CN106399350B - Porcine circovirus type II virus-like particle vaccine and preparation method thereof - Google Patents

Porcine circovirus type II virus-like particle vaccine and preparation method thereof Download PDF

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CN106399350B
CN106399350B CN201610806538.0A CN201610806538A CN106399350B CN 106399350 B CN106399350 B CN 106399350B CN 201610806538 A CN201610806538 A CN 201610806538A CN 106399350 B CN106399350 B CN 106399350B
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porcine circovirus
virus
circovirus type
kluyveromyces marxianus
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CN106399350A (en
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吕红
周峻岗
段进坤
余垚
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Fudan University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10023—Virus like particles [VLP]
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention provides Kluyveromyces marxianus engineering bacteria for recombinant expression of porcine circovirus type II capsid protein, which clones porcine circovirus type II capsid protein genes into an expression vector and obtains porcine circovirus type II virus-like particles through recombinant expression in Kluyveromyces marxianus. The invention also provides a preparation method of the porcine circovirus type II virus-like particle vaccine, which is characterized in that porcine circovirus type II virus-like particles are obtained through high-density fermentation of recombinant kluyveromyces marxianus and the vaccine is prepared through separation and purification. The porcine circovirus type II virus-like particle vaccine provided by the invention can obtain a high-level serum IgG antibody by injecting and immunizing once, has the advantages of good immune effect, high safety, simple culture operation, high yield, low production cost, large-scale production amplification and the like, and can be applied to reducing and preventing relevant clinical symptoms caused by infecting the porcine circovirus PCV 2.

Description

Porcine circovirus type II virus-like particle vaccine and preparation method thereof
Technical Field
The invention relates to a recombinant porcine circovirus type II virus-like particle vaccine and a preparation method thereof, in particular to a method for preparing the porcine circovirus type II virus-like particle vaccine by using Kluyveromyces marxianus with high yield and low cost and the porcine circovirus type II virus-like particle vaccine with good immune effect and high safety.
Background
Porcine Circovirus (PCV) is one of the smallest animal viruses found to date, belonging to the genus circovirus of the family circoviridae, is an unencapsulated single-stranded circular negative-strand DNA virus, is 20-face-symmetric, has a diameter of 17-20nm, and is known to have two serotypes, namely Porcine circovirus type I (PCV1) and Porcine circovirus type II (PCV2), of which PCV1 is a non-pathogenic virus and PCV2 is a pathogenic virus.
PCV is extremely harmful to the pig industry and widely popular worldwide. The pig infected with the traditional Chinese medicine is manifested by emaciation, swollen lymph nodes, asthma and diarrhea, and also can cause reproductive disturbance diseases of sows. PCV also can destroy the immune system of the infected pig body, cause immunosuppression and cause vaccine immune failure. Clinically, PCV is often mixed with swine fever, highly pathogenic porcine reproductive and respiratory syndrome, porcine parvovirus and the like to infect, so that the infection rate and the death rate of the swine herd are high. At present, no specific medicine for the disease exists, and vaccination still is the most effective measure for preventing and controlling PCV disease.
More seriously, PVC2 in the pig industry of China shows the condition of early infection day (the piglets in the delivery room can be infected). PCV2 has therefore been recognized as an important pathogen in the swine industry worldwide, causing immunosuppression in swine, causing double or triple infections, high morbidity and mortality, significant harm to the swine industry, and significant economic losses. At present, the PCV2 vaccine becomes a swine disease vaccine variety with the largest and the largest dosage after the vaccines of swine fever, foot and mouth disease, blue ear disease, pseudorabies and the like are carried out, and the market demand is large.
The commercial vaccines mainly comprise inactivated vaccines and subunit vaccines (viroid particle vaccines), have good immune effect, and have important significance for preventing and controlling PCV diseases. Among them, the subunit vaccine is the first choice vaccine for clinical use because of its higher safety and definite immune effect.
