transforming to obtain the monoclonal strain.

1b.5 plasmid extraction and double enzyme digestion identification

1b.5.1 plasmid extraction

(1) Picking the monoclonal strain from the transformation plate of step 1b.4(5) with a 10. mu.L pipette tip into 5mL of a liquid LB medium containing benzyl amine resistance, and shaking overnight at 37 ℃ and 220 rpm/min;

(2) transferring the bacterial liquid into a 1.5mL EP tube, centrifuging for 2min at room temperature and 12000rpm/min, and removing the supernatant;

(3) adding 250 mu L of plasmid extraction reagent P1buffer into the EP tube in the step (2) to completely suspend the thalli;

(4) adding 250 mu L of plasmid extraction reagent P2buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4 min;

(5) adding 350 mu L of plasmid extraction reagent P3buffer into the solution in the step (4), immediately and gently inverting the centrifuge tube for 5-10 times and uniformly mixing; standing at room temperature for 2-4 min;

(6) centrifuging the solution in the step (5) for 10min at room temperature and 14000 rpm/min;

(7) transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging for 30s at room temperature and 12000rpm/min, and pouring out liquid in a collecting pipe;

(8) adding 500 μ L Buffer DW1 into the center of the adsorption column, centrifuging at 12000rpm/min at room temperature for 30s, and removing liquid from the collection tube;

(9) adding 500 μ L wash solution into the center of the adsorption column, centrifuging at 12000rpm for 30s at room temperature, pouring off the liquid in the collection tube, and repeating once;

(10) air adsorbing column, centrifuging at 12000rpm/min at room temperature for 2 min;

(11) the adsorption column was placed in a clean 1.5mL centrifuge tube, 30. mu.L of Elution buffer was added to the center of the adsorption membrane, the mixture was allowed to stand at room temperature for 5min, centrifuged at 12000rpm/min at room temperature for 2min, and the DNA solution in the tube was stored.

1b.5.2 double restriction enzyme identification

(1) Marking a 1.5mL EP tube which needs to be used, and loading according to the fifth table, wherein the DNA sample in the fifth table is the DNA solution finally obtained in the step 1b.5.1 (11);

TABLE five 20. mu.L reaction System

(2) Putting the EP tube 20 mu L reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ for water bath for 2 h;

(3) carrying out agarose gel electrophoresis on the double enzyme digestion system sample in the step (2), and checking whether the size of the inserted fragment is correct; the results are shown in FIG. 2: the enzyme digestion identification construction is correct;

(4) the recombinant plasmid with correct insert fragment is selected and sent to a sequencing company for sequencing, the recombinant plasmid is sent to Jinzhi biotechnology limited for determination, and the sequence of the subunit F protein coding gene is shown as SEQ ID NO. 3.

1b.6, large extraction of endotoxin-free plasmid

1b.6.1, endotoxin-free plasmid extraction

(1) Inoculating the clone with the correct sequencing in the step 1b.5.2(4) into 100mL of a culture medium containing benzyl alcohol resistance, and culturing for 15h by a constant temperature shaking table at 37 ℃ and at 220 rpm/min;

(2) transferring the bacterial liquid cultured in the step (1) into a 50mL centrifuge tube, centrifuging for 5min at room temperature and 8000rpm/min, collecting thalli, and discarding a supernatant culture medium;

(3) adding 8mL of plasmid extraction reagent P1buffer into the centrifuge tube in the step (2), and fully resuspending the thalli by using a pipette;

(4) adding 8mL of plasmid extraction reagent P2buffer into the centrifuge tube in the step (3), immediately warming and inverting the centrifuge tube for 6-8 times, and standing for 5min at room temperature;

(5) adding 8mL of plasmid extraction reagent P4buffer into the centrifuge tube in the step (4), immediately turning upside down for 6-8 times, fully and uniformly mixing until white flocculent precipitate appears in the solution, standing at room temperature for about 10min, and centrifuging at room temperature and 8000rpm/min for 5-10min to separate the white precipitate to the bottom of the tube;

(6) carefully transferring all the supernatant obtained in the step (5) into a filter CS1, slowly pushing the filter, and collecting the filtrate in a clean 50mL centrifuge tube;

(7) column balancing: adding 2.5mL of equilibrium liquid BL into adsorption column CP6 (placing adsorption column CP6 into 50mL collection tube), centrifuging at room temperature and 8000rpm/min for 2min, pouring off waste liquid in the collection tube, and placing adsorption column CP6 back into the collection tube;

(8) adding isopropanol with the volume 0.3 times that of the filtrate in the step (6), turning upside down, uniformly mixing, transferring to an adsorption column CP6, centrifuging for 2min at room temperature and 8000rpm/min, pouring off liquid in a collecting pipe, and putting the adsorption column CP6 into the same collecting pipe again;

(9) adding 10mL of rinsing liquid PW buffer into the adsorption column CP6 in the step (8), centrifuging for 2min at room temperature and 8000rpm/min, discarding waste liquid in the collection tube, and replacing the adsorption column into the collection tube;

(10) repeating the operation step (9) once;

(11) adding 3mL of absolute ethyl alcohol into the adsorption column CP6 in the step (10), centrifuging for 2min at room temperature and 8000rpm/min, and pouring out waste liquid;

(12) putting the adsorption column CP6 obtained in the step (11) back into the collection tube again, centrifuging for 5min at room temperature and 8000rpm/min, uncovering the adsorption column CP6, and standing at room temperature for several minutes for air drying;

(13) putting the adsorption column in the step (12) into a clean 50mL centrifuge tube, adding 1-2mL TBbuffer in the center of an adsorption membrane, standing for 5min at room temperature, centrifuging for 2min at room temperature and 8000rpm/min, transferring all eluent in the 50mL centrifuge tube into a clean 1.5mL centrifuge tube, wherein the eluent is DNA solution of pEE12.4-OPTI-F recombinant plasmid, the chromatogram of the recombinant plasmid is shown in figure 3, and storing at-20 ℃ after the concentration of the recombinant plasmid is measured;

(14) 1-2. mu.L of the DNA solution obtained in step (13) was subjected to agarose gel electrophoresis and the data of the electrophoresis results were saved, and the electrophoretogram is shown in FIG. 4.

