CN115845041A - Duck circovirus bivalent subunit vaccine and preparation method thereof - Google Patents

Abstract

The invention provides a duck circovirus bivalent subunit vaccine and a preparation method thereof, wherein antigens of the vaccine are duck circovirus type I recombinant Cap protein and type II recombinant Cap protein, nucleotide sequences of the vaccine are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the two proteins are expressed by Kluyveromyces marxianus engineering bacteria. The protein has the advantages of good immune effect, high safety, simple culture operation, high yield, low cost, large-scale production and the like, and can be applied to reducing and preventing relevant clinical symptoms caused by the duck circovirus infection and the mixed infection virus.

Description

Duck circovirus bivalent subunit vaccine and preparation method thereof

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a duck circovirus bivalent subunit vaccine and a preparation method thereof.

Background

Duck circovirus (Du CV) belongs to the family of Circoviridae, the genus circovirus (Circoviridae), has no membrane, and has a single-stranded circular DNA structure. The clinical manifestations of infected duck circovirus include disordered feather, growth and development retardation, weight loss, immunosuppression, weakened immune function and multiple organ growth and development failure, and can also cause secondary infection or multiple infections, so that the disease condition is serious, the death rate is increased, and serious economic loss is caused. The duck circovirus has good stability, strong capacity of resisting external environment and rapid adaptation to environment, and can survive for a period of time in acidic environment and high-temperature environment, so that the virus is difficult to completely eliminate once infecting a host. At present, a cell line capable of being cultured and propagated in vitro is not found for duck circovirus, and the duck circovirus is difficult to be cultured in vitro, so that the research on the duck circovirus is limited, and the research and development are incomplete due to late discovery of the duck circovirus, so that an effective vaccine is not available at present, and the prevention, control and treatment of the duck circovirus disease are difficult.

DuCV-1 and DuCV-2 exist in two major genotypes, about 2.0kb, including 6 or more 200bp Open Reading Frames (ORFs), in which the Cap protein is encoded by ORF2, which is the major structural protein constituting the viral nucleocapsid, plays an important role in viral replication and packaging, and is closely related to host immunity and viral infection. Related researches show that the N end of the Cap protein of the circovirus has an NLS sequence region rich in arginine, so that the expression quantity of the full-length Cap protein is very low or even the full-length Cap protein cannot be expressed.

Disclosure of Invention

The invention aims to provide a duck circovirus bivalent subunit vaccine and a preparation method thereof.

The invention aims to provide an expression vector of duck circovirus recombinant Cap protein.

The invention aims to provide an expression strain of duck circovirus recombinant Cap protein.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

the antigens of the subunit vaccine are duck circovirus type I recombinant Cap protein and type II recombinant Cap protein, and the nucleotide sequences of the subunit vaccine are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

The duck circovirus type I recombinant Cap protein and the type II recombinant Cap protein are expressed by a Kluyveromyces marxianus system, and the Kluyveromyces marxianus expression system comprises a Kluyveromyces marxianus expression vector and a Kluyveromyces marxianus auxotrophic strain.

The Kluyveromyces marxianus expression vector comprises: the kit comprises a Kluyveromyces marxianus autonomous replication sequence, a Kluyveromyces marxianus inulinase promoter, a Kluyveromyces marxianus inulinase terminator, an escherichia coli replication sequence, a resistance screening gene and a nutritional deficiency screening gene.

The gene sequence of the Kluyveromyces marxianus expression vector is shown in SEQ ID NO. 3.

The auxotrophic strain of the kluyveromyces marxianus is obtained by knocking out part or all of specific nutritional genes of the kluyveromyces marxianus.

The auxotrophy screening gene is any one or more of URA3 gene, HIS3 gene and ADE2 gene.

The preparation method comprises the following steps:

1) Inserting the optimized nucleotide for coding the duck circovirus I-type Cap protein and the duck circovirus II-type Cap protein into a Kluyveromyces marxianus expression vector to construct a recombinant vector;

2) Transforming the recombinant vector into a Kluyveromyces marxianus auxotrophic strain to construct recombinant yeast;

3) Culturing and fermenting the recombinant yeast, and expressing the duck circovirus I-type Cap protein and the duck circovirus II-type Cap protein;

4) Collecting the culture fermented bacterial liquid, separating and purifying to obtain duck circovirus I-type Cap protein and duck circovirus II-type Cap protein;

5) Mixing the purified duck circovirus I-type Cap protein and the purified duck circovirus II-type Cap protein with a vaccine adjuvant according to a protein concentration ratio of 1.9-1.1, and preparing the bivalent subunit vaccine.