The PCV virus genome is about 1700bp in size and comprises 11 Open Reading Frames (ORFs), wherein the ORFs 1 and 2 are main open reading frames and respectively code a non-structural protein Rep and a structural protein Cap. The Cap protein is the main capsid protein of the virus, contains a plurality of protective antigenic determinants and is the target protein for vaccine design. There are two types of PCV-2Cap protein virosome vaccines currently on the market:
A) one is PCV2 virus particles (prokaryotic expression) prepared by escherichia coli, ORF2 gene (Cap protein) of PCV-2 is inserted into an escherichia coli prokaryotic vector, and the escherichia coli is used for expressing PCV-2Cap protein to prepare subunit vaccine. The colibacillus expression system has the characteristics of high growth speed, easy culture, high protein expression yield and the like, but most of the expressed protein exists in an inactive inclusion body form, and soluble protein can be obtained after a series of operations such as protein denaturation and renaturation. However, prokaryotic expression is easy to generate inclusion bodies without activity, protein renaturation is needed to improve the vaccine effect, the cost is high, the production period is long (each batch consumes about 2-3 months), the efficiency is low, and the immunogenicity of the obtained protein is poor; in particular, proteins expressed in E.coli may remain endotoxin even after purification, and may cause side reactions in immunized animals.
B) Another kind of vaccine for preparing PCV2 viroid particle by baculovirus/insect cell expression system is prepared by inserting ORF2 gene (Cap protein) of PCV-2 into insect baculovirus by baculovirus particle expressed by baculovirus/insect cell expression system (eukaryotic expression), culturing insect cell to obtain recombinant PCV-2Cap protein, purifying, mixing with immunological adjuvant, and clinically immunizing by injection, wherein the eukaryotic expression process has high requirement, and the product may be mixed with baculovirus particle to produce side reaction, so that certain biological safety risk exists.
Disclosure of Invention
Aiming at the problems of the traditional porcine circovirus type II virus-like particle vaccine and the preparation method thereof, the invention provides a novel preparation method of the recombinant porcine circovirus type II virus-like particle vaccine, the preparation method is to express the porcine circovirus type II virus-like particles through Kluyveromyces marxianus recombination, and the recombinant strain provided by the invention is utilized to realize the production and the application of the porcine circovirus type II vaccine with high yield and low cost.
In a first aspect of the present invention, there is provided a recombinant vector constructed by inserting a nucleotide encoding a porcine circovirus capsid protein (hereinafter also referred to as "porcine circovirus capsid protein Cap" or "Cap protein") into a Kluyveromyces marxianus expression vector.
The second aspect of the invention provides a Kluyveromyces marxianus recombinant engineering strain, which is constructed by transforming Kluyveromyces marxianus auxotrophic strain with the recombinant vector.
In a third aspect of the invention, there is provided a method for preparing a porcine circovirus type II virus-like particle vaccine, preferably an injectable vaccine, comprising:
inserting nucleotide for coding porcine circovirus capsid protein into a Kluyveromyces marxianus expression vector to construct the recombinant vector;
transforming the recombinant vector into a Kluyveromyces marxianus auxotrophic strain to construct a Kluyveromyces marxianus recombinant engineering strain;
culturing and fermenting the recombinant yeast, and expressing the porcine circovirus capsid protein;
self-assembling porcine circovirus capsid protein into porcine circovirus type II virus-like particles in yeast cells; collecting the cultured and fermented bacteria to obtain porcine circovirus type II virus-like particles;
and (3) preparing the porcine circovirus II virus-like particle injection vaccine by taking the porcine circovirus II virus-like particle as an active ingredient or one of the active ingredients.
Wherein the porcine circovirus capsid protein is preferably porcine circovirus type II capsid protein.
More preferably, the porcine circovirus type II virus-like particle is used as an active ingredient or one of the active ingredients, and is emulsified with an adjuvant to prepare the porcine circovirus type II virus-like particle injection vaccine.