1 c: establishment of transfection of CHO-K1 cells with pEE12.4-OPTI-F recombinant plasmid and monoclonal screening

1c.1, CHO-K1 cell transfection

(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 medium (containing 10 wt% serum and 1 wt% double antibody) and PBS buffer solution are placed in a 37 ℃ water bath to be preheated to 37 ℃;

(2) from 37 ℃ CO₂Taking CHO-K1 cells (10cm cell culture dish) out of the cell culture box, discarding the supernatant culture medium, washing the cells once with 8mL of pre-warmed PBS buffer solution, discarding the PBS buffer solution;

(3) adding 1-2mL of 0.25 wt% of trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and presenting single cells;

(4) the digestion reaction was stopped by adding 4mL of DMEM/F12 medium (containing 10 wt% serum, 1 wt% double antibody) and the cells were pipetted off;

(5) transferring the digested cells into a 15mL centrifuge tube, centrifuging at normal temperature for 5min at 200 g;

(6) resuspend cells in DMEM/F12 medium (containing 10 wt% serum, 1 wt% double antibody) and count;

(7) dilute cells to 2 × 10⁵2mL of the mixed cells were added to a six-well plate, which was placed at 37 ℃ in CO ₂5% by volume of CO₂Incubating in a cell incubator overnight;

(8) taking out the six-hole plate in the step (7), and observing the cell state: when the cell intersection degree reaches 80-90%, the transfection can be started, and before the transfection, the culture medium is changed into a DMEM/F12 culture medium without antibiotics or serum, wherein the culture medium is 2 mL/hole;

(9) diluting the recombinant plasmid: the recombinant plasmid was diluted with OPTI-MEM medium, and 2.5. mu.g of the recombinant plasmid was added to 125. mu.L of the OPTI-MEM medium, followed by 2.5. mu.L of PLUS^TMMixing agent, standing at room temperature for 5 min;

(10) dilution of Lipofectamine LTX: to 125. mu.L of OPTI-MEM medium was added 9. mu.L of Lipofectamine LTX, followed by 2.5. mu.L of PLUS^TMMixing agent gently, standing at room temperature for 5 min;

(11) gently mixing the mixture obtained in the step (10) and the mixture obtained in the step (11), standing at room temperature for 5min, and then dropwise adding the mixture into a six-hole plate for uniform distribution;

(12) placing the six-hole plate at 37 ℃ and CO ₂5% by volume of CO₂Culturing for 4-6h in a cell culture box;

(13) liquid changing: the supernatant medium was discarded, 2mL of DMEM/F12 medium (containing 10 wt% serum and 1 wt% diabody) was added, and the six-well plate was placed at 37 ℃ in CO ₂5% by volume of CO₂Culturing in a cell culture box.

1c.2 pressure screening

Pressurization was started 24h after transfection: CO from step 1c.1(11)₂Six-well plate cells were removed from the cell incubator, the supernatant medium was discarded, 2mL of DMEM/F12 (containing 10 wt% serum + 25. mu.M MSX) was added, the pressure was increased for 7 days, cells were observed in the middle, and the dead cells were replaced with more fluid.

1c.3 monoclonal screening

(1) Pressurizing and screening the cells in the step 1c.2 until all negative control cells die, about 7 days, and starting monoclonal screening;

(2) taking out the six-hole plate, discarding the culture medium, washing with PBS buffer solution once, adding 300 mu L0.25wt% of tryptsin-EDTA, digesting at room temperature for about 2min, adding 2mL of DMEM/F12 culture medium (containing 10 wt% of serum and 25 mu M MSX) to terminate the digestion reaction, and blowing off the cells by using a pipette;

(3) transferring the digested cells into a 15mL centrifuge tube, centrifuging at normal temperature for 5min at 200 g;

(4) resuspend cells in DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) and count;

(5) plate paving: thin paperReleasing cells to 5/mL, adding 200 μ L of the mixed cells into a 96-well plate, standing at 37 deg.C and CO ₂5% by volume of CO₂Incubating for 4-6h in a cell incubator;

(6) wells to record individual cells;

(7) when the hole of a single cell in a 96-hole plate grows up, discarding the culture medium, washing with PBS buffer solution once, adding 100 mu L of 0.25 wt% of trypsin-EDTA, digesting at room temperature for about 2min, adding 2mL of DMEM/F12 culture medium (containing 10 wt% of serum and 25 mu M MSX) to stop the digestion reaction, and blowing off the cell by using a pipette; and transferring the cell sap to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by ELISA, and continuously performing expanded culture and freezing storage on the high-efficiency expressed positive clone.

1 d: CHO-K1 cell strain acclimatized to suspension culture

(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) was preheated to 37 ℃ in a 37 ℃ water bath;

(2) taking out the cells (10cm cell culture dish) obtained in the step 3.3(7), discarding the supernatant culture medium, washing the cells once with 8mL of pre-warmed PBS buffer, and discarding the PBS buffer;

(3) adding 1-2mL of 0.25 wt% of trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and presenting single cells;

(4) the digestion was stopped by adding 4mL of DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) and the cells were blown off with a pipette;

(5) transferring the digested cells into a 15mL centrifuge tube, centrifuging at normal temperature for 5min at 200 g;

(6) suspending the cells in 100 wt% DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) and counting;

(7) dilute cells to 5 × 10⁵Inoculating 30mL of the suspension cell-containing culture medium obtained in step (6) into a 125mL shake flask; the cell culture flask was placed at 37 ℃ and CO ₂5% by volume of CO₂Incubating overnight at 120rpm/min on an orbital shaker in a cell incubator;

(8) wiping the biological safety cabinet table board with 75% alcohol for sterilization, and performing ultraviolet irradiation for 30 min;

(9) counting the cell density and activity every 24 h;

(10) performing second-generation culture when the survival rate of the cells reaches 94-97% after the first-generation cells are cultured for one time;

(11) preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; 100 wt% DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) and EX-CELL 302 medium were placed in CO ₂5% by volume of CO₂Preheating to 37 ℃ in a cell culture box;

(12) from CO in step (11)₂Taking out cells from a cell incubator, transferring the cells to a 50mL centrifuge tube, centrifuging the cells for 5min at normal temperature by 200 g;

(13) DMEM/F12 medium (containing 10 wt% serum + 25. mu.M MSX) and EX-CELL 302 medium were mixed at a ratio of 1:1 mixing, resuspending the cells, and counting;

(14) dilute cells to 5 × 10⁵Inoculating 30mL of the suspension cell-containing culture medium obtained in step (13) in a 125mL shake flask; the cell culture flask was placed at 37 ℃ and CO₂CO at a concentration of 5% by volume₂Incubating overnight at 120rpm/min on an orbital shaker in a cell incubator;

(15) wiping the biological safety cabinet table board with 75 wt% alcohol for sterilization, and performing ultraviolet irradiation for 30 min;

(16) counting the cell density and activity every 24 h;

(17) the survival rate of the cells obtained after the second generation culture is twice is more than 95 percent; the cell survival rate obtained after three times of culture of the third to the sixth generation is more than 95 percent; after 7 weeks, the cells were seeded for 3 days and propagated for three generations with a cell density of 1X 10⁶one/mL, with a cell viability of 95%, which cells are considered to have been adapted to suspension culture; cell seeding density is reduced to 3X 10⁵Per mL;

(18) through domestication, 10H4 cell strains and 10H9 cell strains in CHO-K1 cell strains meet the requirements, which shows that the 10H4 cell strains and the 10H9 cell strains are successfully domesticated.