The vaccine adjuvant is selected from any one of oil-in-water, water-in-oil and water-in-oil-in-water adjuvants.

Compared with the prior art, the invention has the following advantages:

the invention overcomes the technical defects of low expression quantity, low duck circovirus activity and the like of an expression method.

The optimized duck circovirus recombinant Cap protein is expressed by using a Kluyveromyces marxianus expression system, the expressed protein content is high, and the method is suitable for large-scale production.

The Kluyveromyces marxianus adopted by the invention is a food safety grade yeast, has the advantages of fast growth, high density, high temperature resistance, utilization of various carbon sources and the like, is an industrial protein expression strain with great potential, has the characteristics of stable genetic property, simple operation, easy high-density culture, high protein yield, low production cost, suitability for industrial large-scale production and the like, has the advantages of foreign protein post-translational processing and the like which are not possessed by a prokaryotic expression system, and simultaneously avoids the phenomena of instability, easy plasmid loss, excessive glycosylation and the like of other yeast expression strains.

The bivalent subunit vaccine of duck circovirus provided by the invention can reduce and prevent relevant clinical symptoms caused by DuCV infection, reduce the occurrence of mixed infection, and can obtain a high-level serum IgG antibody by injecting and immunizing once.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a double restriction enzyme identification electrophoresis diagram of synthesized Cap gene; wherein, 1: recombinant plasmid pUKD-N115/CapI; lane 2: recombinant plasmid pUKD-N115/CapII;

FIG. 2 is a schematic diagram of a duck circovirus type I Cap protein Kluyveromyces marxianus recombinant vector pUKD-N115/CapI;

FIG. 3 is a schematic diagram of a duck circovirus type II Cap protein Kluyveromyces marxianus recombinant vector pUKD-N115/CapII;

FIG. 4 shows the expression of the type I and type II Cap proteins of duck circovirus in Kluyveromyces marxianus; wherein 1 is yeast control; 2 is recombinant yeast for expressing DuCV-CapI; 3 is recombinant yeast for expressing DuCV-CapII;

FIG. 5 is a graph of the levels of antibodies produced by duck circovirus bivalent subunit vaccine stimulated mice.

Detailed Description

The following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the following examples, including the carrier, enzyme, buffer solution and other reagent raw materials, were all from commercial products. The Kluyveromyces marxianus preservation number used in the invention is CGMCC No.10621, the strain is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the preservation unit address is as follows: the microbial research institute of the national academy of sciences No.3, xilu No.1, beijing, chaoyang, and the preservation date is 2015, 3 months and 13 days.

Example 1 Synthesis construction of recombinant expression vectors pUKD-N115/CapI and pUKD-N115/CapII for Cap protein of Duck circovirus

Coding duck circovirus I-type Cap protein and duck circovirus II-type Cap protein with gene sequence numbers of MN068356.1 and MT646348.1 published on NCBI, carrying out codon optimization on the duck circovirus I-type Cap gene sequence and the duck circovirus II-type Cap gene sequence according to the codon preference of Kluyveromyces marxianus, and artificially synthesizing the optimized Cap gene sequences, wherein the optimized nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No. 2.

Constructing a Kluyveromyces marxianus expression vector pUKD-N115, which comprises an autonomous replication sequence of Kluyveromyces marxianus, an inulinase promoter sequence of Kluyveromyces marxianus, an inulinase terminator sequence of Kluyveromyces marxianus and an auxotrophy selection marker gene URA3 (KmURA 30 RF) of Kluyveromyces marxianus. Then, the pUKD-N115 vector is digested by SmaI and NotI restriction enzymes, the synthesized recombinant expression vectors pUKD-N115/CapII and pUKD-N115/CapII are subjected to double digestion, nucleic acid fragments are separated by electrophoresis of 1% agarose gel, and the recombinant vector can be digested into two fragments with different sizes, and the result is shown in figure 1.

Example 2 expression of the Cap protein of Duck circovirus in Kluyveromyces marxianus genetically engineered bacteria Fim-lura 3. Delta. -pUKD-N115/CapI and Fim-lura 3. Delta. -pUKD-N115/CapII.

Kluyveromyces marxianus uracil auxotrophic strain Fim-lura3 delta has a preservation number of: CGMCC No.10621, fim-lura3 delta inoculated in glass test tube containing 3mL YEPD culture medium, shaking table at 30 ℃ overnight to OD ₆₀₀ Is 12-15. Collecting thallus, and treating with LiAc-TEThe solution (100mM LiAc, l0mM Tris-HCl,1mM EDTA) was washed.