More preferably, the separation and purification comprises: and (3) collecting the collected bacteria for culture and fermentation, collecting the released virus-like particles after breaking, and purifying the porcine circovirus type II virus-like particles by gel chromatography.
The fourth aspect of the invention provides a porcine circovirus type II virus-like particle vaccine, preferably an injection vaccine, and porcine circovirus type II virus-like particles obtained by culturing, fermenting, separating and purifying the Kluyveromyces marxianus recombinant engineering strain are active ingredients or one of the active ingredients. More preferably by the process of the third aspect of the invention.
In a preferred embodiment of the present invention, any of the vaccines above may be one or more of liquid injection, powder injection, and tablet injection, and is preferably liquid injection.
Wherein, the vaccine can comprise an adjuvant which can be any one or more of but not limited to oil-in-water, water-in-oil and water-in-oil-in-water adjuvants.
For example, the adjuvant may be, but is not limited to, any one or more of ISA201 water-in-oil-in-water mineral oil adjuvant, IMS1313 water-soluble nano agent, GEL01 novel nano agent, and preferably is IMS1313 water-soluble nano agent.
In a preferred embodiment of the present invention, the porcine circovirus type II virus-like particle is an active ingredient or one of the active ingredients, and is prepared into injection powder, injection tablet, or liquid injection by adding adjuvant.
In a preferred embodiment, the porcine circovirus capsid protein amino acid sequence comprises (preferably, is):
1) the amino acid sequence of SEQ ID No. 1; or
2) The protein which is derived from the amino acid sequence of SEQ ID No.1 and has the activity of porcine circovirus nucleocapsid Cap protein and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of SEQ ID No. 1.
In a preferred embodiment, the nucleotide sequence encoding the porcine circovirus capsid protein is preferably the sequence shown in SEQ id No. 2.
In a preferred embodiment, the method is performed by codon-optimizing the nucleotide encoding the porcine circovirus capsid protein according to the codon preference of kluyveromyces marxianus, more preferably under conditions that ensure the sequence encoding the porcine circovirus capsid protein is identical.
In a preferred embodiment, the Kluyveromyces marxianus expression vector comprises a Kluyveromyces marxianus inulinase promoter gene and a terminator gene.
In a preferred embodiment, the kluyveromyces marxianus expression vector auxotrophy selection marker gene.
In a more preferred embodiment of the present invention, the recombinant vector comprises a kluyveromyces marxianus autonomously replicating sequence, a kluyveromyces marxianus inulinase promoter gene sequence, a gene sequence encoding a porcine circovirus nucleocapsid Cap protein, a kluyveromyces marxianus inulinase terminator gene sequence, and an auxotrophic screening marker gene sequence. Coli replication sequences and resistance selection genes may also be included.
In a more preferred embodiment, the transcriptional translation of porcine circovirus capsid protein is controlled in the recombinant vector by a Kluyveromyces marxianus constitutive inulinase promoter and terminator.
In a more preferred embodiment, the DNA sequence of the Kluyveromyces marxianus expression vector is SEQ ID No. 5.
The auxotrophic strain of the Kluyveromyces marxianus is obtained by knocking out part or all of specific nutritional genes of the Kluyveromyces marxianus. The specific nutrition gene is preferably any one or more selected from URA3 gene, HIS3 gene and ADE2 gene, and is preferably URA3 gene.
Wherein, in a more preferred embodiment, the Kluyveromyces marxianus auxotrophic strain is incapable of growing in Uracil auxotrophic medium.
In a preferred embodiment of the present invention, the Kluyveromyces marxianus is preferably CGMCC No. 10621. The strain is preserved in China general microbiological culture Collection center, 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.
In a preferred embodiment of the invention, the transformation may be any one or more of an electrical transformation or a chemical transformation.
In a preferred embodiment of the present invention, the culture medium for culturing the recombinant yeast contains glucose, ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate.