1 e: cell shake flask fermentation

(1) Preparation of a subculture medium: placing 60 wt% of CD-CHO culture medium and 40 wt% of Ex-cell 302 culture medium in a 37 ℃ water bath to preheat to 37 ℃;

(2) taking out the 10H4 cell line and the 10H9 cell line suspension-cultured in the step 1d (17), and counting;

(3) diluting the 10H4 cell line and the 10H9 cell line in the step (2) to 2.5X 10 cells respectively⁵Per mL-3.5X 10⁵One cell/mL, two cell lines were inoculated into 30mL of the subculture medium of step (1) in a 125mL shake flask, the flask was placed at 37 ℃ and CO was added₂CO at a concentration of 5% by volume₂Incubating overnight at 100rpm/min in a constant temperature shaking table;

(4) counting the cell density and activity every 24h, measuring glucose, and adding the glucose to 4g/L when the blood sugar is lower than 2 g/L; taking 1mL of sample every day, and using the supernatant for detecting the protein expression condition;

(5) feeding (about day four): 70g/L of CB5 is supplemented, and the addition amount is 10 percent of the original culture medium;

(6) culture temperature adjustment (fifth day): introducing CO₂Adjusting the temperature of the incubator to 32 ℃;

(7) refeeding (day nine): 70g/L of CB5 is supplemented, and the addition amount is 10 percent of the original culture medium;

(8) on the twelfth day, 10H4 cell culture broth and 10H9 cell culture broth were harvested, respectively.

1 f: protein purification

(1) Collecting the 10H4 cell culture fluid and 10H9 cell culture fluid (each batch is about 100ml), centrifuging for 30min at 4 ℃ and 8000g, taking supernatant, filtering through a 0.8 mu m filter membrane, loading, reserving 80 mu L of sample, adding 20 mu L of 5 xSDS-sample buffer solution for SDS-PAGE detection, and determining the concentration and purity of the sample before purification;

(2) column balancing: balancing 2-3CV (column volume) with ultrapure water, and discharging ethanol preservation solution; then Buffer A (20mM NaH) is used₂PO₄(pH 7.4), 500mM NaCl) at an equilibrium rate of 4-7mL/min for 2-3 CV;

(3) loading: preparing a first 5mL pre-packed column, loading at a Flow rate of 1mL/min (adjusting the loading Flow rate according to the volume of the pre-packed column), keeping the Flow for 5min, collecting Flow Through (FT), taking 80 mu L of sample, adding 20 mu L of 5 xSDS-sample buffer solution, and using the sample for SDS-PAGE detection to determine the adsorption effect of the target protein on the pre-packed column;

(4) washing: with 4 wt% Buffer B (20mM NaH)₂PO₄(pH 7.4), washing the column with 500mM NaCl and 20mM imidazole) at a flow rate of 4mL/min, and washing the protein which is not combined with the column and the hybrid protein with weak combination ability until the OD value baseline at the wavelength of 280nm is stable;

(5) and (3) elution: with 50 wt% Buffer B (20mM NaH)₂PO₄(pH 7.4), 500mM NaCl, 20mM imidazole) eluting the target protein until the OD value of the target protein at the wavelength of 280nm is flush with the base line, the elution speed is 2mL/min, collecting the target protein according to the specification of 10 mL/tube, taking 80 mu L of sample, adding 20 mu L of 5 xSDS-sample buffer solution for SDS-PAGE detection, and determining the elution effect of the target protein;

(6) washing: with 100 wt% Buffer B (20mM NaH)₂PO₄(pH 7.4), 500mM NaCl, 500mM imidazole) wash at 4mL/min flow rate, rinse 2-3CV until UV baseline wash-out; then balancing 2-3CV by using ultrapure water, preserving the HisTrap excel column, and balancing 2-3CV by using 20% ethanol preservation solution to obtain imidazole eluent containing target protein;

(7) and (3) dialysis liquid change: pouring the imidazole eluent containing the target protein obtained in the step (6) into a dialysis bag, dialyzing 1000 times by using 1 XPBS buffer solution to obtain a dialyzed sample, and taking 80 mu L of sample to be reserved for SDS-PAGE detection so as to determine the concentration and purity of the purified target protein;

(8) and (3) degerming and filtering: passing the dialyzed sample obtained in step (7) through a low protein binding needle filter with a pore size of 0.22 μm in a biosafety cabinet; in addition, if the protein content in the dialyzed sample is too high, the dialyzed sample can be filtered by using a sterilized Nalgene filter with a 0.22 μm filter membrane, and the filtered protein solution sample is stored in a refrigerator at minus 80 ℃;

(9) protein concentration determination: the concentration of the protein after the sterilization and filtration is measured by adopting a BCA method, the concentration of the protein purified by the 10H4 cell strain and the 10H9 cell strain is 1.1mg/mL-1.3mg/mL, and the volume of the protein purified by the 10H4 cell strain and the 10H9 cell strain is about 30 mL; by calculation (protein yield: protein concentration: protein volume/volume of fermentation supernatant taken), the protein yields of 10H4 cell line and 10H9 cell line were 1.1g/L to 1.3g/L, respectively.

1 g: identification of subunit F proteins

1g.1, SDS-PAGE detection

The protein purified in step 1F was subjected to SDS-PAGE using a sample having a subunit F protein concentration of 2. mu.g/well, and the results are shown in FIG. 5. As can be calculated from the figure, the purified subunit F protein has an SDS-PAGE purity of 92% and a molecular weight of about 52KD (F)₀)，40KD(F₁) The band at 52KD is weaker, and the band at 40KD is stronger.