The vector DNA, recombinant vector pUKD-N115/CapI or pUKD-N115/CapII, PEG solution (40% wt PEG4000, 100m LiAc, l0mM Tris-HCl pH 7.5,1mM EDTA) and final concentration l0mM DTT were added to the cells in this order, mixed well, immediately separated in 30 ℃ water bath 15min,47 ℃ water bath 15min,8000rpm, discarded the supernatant, suspended cells in 100uL sterile water, coated with SD plate (0.67% aminoyeast-free nitrogen source YNB,2% glucose, 2% agar) and cultured at 30 ℃ for 2-4 days until the clone was formed.

Selecting a single clone from a selective medium plate, culturing the single clone in a test tube of 3mL YEPD medium at 30 ℃ overnight by a shaking table, extracting a transformant genome by a conventional yeast genome extraction method, and carrying out PCR verification by using universal primers DuCVN125-F (5 'TATAATGTCGCTGTGACC AGGC 3') and DuCVN125-R (5 'CAGCAATTAA ATCCGGGTA AG 3') to amplify a positive clone with a band of about 1200bp, namely the genetically engineered bacteria Fim-lura3 delta-pUKD-N115/CapI and Fim-lura3 delta-pUKD-N115/CapII transferred into the DuCV Cap protein.

The constructed engineering bacteria Fim-lura3 delta-pUKD-N115/CapI, fim-lura3 delta-pUKD-N115/CapII and the control bacteria Fim-lura3 delta are respectively inoculated in YP culture medium (1% Yeast Extract and 2% glucose) with 50mL, cultured for 54h at 30 ℃, centrifuged and collected cells, the cells are broken by adopting a high-pressure homogenization method, protein electrophoresis SDS-PAGE (polyacrylamide gel electrophoresis, PAGE) is carried out on cell lysate, the recombinant expression nucleocapsid protein Cap is basically consistent with the theoretical molecular weight of 28kD, and the result is shown in figure 4.

Kluyveromyces marxianus genetically engineered bacteria Fim-lura3 delta-pUKD-N115/CapI and Fim-lura3 delta-pUKD-N115/CapII are inoculated on a YEPD solid culture medium and cultured for 2 days at 30 ℃ for activation. Then transferring to synthetic medium or complete medium (1% yeast extract in 2% glucose) containing glucose, sulfuric acid, potassium dihydrogen phosphate and magnesium sulfate, and culturing at 30 deg.C under shaking (180-220 rpm) for 18-24h.

Inoculating the seed solution into a 100L fermentation tank containing 35L culture medium at a ratio of 10%, ventilating and stirring during fermentation, supplementing with dissolved oxygen in full feed flow mannerControlling the temperature at 30-35 deg.C, pH at 5.5-6.0, and fermenting for 60 hr ₆₀₀ The expression level of the target protein is up to 400, and the expression level of the target protein is close to 1g/L.

Example 3 purification of the proteins of the Duck circovirus cap I and cap II and preparation of the subunit vaccine

And (4) collecting thalli by high-speed centrifugation of yeast liquid obtained by high-density fermentation. After the thalli are re-suspended by PBS buffer solution with the volume of 2 times, a high-pressure homogenizer is adopted to break the cells, and the breaking pressure is 1000-1200bar. Clarifying the bacteria-breaking liquid in a centrifugal mode, collecting supernatant, and then concentrating twice through a membrane package. The prepared cap I and cap II protein solutions are mixed in equal proportion, then 2 times of oil adjuvant is added, the duck circovirus bivalent subunit vaccine is prepared after full emulsification, and the bivalent vaccine prepared by adopting oil-in-water, water-in-oil-in-water and other adjuvant forms is used for the following immunity test.

Example 4 immunoassay

30 Balb/c mice at 6 weeks of age were purchased and randomly divided into 3 groups of 10 mice each. The first group was blank control group, and no immunization was performed; the second group was a negative control group, which was immunized subcutaneously with a blank vaccine (0.2 ml/tube) containing no antigen component; the third group was experimental group, which was injected subcutaneously with duck circular subunit vaccine (0.2 ml/mouse). 0.1ml of blood was collected from all mice at 14 days, 21 days and 28 days after immunization, respectively, and the serum obtained by the isolation was stored in a-20 ℃ refrigerator.