In a preferred embodiment of the invention, the Kluyveromyces marxianus recombinant engineering strain is subjected to high-density fermentation to recombinantly express porcine circovirus type II virus particles; collecting fermented yeast cells, and purifying porcine circovirus type II virus-like particles through high-pressure homogenization, crushing, centrifugal clarification and molecular sieve gel filtration; and adding an adjuvant into the purified virus-like particle suspension, and emulsifying to prepare the porcine circovirus type II vaccine.
The porcine circovirus type II virus-like particle injection vaccine is preferably used for preparing a medicament and/or a vaccine for preventing and/or treating diseases caused by the porcine circovirus type II virus, and is preferably an injection medicament and/or a vaccine.
Wherein, the diseases caused by the porcine circovirus type II virus include but are not limited to any one or more of postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, piglet congenital tremor and sow dysgenesis.
In a preferred embodiment of the present invention, the porcine circovirus type II virus-like particle injection vaccine can also include an adjuvant, and the adjuvant is preferably any one or more of a solvent, a cosolvent, a solubilizer, an emulsifier, a suspending agent, a disintegrant, a wetting agent, a diluent, a flocculant or a deflocculant, an antifoaming agent or a foaming agent, a thickener, an antioxidant, a preservative, a metal band complexing agent, a buffer, a pH regulator, an analgesic, and an osmotic pressure regulator.
The porcine circovirus type II virus-like particle vaccine provided by the invention can reduce and prevent relevant clinical symptoms caused by PCV2 infection, can obtain a high-level serum IgG antibody by injecting immunization once, has the advantages of good immunization effect, high safety, simple culture operation, high yield, low production cost, large-scale production amplification and the like, and can be applied to reducing and preventing relevant clinical symptoms caused by PCV2 infection.
Drawings
FIG. 1 is a PCR-amplified DNA electrophoresis of Cap gene;
FIG. 2 is a schematic diagram of the Cap protein Kluyveromyces marxianus recombinant vector pUKD-N115/Cap;
FIG. 3 shows the expression of porcine circovirus type II Cap protein in Kluyveromyces marxianus; wherein, lane 1: fim-1ura3 Δ; lane 2: fim-1ura3 delta-pUKD-N115/Cap recombinant yeast whole cell lysate; lane 3: fim-1ura3 delta-pUKD-N115/Cap recombinant yeast hydrolysate supernatant;
FIG. 4 shows Western Blot detection of expression of porcine circovirus type II Cap protein by Kluyveromyces marxianus; wherein, lane 1: fim-1ura3 Δ; lanes 2 and 3: fim-1ura3 delta-pUKD-N115/Cap recombinant yeast;
FIG. 5 shows the results of high density fermentation of recombinant yeast Fim-1ura 3. delta. -pUKD-N115/Cap in different media, wherein a: biomass over time in different media; b, performing PCR detection on Cap expression obtained by different culture media; c and d are the yields of Cap protein in different media;
FIG. 6 is an electron microscopic observation photograph of PCV virus-like particles in Kluyveromyces marxianus expressing cells (A) and after purification (B);
FIG. 7 shows the purification of AKTA expressing PCV virus-like particles in K.marxianus;
FIG. 8 shows SDS-PAGE electrophoretic detection of purified sample AKTA expressing PCV virus-like particles in K.marxianus, wherein, lane 1: f4 fermented Fim-1ura3 delta-pUKD-N115/Cap cell lysis supernatant; lane 2: f4 fermented Fim-1ura3 delta-pUKD-N115/Cap cell lysis precipitate; lane 3: purifying the collected 40-50ml fractions with AKTA; lane 4: purifying the collected 50-60ml fractions with AKTA; lane 5: purifying the collected 60-70ml fractions with AKTA; lane 6: purifying the collected 70-80ml fractions with AKTA; lane 7: purifying the collected 80-90ml fractions with AKTA; lane 8: purifying the collected 90-100ml fractions with AKTA; lane 9: AKTA purification of the collected 100-fold 110ml fractions;
FIG. 9 shows the Cap protein immunoblot Western Blot detection of purified samples of AKTA expressing PCV virus-like particles from Kluyveromyces marxianus;
FIG. 10 shows the effect of adjuvant on the immune effect of porcine circovirus type II vaccine prepared from Kluyveromyces marxianus;
FIG. 11 shows the effect of different doses of Kluyveromyces marxianus vaccine on porcine circovirus type II immunized mice.