1g.2 Werstren Blot assay

The purified protein of step 1f was subjected to the Werstern Blot assay, the results of which are shown in FIG. 6. The subunit F protein (labeled 1 in the figure) concentration in the samples used was 2. mu.g/well; the primary antibody is derived from the serum of sheep immunized by the weak vaccine of the peste des petits ruminants, and the dilution ratio is 1: 100, respectively; the secondary antibody is an HRP-labeled donkey anti-sheep IgG secondary antibody, and the dilution ratio is 1: 5000. as can be seen from the figure, the serum specifically binds to the subunit F protein of the present invention, and thus, the subunit F protein produced by the present invention has excellent immunogenicity.

1g.3, Elisa test

(1) Coating: on an ELISA plate, the purified subunit F protein was diluted to 0.5. mu.g/ml with a coating solution (50mM carbonate buffer, pH 9.5), each antigen was coated in 8 wells (4 wells with serum sample and 4 wells with blocking solution as control), each antigen was added in 100. mu.l/well, sealed with a sealing film, and then left overnight in a refrigerator at 4 ℃;

(2) washing: taking out the enzyme label plate in the step (1) from the refrigerator, and washing the plate for 5 times by using PBST buffer solution;

(3) and (3) sealing: adding 200 μ l of sealing solution (5 wt% skimmed milk) into each well containing subunit F protein, sealing with sealing film, and incubating at 37 deg.C for 2 hr;

(4) serum dilution: diluting positive serum of sheep immunized by attenuated vaccine of Peste des petits ruminants by 100 times with confining liquid;

(5) washing: the same (2);

(6) sample adding: adding diluted serum, simultaneously using confining liquid as negative control, and incubating for 1h at 37 ℃;

(7) washing: the same (2);

(8) adding a secondary antibody: adding 100 mu l of diluted (dilution ratio of 1:5000) donkey anti-sheep IgG secondary antibody marked by HRP into each hole, and incubating for 0.5h at 37 ℃;

(9) washing: the same (2);

(10) color development: adding 100 mul of TMB color development solution into each hole under the condition of keeping out of the sun, and incubating for 10min at 37 ℃;

(11) and (4) terminating: add 50. mu.l stop solution (2M H) to each well₂SO₄) Terminating the reaction;

(12) and (3) detection: measuring the OD value of the sample at the wavelength of 450nm, and analyzing the data;

(13) the results are shown in table six below: the coating subunit F protein can be specifically combined with serum, and the mean value of OD450 is 0.918; neither the coating subunit F protein specifically bound to the blocking solution, and the mean OD450 was 0.05. This shows that subunit F protein can be used as an antigen of an Elisa kit, has excellent immunogenicity, and can be developed into a diagnostic kit for detecting Peste des petits ruminants infection and immunity after searching for proper coating concentration and serum dilution ratio.

TABLE six Elisa assays for the identification of subunit F proteins

1g.4 stability verification

Diluting the subunit F protein purified in the step 1F to 1mg/ml by using PBS buffer, and dividing into 20 parts, wherein each part is 0.5 ml; wherein 10 parts of the mixture are placed in a refrigerator at 4 ℃, and one part of the mixture is sampled every week and is continuously sampled for 10 times; another 10 parts are placed in a refrigerator at the temperature of 20 ℃ below zero, and one part is sampled every week and is continuously sampled for 10 times; protein concentration was measured by BCA after each sampling and the results are shown in table seven below:

stability of Epheptamer F protein

See table seven, from the change in protein concentration, the protein remained essentially stable during both sets of experiments. To further verify whether the treated protein was degraded, we performed SDS-PAGE with the 10 th sample, and the results are shown in FIG. 7. Wherein, 1 in A of FIG. 7 is Marker, 2 is subunit F protein after 4 ℃ treatment, and the loading amount is 2 mug; in B, 1 is Marker, 2 is subunit F protein after-20 ℃ treatment, and the loading amount is also 2 mug. As can be seen from the figure, the treated sample (10 th sampling) was still stable, and thus it was found that the subunit F protein produced by the present invention had excellent stability.

1 h: vaccine preparation

1h.1 vaccine preparation

(1) Preparing an aqueous phase: according to the content of subunit F protein in the vaccine, PBS buffer solution (or normal saline) is used for diluting the subunit F protein into a plurality of parts with different concentration gradients, such as 50 mug/mL, 100 mug/mL, 200 mug/mL, 400 mug/mL and the like, in the embodiment, the subunit F protein is diluted to 50 mug/mL, namely, the water phase;

(2) preparing an oil phase: according to the total amount of the prepared vaccine, a proper amount of ISA 201VG adjuvant is measured according to the weight ratio of 1:1 and the volume ratio of 46:54 of the antigen phase and the adjuvant;

(3) emulsification: preheating the water phase and the oil phase to 33 ℃, slowly adding the water phase into the oil phase, stirring at 200-500rpm/min for 20-30min, standing at 20 ℃ for 1, and standing at 4 ℃ overnight;

(4) subpackaging and storing: subpackaging as required, and storing at 4 deg.C for use after inspection.

1h.2, vaccine quality inspection

Observing the physical properties by adopting an eye-watching method to observe the appearance (whether the emulsion is milky white or not);

sucking a small amount of vaccine by using a clean straw and dripping the vaccine into cold water, observing (except for the 1 st drop), wherein the vaccine is dispersed in a cloud form and is judged to be a water-in-oil-in-water dosage form;

adding 10mL of vaccine into a centrifuge tube, centrifuging for 15min at the rotating speed of 3000r/min, and judging the vaccine to be stable if the water separated out from the tube bottom is less than or equal to 0.5 mL;

and (4) performing viscosity detection on the vaccine by using a viscometer, wherein the viscosity detection is required to be within 20-50cp, and the vaccine is judged to be qualified.

Example 2: the difference from example 1 is that the coding gene sequence of the subunit F protein is not codon optimized in this example, i.e., the coding gene sequence of the subunit F protein is shown in SEQ ID NO. 2.

Example 3: the difference from example 1 is that the coding gene sequence of subunit F protein is codon optimized in this example, and the specific coding gene sequence is shown in SEQ ID NO. 4.

Example 4: the difference from example 1 is that the coding gene sequence of subunit F protein is codon optimized in this example, and the specific coding gene sequence is shown in SEQ ID NO. 5.

Example 5: the difference from example 1 is that the coding gene sequence of subunit F protein is codon optimized in this example, and the specific coding gene sequence is shown in SEQ ID NO. 6.

Example 6: the difference from example 1 is that the coding gene sequence of subunit F protein is codon optimized in this example, and the specific coding gene sequence is shown in SEQ ID NO. 7.