The detection of the anti-duck circovirus IgG antibody content in mouse serum adopts a self-established ELISA method, and the detection process is as follows:

preparation of coating antigen: expressing and preparing duck circular cap protein by using an escherichia coli system;

antigen coating: diluting the purified recombinant duck circular Cap protein to a final concentration of 1 mug/mL, coating 100 mug/well on a 96-well enzyme label plate, and standing overnight at 4 ℃;

and (3) sealing: washing with PBST for 3 times (200. Mu.L/well), adding (5% skim milk) blocking solution, 100. Mu.L/well, and blocking at 37 deg.C for 2h;

adding serum: washing with PBST for 3 times (200 mu L/hole), diluting the mouse negative serum and the serum to be detected by 1 time to 200 times respectively, adding an enzyme label plate into 100 mu L/hole, and incubating for 1h at 37 ℃;

adding enzyme-labeled secondary antibody, washing with PBST for 3 times (200. Mu.L/hole), diluting secondary antibody of HRP-IgG goat anti-mouse with 1;

substrate color development: washing with PBST for 3 times (200. Mu.L/well), adding TMB substrate at 100. Mu.L/well for color development, and incubating at room temperature for 5min;

and (3) terminating the reaction: 50 mu L/hole, 2mol/L H is added ₂ SO ₄ Terminating the reaction;

determination of OD _450nm Value OD read by microplate reader _450nm Absorbance values and calculating the average value.

The mouse immune vaccine generates serum IgG antibody at the beginning of 2-3 weeks, no specific antibody is detected in the serum of the immune mouse after 14D injection, the specific antibody appears in the serum of the immune mouse after 21D injection, and the measured OD _450nm 0.227, the antibody level in the immunized group was significantly increased after immunization 28D, OD _450nm Is 0.463. After 4 weeks, the antibody is continuously increased (figure 5), which shows that the duck circular subunit vaccine prepared by the invention has high immune effect. Vaccines prepared with the different forms of adjuvant of example 3 also had similar immunising effects.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (8)

1. The duck circovirus bivalent subunit vaccine and the preparation method thereof are characterized in that the antigens of the subunit vaccine are duck circovirus type I recombinant Cap protein and type II recombinant Cap protein, and the nucleotide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

2. The duck circovirus bivalent subunit vaccine and the preparation method thereof as claimed in claim 1, wherein the duck circovirus type I and type II recombinant Cap proteins are expressed by a Kluyveromyces marxianus system, and the Kluyveromyces marxianus expression system comprises a Kluyveromyces marxianus expression vector and a Kluyveromyces marxianus auxotrophic strain.

3. The duck circovirus bivalent subunit vaccine and the preparation method thereof as claimed in claim 2, wherein said kluyveromyces marxianus expression vector comprises: the kit comprises a Kluyveromyces marxianus autonomous replication sequence, a Kluyveromyces marxianus inulase promoter, a Kluyveromyces marxianus inulase terminator, an escherichia coli replication sequence, a resistance screening gene and an auxotrophy screening gene.

4. The duck circovirus bivalent subunit vaccine as claimed in claim 2, wherein the gene sequence of the kluyveromyces marxianus expression vector is shown in SEQ ID No. 3.

5. The duck circovirus bivalent subunit vaccine as claimed in claim 2, wherein the auxotrophic strain of kluyveromyces marxianus is obtained by knocking out part or all of specific nutritional genes of kluyveromyces marxianus.

6. The divalent subunit vaccine of duck circovirus and the preparation method thereof as claimed in claim 5, wherein the auxotrophic screening gene is any one or more of URA3 gene, HIS3 gene and ADE2 gene.

7. The divalent subunit vaccine of duck circovirus and preparation method thereof according to claim 2, wherein the preparation method of the vaccine comprises:

1) Inserting the optimized nucleotide for coding the duck circovirus I-type Cap protein and the duck circovirus II-type Cap protein into a Kluyveromyces marxianus expression vector to construct a recombinant vector;

2) Transforming the recombinant vector into a Kluyveromyces marxianus auxotrophic strain to construct recombinant yeast;

3) Culturing and fermenting the recombinant yeast, and expressing the duck circovirus I-type Cap protein and the duck circovirus II-type Cap protein;

4) Collecting the culture fermented bacterial liquid, separating and purifying to obtain duck circovirus type I Cap protein and duck circovirus type II Cap protein;

5) Mixing the purified duck circovirus I-type Cap protein and the purified duck circovirus II-type Cap protein with a vaccine adjuvant according to a protein concentration ratio of 1.9-1.1, and preparing the bivalent subunit vaccine.

8. The divalent subunit vaccine of duck circovirus and preparation method thereof as claimed in claim 7, wherein said vaccine adjuvant is selected from any one of oil-in-water, water-in-oil, and water-in-oil-in-water adjuvants.

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