Detailed Description
Example 1 construction of PCV capsid protein recombinant expression vector pUKD-N115/Cap
In this example, the gene encoding the amino acid sequence of porcine circovirus capsid protein (also referred to as "porcine circovirus Cap protein", or "Cap protein" in the context of the present invention) has the sequence of SEQ ID No. 2. According to the codon preference of Kluyveromyces marxianus, codon optimization is carried out on the Cap gene sequence, and the optimized Cap gene sequence is artificially synthesized.
The amino acid sequence of the encoded Cap protein can be shown as SEQ ID No. 1.
The Cap gene was amplified using PCR amplification with PCVN125-F (5'-GGGGCCCGGG ACATATCCAAGGAGGCGTTTCAGAAG-3') and PCVN125-R (5'-GGGGGCGGCCGCTCACTTAGGGTTAAGTGGAGGGTCCTTAAG-3') as primers. After electrophoresis on a 1% agarose gel, a fragment of about 700kb was recovered by using a DNA gel kit (see FIG. 1).
Provides a pUKD-N115 vector, wherein the pUKD-N115 vector comprises a Kluyveromyces marxianus autonomous replication sequence, a Kluyveromyces marxianus inulinase promoter sequence, a Kluyveromyces marxianus inulinase terminator sequence and a Kluyveromyces marxianus auxotroph gene URA3(KmURA3ORF) as shown in SEQ ID No. 5.
The preparation method of the pUKD-N115 carrier can adopt the method for preparing the PUKDN115 carrier disclosed in the embodiment of the Chinese patent application CN 105063080A. The pUKD-N115 vector was digested with two restriction enzymes Sma I and Not I, electrophoresed on 1% agarose gel, and a vector fragment of about 11.3kb was recovered using a DNA gel kit.
The Cap gene fragment and the vector fragment were ligated by the Gibson Assembly method using the Gibson Assembly traceless ligation System (product No. E2611S/L) of NEB.
Escherichia coli DH5 α is transformed, transformants are screened on Luria-Bertani solid plates containing 100 mu g/ml ampicillin, the transformants are identified by sequencing to construct a recombinant vector pUKD-N115/Cap, as shown in figure 2, a gene sequence (PCV2CAP) for coding Cap protein is inserted into the recombinant vector, and the recombinant vector also comprises an inulinase promoter, an inulinase terminator, a defect screening marker URA3(KmURA3) and a promoter thereof.
Example 2 construction of PCV capsid protein recombinant Kluyveromyces marxianus genetically engineered bacterium Fim-1ura3 delta-pUKD-N115/Cap
The Kluyveromyces marxianus uracil auxotrophic strain Fim-1ura3 delta is provided, and the preparation method of the strain can adopt the construction method of the auxotrophic strain Fim-1(ura3 delta) disclosed in example 1 of Chinese patent publication document CN 105112313A.
Fim-1ura 3. delta. was inoculated into a glass tube containing 3mL YEPD medium and shaken overnight at 30 ℃ until OD600 was 12-15. The cells were collected and washed with LiAc-TE solution (100mM LiAc, 10mM Tris-HCl,1mM EDTA).
The carrier DNA, the recombinant vector pUKD-N115/Cap, PEG solution (40% PEG4000, 100mM LiAc, 10mM Tris-HCl pH 7.5, 1mM EDTA) and DTT with the final concentration of 10mM are added into the thalli in sequence, after fully and uniformly mixing, water bath at 30 ℃ is carried out for 15min, water bath at 47 ℃ is carried out for 15min, instant separation is carried out at 8000rpm, supernatant is discarded, 100 microliter of sterile water is added to suspend the thalli, SD plates (0.67% of aminofree yeast nitrogen source YNB, 2% glucose and 2% agar) are coated, and the thalli are cultured at 30 ℃ for 2-4 days until cloning is formed.