Example 7: the difference from example 1 is that the eukaryotic expression vector used in this example was pEE6.4, and the cell line was DG44 among CHO cell lines.

Example 8: the difference from example 1 is that the eukaryotic expression vector used in this example was pGL4.13, and the cell line was CHO-K1 among CHO cell lines.

Example 9: the difference from example 1 is that the eukaryotic expression vector used in this example was pcDNA3.1 and the cell line was the CHO-S cell line among CHO cell lines.

Example 10: the difference from example 1 is that the eukaryotic expression vector used in this example was pEE12.4, and the cell line was a CHO-S cell line among CHO cell lines.

Example 11: the differences from example 1 are that the eukaryotic expression vector used in this example was pEE12.4, the cell line was HEK293, and the subunit F protein was prepared without acclimatization as in step 1 d.

Example 12: the differences from example 1 are that the eukaryotic expression vector used in this example was pEE12.4, the cell line was 293T/17 cell line, and the subunit F protein was prepared without acclimatization as in step 1 d.

Example 13: the difference from example 1 is that the eukaryotic expression vector used in this example was pCEP4, and the cell line was DG44 among CHO cell lines.

Comparative example: the difference from example 1 is that the coding gene sequence of the F protein of peste des petits ruminants virus in this comparative example is GenBank: 5532-7172 genome sequence in KM 091959.1.

The expression yields and protein purities of the subunit F proteins in examples 2 to 13 and comparative examples were measured, and the expression results are shown in Table eight below.

TABLE eighthly yield and purity of subunit F protein for example 2-example 13 and comparative examples

Referring to the table eight, the protein yield of the invention is 0.6g/L-1.3g/L, the protein purity is higher than 70%, which is obviously superior to the protein yield of 0.0g/L-0.01g/L and the protein purity of 23% in the comparative example, so that the subunit F protein coding gene constructed by the invention can efficiently secrete and express the subunit F protein in a cell strain, and the protein purity of the obtained subunit F protein is higher.

The protein yield of example 2-example 6 is 0.8g/L-1.0g/L, the protein purity is 75% -86%, while the protein yield of example 1 is 1.1g/L-1.3g/L, the protein purity is 92%, therefore example 1 is the preferred example of example 1-example 6, therefore, it can be obtained that when the coding gene sequence of subunit F protein is SEQ ID NO.3, the corresponding protein yield and protein purity reach the optimal values.

The protein yield of example 7-example 13 is 0.6g/L-0.9g/L, the protein purity is 71% -82%, which is lower than the detection result of example 1. Therefore, when the eukaryotic expression vector is selected from pEE12.4, and the cell strain is selected from one of a CHO cell strain, a HEK293 cell strain and a 293T/17 cell strain, the expression yield and the protein purity of the subunit F protein are both high; particularly, when the eukaryotic expression vector is pEE12.4 and the cell strain is CHO-K1, the expression quantity and the protein purity of the subunit F protein reach the optimal values.

In conclusion, the recombinant plasmid constructed by the invention can be effectively expressed in an engineered cell strain to obtain the high-yield and high-purity Peste des petits ruminants virus subunit F protein, the Peste des petits ruminants virus subunit F protein has good specificity and stability, can be produced in a large scale, effectively reduces the production cost of the Peste des petits ruminants virus subunit F protein, and simultaneously can be better applied to a Peste des petits ruminants virus subunit vaccine or a diagnostic reagent. Therefore, the subunit F protein has the characteristics of high secretion expression efficiency, high protein purity, easiness in purification, low production cost, high safety performance and the like.

The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.

Sequence listing

Sequence listing

<110> Zhejiang Hilon Biotechnology Ltd

<120> subunit F protein of peste des petits ruminants virus, preparation method and application thereof

<130> P010PTA01F1800033

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 491

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Thr Arg Val Ala Ile Leu Thr Phe Leu Phe Leu Phe Pro Asn Val