Selecting a monoclonal from a selective culture medium plate, carrying out shaking table overnight culture in a test tube of 3mL YEPD culture medium at 30 ℃, extracting a transformant genome by adopting a conventional yeast genome extraction method, carrying out PCR verification by using the PCVN125-F and PCVN125-R primers, and amplifying a positive clone with a band of about 700bp, namely the genetically engineered bacterium Fim-1ura3 delta-pUKD-N115/Cap which is transferred into PCV Cap protein.
Example 3 expression of PCV Cap protein in Kluyveromyces marxianus genetically engineered bacterium Fim-1ura3 delta-pUKD-N115/Cap
The constructed engineering bacteria Fim-1ura3 delta-pUKD-N115/Cap and the control bacteria Fim-1ura3 delta are respectively inoculated in YP culture medium (1% Yeast Extract and 2% glucose) filled with 50ml, cultured at 30 ℃ and 220rpm for 96h, centrifuged to collect cells, the cells are crushed by a high-pressure homogenization method, cell lysate is subjected to protein electrophoresis SDS-PAG (polyacrylamide gel electrophoresis, PAGE) and PCV antibody immunoblotting Western Blot detection, and the expression level of Porcine Circovirus (PCV) Cap protein in Kluyveromyces is analyzed. Western Blot adopts PCV-resistant pig serum polyclonal antibody and goat HRP-resistant enzyme-labeled secondary antibody.
From the results of protein electrophoresis detection and Western Blot detection of cell lysate (as shown in FIG. 3 and FIG. 4), it can be clearly found that the Porcine Circovirus (PCV) nucleocapsid protein Cap protein after codon optimization is efficiently expressed in Kluyveromyces lactis, and the nucleocapsid protein Cap protein which is recombinantly expressed is basically consistent with the theoretical molecular weight of 28 kD.
Example 4 Kluyveromyces marxianus genetically engineered bacterium Fim-1ura3 delta-pUKD-N115/Cap high-density fermentation
Kluyveromyces marxianus genetically engineered bacterium Fim-1ura3 delta-pUKD-N115/Cap is inoculated on a YEPD solid culture medium and cultured for 2 days at 30 ℃ for activation. Then transferring the culture medium into a synthetic medium or a complete medium (1% yeast extract and 2% glucose) containing glucose (or sucrose), ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate and the like, and culturing for 18-24h at 30 ℃ with shaking (180-220 rpm).
Inoculating the seed solution into a 5L fermentor containing 2L culture medium at a ratio of 10%, ventilating and stirring during fermentation, feeding with whole material flow, controlling dissolved oxygen at 20-30%, controlling temperature at 30-35 deg.C, controlling pH at 5.5-6.0, and controlling fermentation time at 80 h.
Referring to FIG. 5, the recombinant strain Fim-1ura3 delta-pUKD-N115/Cap has higher expression level of biomass and Cap protein in complete medium (F1 pot and F2 pot) than synthetic medium (F3-F5 pot) (FIGS. 5a and b). Referring to FIGS. 5c and d, the yield of Cap protein was about 2.5g/L and the dry cell weight was 106 g/L; the tank F3 used sucrose as carbon source, and its yield of Cap protein was significantly lower than that of the tanks F4 and F5 using glucose as carbon source, and about 0.5g/L (see FIG. 5 d). Meanwhile, the culture mediums of the F4 tank and the F5 tank both adopt glucose, and the F5 tank is added with more vitamins such as riboflavin and inositol than the F4 tank. The expression level of the Cap protein from the F4 tank was about 2.0g/L, whereas the expression level of the Cap protein from the F5 tank was reduced to about 0.9g/L (FIG. 5 d).