1 5 10 15

Val Ala Cys Gln Ile His Trp Gly Asn Leu Ser Lys Ile Gly Ile Val

20 25 30

Gly Thr Gly Ser Ala Ser Tyr Lys Val Met Thr Arg Pro Ser His Gln

35 40 45

Thr Leu Val Ile Lys Leu Met Pro Asn Ile Thr Ala Ile Asp Asn Cys

50 55 60

Thr Lys Ser Glu Ile Ala Glu Tyr Lys Arg Leu Leu Ile Thr Val Leu

65 70 75 80

Lys Pro Val Glu Asp Ala Leu Ser Val Ile Thr Lys Asn Val Arg Pro

85 90 95

Ile Gln Thr Leu Thr Pro Gly Arg Arg Thr Arg Arg Phe Ala Gly Ala

100 105 110

Val Leu Ala Gly Val Ala Leu Gly Val Ala Thr Ala Ala Gln Ile Thr

115 120 125

Ala Gly Val Ala Leu His Gln Ser Leu Met Asn Ser Gln Ala Ile Glu

130 135 140

Ser Leu Lys Thr Ser Leu Glu Met Ser Asn Gln Ala Ile Glu Glu Ile

145 150 155 160

Arg Leu Ala Asn Lys Glu Thr Ile Leu Ala Val Gln Gly Val Gln Asp

165 170 175

Tyr Ile Asn Asn Glu Leu Val Pro Ser Val His Arg Met Ser Cys Glu

180 185 190

Leu Val Gly His Lys Leu Gly Leu Lys Leu Leu Arg Tyr Tyr Thr Glu

195 200 205

Ile Leu Ser Ile Phe Gly Pro Ser Leu Arg Asp Pro Ile Ser Ala Glu

210 215 220

Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly Asp Ile Asn Lys

225 230 235 240

Ile Leu Asp Lys Leu Gly Tyr Ser Gly Gly Asp Phe Leu Ala Ile Leu

245 250 255

Glu Ser Lys Gly Ile Lys Ala Arg Val Thr Tyr Val Asp Thr Arg Asp

260 265 270

Tyr Phe Ile Ile Leu Ser Ile Ala Tyr Pro Thr Leu Ser Glu Ile Lys

275 280 285

Gly Val Ile Val His Lys Ile Glu Ala Ile Thr Tyr Asn Ile Gly Ala

290 295 300

Gln Glu Trp Tyr Thr Thr Ile Pro Lys Tyr Val Ala Thr Gln Gly Tyr

305 310 315 320

Leu Ile Ser Asn Phe Asp Glu Thr Ser Cys Val Phe Thr Pro Glu Gly

325 330 335

Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser Pro Leu Leu Gln

340 345 350

Glu Cys Phe Arg Gly Ser Thr Lys Ser Cys Ala Arg Thr Leu Val Ser

355 360 365

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

370 375 380

Asn Cys Ala Ser Val Leu Cys Lys Cys Tyr Thr Thr Glu Thr Val Ile

385 390 395 400

Ser Gln Asp Pro Asp Lys Leu Leu Thr Val Val Ala Ser Asp Lys Cys

405 410 415

Pro Val Val Glu Val Asp Gly Val Thr Ile Gln Val Gly Ser Arg Glu

420 425 430

Tyr Pro Asp Ser Val Tyr Leu His Lys Ile Asp Leu Gly Pro Ala Ile

435 440 445

Ser Leu Glu Lys Leu Asp Val Gly Thr Asn Leu Gly Asn Ala Val Thr

450 455 460

Arg Leu Glu Asn Ala Lys Glu Leu Leu Asp Ala Ser Asp Gln Ile Leu

465 470 475 480

Lys Thr Val Lys Gly Val Pro Phe Ser Gly Asn

485 490

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<213> Artificial Sequence (Artificial Sequence)

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atgacgcggg tcgcaatttt gacatttctg tttcttttcc caaatgttgt tgcgtgtcag 60

attcactggg gcaatctatc caagatcggg attgtaggaa cagggagtgc cagctacaag 120

gtgatgacta ggccaagcca ccagactctg gttataaagt taatgccaaa tataacggcc 180

atcgacaatt gtacaaagtc agagattgca gagtacaaga gattgctgat cacagtgtta 240

aagcctgtag aggatgctct gtcggtgata accaagaatg taagaccaat tcaaactcta 300

acacctgggc gtagaacccg ccgttttgct ggagctgttc tggccggggt agcacttgga 360

gttgcgacag ccgctcagat aactgcagga gtcgcccttc atcaatcatt gatgaactcc 420

caagcaattg agagtttaaa aaccagtctt gagatgtcga atcaggcaat agaagaaatc 480

agacttgcaa ataaggagac catactggca gtacagggcg tccaggatta tatcaacaat 540

gagctcgtcc cttctgttca tagaatgtca tgcgagctgg taggtcacaa gctcggcctc 600

aagctcctta ggtactacac cgagatcctg tccatattcg ggcccagtct tcgagacccg 660

atatctgccg aaatatcaat ccaggcactt agttatgcat taggcggaga cattaataaa 720

atcctggaca agcttgggta tagcggtggg gatttccttg ccatcctaga aagcaaggga 780

ataaaggccc gggttacata tgtggacaca agagattact ttataatcct tagcatcgcc 840

tacccaacct tatctgagat caagggagtg atagttcaca agatagaagc tataacatac 900

aacattgggg cacaggagtg gtatactact atccctaaat atgtagccac tcaggggtat 960

ctgatatcga actttgatga gacgtcatgc gtattcactc cagaggggac agtttgcagc 1020

cagaatgcgt tgtacccaat gagcccattg cttcaggaat gtttcagggg gtcaacaaaa 1080

tcgtgcgcca gaaccttagt ttcagggacc ataagtaata gatttatcct atcaaaaggg 1140

aacctgattg caaattgtgc gtcagttttg tgcaaatgtt acacaacaga gacagttatc 1200

agccaagatc ctgacaaact actaactgtt gtagcatccg acaagtgtcc tgtagttgag 1260

gtggatggag tgacaataca ggtcggcagt cgagagtatc cggattctgt atacttacac 1320

aaaatagact taggtccagc catctcccta gaaaaactgg atgtaggcac caatttaggc 1380

aatgcagtca caagactgga gaatgcaaag gagctcctag atgcatcaga ccaaatactg 1440

aagactgtta aaggggtacc tttcagtggg aat 1473

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgaccaggg tggctatcct gacattcctg tttctgttcc caaacgtggt ggcctgccag 60

atccactggg gcaatctgtc caagatcggc atcgtgggca ccggctccgc tagctataaa 120

gtgatgacca gacccagcca tcagaccctg gtcatcaagc tgatgcctaa catcaccgct 180

atcgacaatt gtacaaagtc tgagatcgcc gagtacaaga gactgctgat caccgtgctg 240

aagcccgtgg aggatgccct gtccgtgatc acaaagaacg tgcgccctat ccagaccctg 300

acaccaggca ggcggaccag acgctttgct ggagctgtgc tggctggagt ggctctgggc 360

gtggctaccg ctgctcagat cacagctgga gtggctctgc accagagcct gatgaactcc 420

caggccatcg agtccctgaa gaccagcctg gagatgtcta accaggccat cgaggagatc 480

aggctggcta ataaggagac aatcctggcc gtgcagggcg tgcaggacta catcaacaat 540

gagctggtgc ctagcgtgca ccggatgtct tgcgagctgg tgggccataa gctgggcctg 600

aagctgctga ggtactatac cgagatcctg tccatcttcg gaccaagcct gagggaccca 660

atctccgctg agatcagcat ccaggccctg agctatgctc tgggcggcga catcaacaag 720

atcctggata agctgggtta cagcggcggc gattttctgg ctatcctgga gtctaagggc 780

atcaaggcca gggtgaccta cgtggacaca cgggattatt tcatcatcct gtccatcgct 840

tatccaaccc tgagcgagat caagggcgtg atcgtgcaca agatcgaggc tatcacatac 900

aatatcggcg cccaggagtg gtacaccaca atccccaagt atgtggccac ccagggctac 960

ctgatctcta actttgacga gacatcctgc gtgttcaccc ctgagggcac cgtgtgctct 1020

cagaatgctc tgtatcccat gtcccctctg ctgcaggagt gctttagagg ctctaccaag 1080

tcctgtgccc gcaccctggt gtctggcaca atctccaaca ggttcatcct gtccaagggc 1140

aacctgatcg ccaattgcgc tagcgtgctg tgcaagtgtt acaccacaga gaccgtgatc 1200

tctcaggacc ctgataagct gctgacagtg gtggcctccg acaagtgtcc agtggtggag 1260

gtggatggcg tgaccatcca agtgggcagc agagagtacc cagactccgt gtacctgcat 1320

aagatcgatc tgggccccgc tatctctctg gagaagctgg atgtgggcac caacctgggc 1380

aatgccgtga cacgcctgga gaacgccaag gagctgctgg acgctagcga tcagatcctg 1440

aagacagtga agggcgtgcc cttttctggc aat 1473

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<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgactagag ttgctattct tactttcctt ttccttttcc caaatgttgt tgcttgtcaa 60

attcattggg gaaatctttc taaaattgga attgttggaa ctggatctgc ttcttataaa 120

gttatgacta gaccatctca tcaaactctt gttattaaac ttatgccaaa tattactgct 180

attgataatt gtactaaatc tgaaattgct gaatataaaa gacttcttat tactgttctt 240

aaaccagttg aagatgctct ttctgttatt actaaaaatg ttagaccaat tcaaactctt 300

actccaggaa gaagaactag aagattcgct ggagctgttc ttgctggagt tgctcttgga 360

gttgctactg ctgctcaaat tactgctgga gttgctcttc atcaatctct tatgaattct 420

caagctattg aatctcttaa aacttctctt gaaatgtcta atcaagctat tgaagaaatt 480

agacttgcta ataaagaaac tattcttgct gttcaaggag ttcaagatta tattaataat 540

gaacttgttc catctgttca tagaatgtct tgtgaacttg ttggacataa acttggactt 600

aaacttctta gatattatac tgaaattctt tctattttcg gaccatctct tagagatcca 660

atttctgctg aaatttctat tcaagctctt tcttatgctc ttggaggaga tattaataaa 720

attcttgata aacttggata ttctggagga gatttccttg ctattcttga atctaaagga 780

attaaagcta gagttactta tgttgatact agagattatt tcattattct ttctattgct 840

tatccaactc tttctgaaat taaaggagtt attgttcata aaattgaagc tattacttat 900

aatattggag ctcaagaatg gtatactact attccaaaat atgttgctac tcaaggatat 960

cttatttcta atttcgatga aacttcttgt gttttcactc cagaaggaac tgtttgttct 1020

caaaatgctc tttatccaat gtctccactt cttcaagaat gtttcagagg atctactaaa 1080

tcttgtgcta gaactcttgt ttctggaact atttctaata gattcattct ttctaaagga 1140

aatcttattg ctaattgtgc ttctgttctt tgtaaatgtt atactactga aactgttatt 1200

tctcaagatc cagataaact tcttactgtt gttgcttctg ataaatgtcc agttgttgaa 1260

gttgatggag ttactattca agttggatct agagaatatc cagattctgt ttatcttcat 1320

aaaattgatc ttggaccagc tatttctctt gaaaaacttg atgttggaac taatcttgga 1380

aatgctgtta ctagacttga aaatgctaaa gaacttcttg atgcttctga tcaaattctt 1440

aaaactgtta aaggagttcc attctctgga aat 1473

<210> 5

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgacccgcg tggccatcct gaccttcctg ttcctgttcc ccaacgtggt ggcctgccag 60

atccactggg gcaacctgtc caagatcggc atcgtgggca ccggctccgc ctcctacaag 120

gtgatgaccc gcccctccca ccagaccctg gtgatcaagc tgatgcccaa catcaccgcc 180

atcgacaact gcaccaagtc cgagatcgcc gagtacaagc gcctgctgat caccgtgctg 240

aagcccgtgg aggacgccct gtccgtgatc accaagaacg tgcgccccat ccagaccctg 300

acccccggcc gccgcacccg ccgcttcgcc ggcgccgtgc tggccggcgt ggccctgggc 360

gtggccaccg ccgcccagat caccgccggc gtggccctgc accagtccct gatgaactcc 420

caggccatcg agtccctgaa gacctccctg gagatgtcca accaggccat cgaggagatc 480

cgcctggcca acaaggagac catcctggcc gtgcagggcg tgcaggacta catcaacaac 540

gagctggtgc cctccgtgca ccgcatgtcc tgcgagctgg tgggccacaa gctgggcctg 600

aagctgctgc gctactacac cgagatcctg tccatcttcg gcccctccct gcgcgacccc 660

atctccgccg agatctccat ccaggccctg tcctacgccc tgggcggcga catcaacaag 720

atcctggaca agctgggcta ctccggcggc gacttcctgg ccatcctgga gtccaagggc 780

atcaaggccc gcgtgaccta cgtggacacc cgcgactact tcatcatcct gtccatcgcc 840

taccccaccc tgtccgagat caagggcgtg atcgtgcaca agatcgaggc catcacctac 900

aacatcggcg cccaggagtg gtacaccacc atccccaagt acgtggccac ccagggctac 960

ctgatctcca acttcgacga gacctcctgc gtgttcaccc ccgagggcac cgtgtgctcc 1020

cagaacgccc tgtaccccat gtcccccctg ctgcaggagt gcttccgcgg ctccaccaag 1080

tcctgcgccc gcaccctggt gtccggcacc atctccaacc gcttcatcct gtccaagggc 1140

aacctgatcg ccaactgcgc ctccgtgctg tgcaagtgct acaccaccga gaccgtgatc 1200

tcccaggacc ccgacaagct gctgaccgtg gtggcctccg acaagtgccc cgtggtggag 1260

gtggacggcg tgaccatcca ggtgggctcc cgcgagtacc ccgactccgt gtacctgcac 1320

aagatcgacc tgggccccgc catctccctg gagaagctgg acgtgggcac caacctgggc 1380

aacgccgtga cccgcctgga gaacgccaag gagctgctgg acgcctccga ccagatcctg 1440

aagaccgtga agggcgtgcc cttctccggc aac 1473

<210> 6

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgacccgcg tggccatcct gaccttcctg ttcctgttcc ccaacgtggt ggcctgccag 60