Example 5 purification and characterization of recombinant porcine circovirus type II Virus-like particles (VLPs)
The Porcine Circovirus (PCV) nucleocapsid protein Cap protein is observed by an electron microscope to be self-assembled virus-like particles in the expression of Kluyveromyces marxianus (as shown in FIG. 6A).
Fim-1ura3 delta-pUKD-N115/Cap recombinant yeast cells obtained by high-density fermentation in a fermentation tank are deionized and washed, and then are treated with PBS buffer solution (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4，1.4mM KH2PO4) Resuspending, height by volumeAfter crushing and centrifugation, the supernatant was collected, and after 0.2. mu.M membrane filtration, the supernatant was applied to a Sephacryl gel HiPrep 26/60Sephacryl S-500(GE healthcare) to purify by gel chromatography, eluted with PBS buffer (see FIG. 7), collected in one tube per 10ml, and examined by protein electrophoresis SDS-PAGE (see FIG. 8) and Western Blot (see FIG. 9), and the porcine circovirus PCV virus-like particles were found to elute in a volume of 60 to 90 ml.
The purity of the virus-like particles purified by gel chromatography reaches about 95%, and the virus-like particles are still maintained by the nucleocapsid Cap protein in the purified sample through the purification process observed by an electron microscope (as shown in figure 6B).
Example 6: preparation of recombinant porcine circovirus II type vaccine and immune effect analysis
And (3) measuring the concentration of virus-like particles (VLP) of the recombinant porcine circovirus obtained by purification by using a Bradford protein concentration measuring kit, adjusting the concentration to 120 mu g/ml by using PBS buffer solution, and mixing the PBS buffer solution with an adjuvant at a ratio of 1: 1 to prepare the recombinant porcine circovirus type II vaccine. Screening of the adjuvant comprises an ISA201 water-in-oil-in-water mineral oil adjuvant, an IMS1313 water-soluble nano agent, a GEL01 novel nano agent and the like.
Healthy female SPF-grade Balb/c mice of 6 weeks are selected and randomly divided into 7 mice each, and are divided into an empty immune white control group (PBS) and a vaccine group (ISA 201 group, IMS1313 group and GEL01 group according to adjuvants) without immune white control group, and are injected subcutaneously at multiple points for immunization, and the dosage is 60 mu g/mouse. After 2 weeks of immunization, the mice are bled from the retroorbital venous plexus, the PCV2 antibody titer in the serum is determined by an ELISA method, and the detection process refers to the use instruction of the porcine circovirus type 2 ELISA antibody detection kit produced by Wuhan's pre-biological products Co.
The results show that PCV cap IgG antibody is generated in the serum of mice after 2 weeks of injection of porcine circovirus II type vaccine prepared by Kluyveromyces marxianus and gradually increases along with time, and the OD450 of PCV cap IgG antibody titer reaches about 0.8 after 35 days (as shown in figure 10). Among the three selected adjuvants, the IMS1313 water-soluble nano agent has small emergency response of mice, and is the first choice adjuvant for preparing Kluyveromyces marxianus recombinant expression porcine circovirus II type vaccines.
The IMS1313 water-soluble nano agent is used to test the influence of different vaccine immunization doses on the concentration of mouse PCV cap IgG antibody, as shown in figure 11, after 2 weeks, 8 mu g/and 3 mu g/dose of protein have produced PCV cap IgG antibody, after 42 days, under the condition of 3 mu g/dose, the titer OD450 of PCV cap IgG antibody has reached about 0.75, and the antibody is still increased at 49 days, namely, low-dose protein can also produce good immune effect, which indicates that the porcine circovirus type II vaccine prepared by Kluyveromyces marxianus has good immunogenicity.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Figure IDA0001110730560000011
Figure IDA0001110730560000021
Figure IDA0001110730560000031
Figure IDA0001110730560000051
Figure IDA0001110730560000061
Figure IDA0001110730560000071
Figure IDA0001110730560000081
Figure IDA0001110730560000091
Figure IDA0001110730560000101
Figure IDA0001110730560000121

Claims (15)

1. A recombinant vector is characterized in that the recombinant vector is constructed by inserting nucleotide coding porcine circovirus type II Cap protein into a Kluyveromyces marxianus expression vector;
the recombinant vector comprises an autonomous replication sequence of Kluyveromyces marxianus, an inulinase promoter sequence of Kluyveromyces marxianus, a gene sequence for coding porcine circovirus type II Cap protein, an inulinase terminator sequence of Kluyveromyces marxianus, an escherichia coli replication sequence, a resistance screening gene and a nutritional defect screening gene, wherein the DNA sequence of the Kluyveromyces marxianus expression vector is SEQ ID No. 5.