atccactggg gcaacctgag caagatcggc atcgtgggca ccggcagcgc cagctacaag 120

gtgatgaccc gccccagcca ccagaccctg gtgatcaagc tgatgcccaa catcaccgcc 180

atcgacaact gcaccaagag cgagatcgcc gagtacaagc gcctgctgat caccgtgctg 240

aagcccgtgg aggacgccct gagcgtgatc accaagaacg tgcgccccat ccagaccctg 300

acccccggcc gccgcacccg ccgcttcgcc ggcgccgtgc tggccggcgt ggccctgggc 360

gtggccaccg ccgcccagat caccgccggc gtggccctgc accagagcct gatgaacagc 420

caggccatcg agagcctgaa gaccagcctg gagatgagca accaggccat cgaggagatc 480

cgcctggcca acaaggagac catcctggcc gtgcagggcg tgcaggacta catcaacaac 540

gagctggtgc ccagcgtgca ccgcatgagc tgcgagctgg tgggccacaa gctgggcctg 600

aagctgctgc gctactacac cgagatcctg agcatcttcg gccccagcct gcgcgacccc 660

atcagcgccg agatcagcat ccaggccctg agctacgccc tgggcggcga catcaacaag 720

atcctggaca agctgggcta cagcggcggc gacttcctgg ccatcctgga gagcaagggc 780

atcaaggccc gcgtgaccta cgtggacacc cgcgactact tcatcatcct gagcatcgcc 840

taccccaccc tgagcgagat caagggcgtg atcgtgcaca agatcgaggc catcacctac 900

aacatcggcg cccaggagtg gtacaccacc atccccaagt acgtggccac ccagggctac 960

ctgatcagca acttcgacga gaccagctgc gtgttcaccc ccgagggcac cgtgtgcagc 1020

cagaacgccc tgtaccccat gagccccctg ctgcaggagt gcttccgcgg cagcaccaag 1080

agctgcgccc gcaccctggt gagcggcacc atcagcaacc gcttcatcct gagcaagggc 1140

aacctgatcg ccaactgcgc cagcgtgctg tgcaagtgct acaccaccga gaccgtgatc 1200

agccaggacc ccgacaagct gctgaccgtg gtggccagcg acaagtgccc cgtggtggag 1260

gtggacggcg tgaccatcca ggtgggcagc cgcgagtacc ccgacagcgt gtacctgcac 1320

aagatcgacc tgggccccgc catcagcctg gagaagctgg acgtgggcac caacctgggc 1380

aacgccgtga cccgcctgga gaacgccaag gagctgctgg acgccagcga ccagatcctg 1440

aagaccgtga agggcgtgcc cttcagcggc aac 1473

<210> 7

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgacccgtg ttgctatcct gaccttcctg ttcctgttcc cgaacgttgt tgcttgccag 60

atccactggg gtaacctgtc taaaatcggt atcgttggta ccggttctgc ttcttacaaa 120

gttatgaccc gtccgtctca ccagaccctg gttatcaaac tgatgccgaa catcaccgct 180

atcgacaact gcaccaaatc tgaaatcgct gaatacaaac gtctgctgat caccgttctg 240

aaaccggttg aagacgctct gtctgttatc accaaaaacg ttcgtccgat ccagaccctg 300

accccgggtc gtcgtacccg tcgtttcgct ggtgctgttc tggctggtgt tgctctgggt 360

gttgctaccg ctgctcagat caccgctggt gttgctctgc accagtctct gatgaactct 420

caggctatcg aatctctgaa aacctctctg gaaatgtcta accaggctat cgaagaaatc 480

cgtctggcta acaaagaaac catcctggct gttcagggtg ttcaggacta catcaacaac 540

gaactggttc cgtctgttca ccgtatgtct tgcgaactgg ttggtcacaa actgggtctg 600

aaactgctgc gttactacac cgaaatcctg tctatcttcg gtccgtctct gcgtgacccg 660

atctctgctg aaatctctat ccaggctctg tcttacgctc tgggtggtga catcaacaaa 720

atcctggaca aactgggtta ctctggtggt gacttcctgg ctatcctgga atctaaaggt 780

atcaaagctc gtgttaccta cgttgacacc cgtgactact tcatcatcct gtctatcgct 840

tacccgaccc tgtctgaaat caaaggtgtt atcgttcaca aaatcgaagc tatcacctac 900

aacatcggtg ctcaggaatg gtacaccacc atcccgaaat acgttgctac ccagggttac 960

ctgatctcta acttcgacga aacctcttgc gttttcaccc cggaaggtac cgtttgctct 1020

cagaacgctc tgtacccgat gtctccgctg ctgcaggaat gcttccgtgg ttctaccaaa 1080

tcttgcgctc gtaccctggt ttctggtacc atctctaacc gtttcatcct gtctaaaggt 1140

aacctgatcg ctaactgcgc ttctgttctg tgcaaatgct acaccaccga aaccgttatc 1200

tctcaggacc cggacaaact gctgaccgtt gttgcttctg acaaatgccc ggttgttgaa 1260

gttgacggtg ttaccatcca ggttggttct cgtgaatacc cggactctgt ttacctgcac 1320

aaaatcgacc tgggtccggc tatctctctg gaaaaactgg acgttggtac caacctgggt 1380

aacgctgtta cccgtctgga aaacgctaaa gaactgctgg acgcttctga ccagatcctg 1440

aaaaccgtta aaggtgttcc gttctctggt aac 1473

Claims (9)

1. Subunit F protein of peste des petits ruminants virus, characterized in that, the amino acid sequence of the subunit F protein is the amino acid sequence shown in SEQ ID NO. 1;

the subunit F protein is connected with one label of poly-His, FLAG, c-myc, HA and poly-Arg at the amino terminal or the carboxyl terminal of the amino acid sequence shown as SEQ ID NO. 1.

2. The subunit F protein of Peste des petits ruminants virus of claim 1, wherein the coding gene sequence of the subunit F protein is shown in SEQ ID No.2, or is obtained by codon optimization of SEQ ID No. 2.

3. The subunit F protein of Peste des petits ruminants virus of claim 2, wherein the coding gene sequence of the subunit F protein is shown in SEQ ID No. 3.

4. A method of producing a subunit F protein of peste des petits ruminants virus according to any one of claims 1 to 3, comprising the steps of:

firstly, constructing an encoding gene sequence of subunit F protein of peste des petits ruminants virus;

secondly, cloning the coding gene sequence of the subunit F protein into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit F protein;

transfecting recombinant plasmids containing subunit F protein coding gene sequences into engineering cells of animals to obtain cell strains;

fourthly, screening out cell strains with high expression from the cell strains obtained in the third step;

fifthly, purifying the highly expressed cell strain obtained in the fermentation culture step to obtain subunit F protein of the peste des petits ruminants virus.

5. The method of claim 4, wherein the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.1.

6. The method of claim 5, wherein the eukaryotic expression vector is pEE12.4 in step (ii).

7. The method for preparing subunit F protein of Peste des petits ruminants virus according to claim 4, wherein in the third step, the cell strain is one of CHO cell strain, HEK293 cell strain and 293T/17 cell strain.

8. The method for preparing subunit F protein of Peste des petits ruminants virus of claim 7, wherein in step (c), the CHO cell line is one of DG44 cell line, DXB11 cell line, CHO-K1 cell line and CHO-S cell line.

9. Use of a subunit F protein of a peste des petits ruminants virus according to any one of claims 1 to 3 for the preparation of a subunit vaccine or diagnostic agent suitable for peste des petits ruminants virus.

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