2. The recombinant vector according to claim 1, wherein transcription and translation of the porcine circovirus type II Cap protein is controlled by a kluyveromyces marxianus constitutive inulinase promoter and terminator.
3. The recombinant vector according to claim 1, wherein the nucleotide sequence encoding the porcine circovirus type II Cap protein is the sequence shown in SEQ ID No. 2.
4. A Kluyveromyces marxianus recombinant engineered strain constructed by transforming the Kluyveromyces marxianus auxotrophic strain with the recombinant vector of claim 1.
5. The Kluyveromyces marxianus recombinant engineered strain as claimed in claim 4, wherein the auxotrophic Kluyveromyces marxianus strain is obtained by knocking out part or all of specific nutritional genes.
6. The Kluyveromyces marxianus recombinant engineering strain as claimed in claim 5, wherein the specific nutrient gene is URA3 gene.
7. The Kluyveromyces marxianus recombinant engineering strain as claimed in claim 5, wherein the Kluyveromyces marxianus is CGMCC No. 10621.
8. A method of preparing a porcine circovirus type II virus-like particle vaccine, comprising:
inserting nucleotide encoding porcine circovirus type II Cap protein into a Kluyveromyces marxianus expression vector to construct the recombinant vector of claim 1;
converting the recombinant vector into a Kluyveromyces marxianus auxotrophic strain to construct recombinant yeast;
culturing and fermenting the recombinant yeast, and expressing Cap protein;
self-assembling the Cap protein into porcine circovirus type II virus-like particles in yeast cells;
collecting the cultured and fermented bacteria, and separating and purifying to obtain porcine circovirus type II virus-like particles;
and (3) preparing the porcine circovirus type II virus-like particle vaccine by taking the porcine circovirus type II virus-like particle as an active ingredient or one of the active ingredients.
9. The method of claim 8, wherein the separating and purifying comprises: and (3) collecting the collected bacteria for culture and fermentation, collecting the released virus-like particles after breaking, and purifying the porcine circovirus type II virus-like particles by gel chromatography.
10. The method according to claim 8, wherein the culture medium for culturing the recombinant yeast contains glucose, ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate.
11. A porcine circovirus type II virus-like particle vaccine, which is characterized in that porcine circovirus type II virus-like particles obtained by culturing, fermenting, separating and purifying the Kluyveromyces marxianus recombinant engineering strain of claim 6 are used as active ingredients or one of the active ingredients.
12. The porcine circovirus type II virus-like particle vaccine of claim 11, wherein the vaccine is selected from one or more of a liquid injection, an injectable powder, and an injectable tablet.
13. The porcine circovirus type II virus-like particle vaccine of claim 11, comprising an adjuvant selected from any one or more of oil-in-water, water-in-oil, and water-in-oil-in-water adjuvants.
14. The porcine circovirus type II virus-like particle vaccine of claim 13, wherein the adjuvant is selected from any one or more of ISA201 water-in-oil-in-water mineral oil adjuvant, IMS1313 water-soluble nano-adjuvant, GEL01 novel nano- agent.
15. The use of the porcine circovirus type II virus-like particle of claim 11 for the preparation of a vaccine for the preparation of a medicament and/or a vaccine for the prevention and/or treatment of a disease caused by porcine circovirus type II virus selected from the group consisting of: any one or more of postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, piglet congenital tremor and sow dysgenesis.
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