WO2019242177A1 - 一种用于预防犬弓形虫感染的疫苗及其制备方法 - Google Patents

一种用于预防犬弓形虫感染的疫苗及其制备方法 Download PDF

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WO2019242177A1
WO2019242177A1 PCT/CN2018/111235 CN2018111235W WO2019242177A1 WO 2019242177 A1 WO2019242177 A1 WO 2019242177A1 CN 2018111235 W CN2018111235 W CN 2018111235W WO 2019242177 A1 WO2019242177 A1 WO 2019242177A1
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pet
protein
rosetta
plyss
cyclophilin
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French (fr)
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侯峰
曹利利
宫鹏涛
李思明
陈星远
丁鹤
王典
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侯峰
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Priority to US17/253,798 priority Critical patent/US11534485B2/en
Priority to EP18923744.9A priority patent/EP3838916B1/en
Priority to JP2021520258A priority patent/JP7008165B2/ja
Publication of WO2019242177A1 publication Critical patent/WO2019242177A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/45—Toxoplasma
    • 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
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the fields of immunology and biology, and relates to a vaccine and a preparation method for the prevention of Toxoplasma gondii infection, in particular to the application of the vaccine in the prevention of Toxoplasmosis canis infection in dogs.
  • Toxoplasmosis is a multi-host protozoan disease that is parasitic in the cells of animals or humans caused by Toxoplasma and Toxoplasma of the genus Toxoplasma. It is a worldwide distribution Of zoonotic parasites, cats are terminal hosts, and humans and various animals are intermediate hosts. Toxoplasmosis has become one of the important public health problems to be solved urgently in China. The main reasons are as follows: (1) The infection rate of human Toxoplasma is extremely high, generally 20% to 50%, especially women and children. The infection rate is even higher. About one third of the world ’s population is infected with Toxoplasma gondii.
  • the infection rate of Toxoplasma gondii in the Chinese population is 5% to 20%. Regardless of the clinical symptoms of pregnant women after infection, about 50% can occur vertical transmission, causing symptoms such as premature delivery, miscarriage, fetal developmental deformity or stillbirth; 6% to 10% of AIDS patients are complicated by toxoplasmosis, and the brain of AIDS patients 50% of inflammation is caused by Toxoplasma gondii infection.
  • Dogs are important intermediate hosts for Toxoplasma gondii, and cats are the only terminal hosts, and they are in close contact with people. , Has become the main source of human toxoplasmosis. (4) Toxoplasma gondii can infect all nucleated cells. The average infection rate of animals (pig, cow, sheep, chicken, duck, goose, etc.) is 15.4%. This caused Toxoplasma gondii to easily flow into the food market through meat, milk, and eggs, becoming a source of human infection, and seriously threatening the public health and safety of animal foods.
  • Toxoplasma gondii vaccine has been around the whole insect vaccine since the 1960s.
  • Whole worm vaccines include inactivated vaccines and live attenuated vaccines, but inactivated vaccines lack immunoprotective effects on mice, so they have no practical application value.
  • Toxoplasma gondii tachyzoites weaken their virulence after treatment with ultraviolet rays, radiation and chemical agents.
  • the Toxoplasma gondii antigen gene is expressed in a high-efficiency expression vector, thereby obtaining a large amount of purified single antigen, which has the advantages of good immunogenicity, high biological safety, and low immune stimulus. Based on the above discussion, it can be found that genetically engineered vaccines are the hope for the development of Toxoplasma gondii vaccine, and they have more development and application value.
  • Toxoplasma gondii can be divided into five stages, namely trophozoites, cysts, schizonts, gametophytes, and oocysts. The first two stages are performed in the intermediate host, and the last three stages are only in the terminal host, which is the cat. Intestines and body surfaces.
  • the development of the parasite is when the cat eats the mature oocysts or animal tissues containing Toxoplasma gondii.
  • the sporozoites or trophozoites in the oocysts will invade the digestive tract of the body and gradually move to the intestine. Epithelial cells and coccidia-shaped development and reproduction within the cells.
  • Dogs are infected with toxoplasmosis due to ingestion of spore-forming oocysts or swallowing of meat and internal organs containing cysts and trophozoites. In addition, they can also pass through damaged skin, respiratory tract, eyes, and placenta. Route infection.
  • the affected dogs are mostly puppies or young dogs less than 1 year old.
  • Pregnant female dogs can be infected with toxoplasmosis to cause miscarriage and premature delivery.
  • Adult dogs are mostly recessive or transient, but there have been reports of deaths.
  • the occurrence of the disease is also related to fecal habit of dogs or predation of rats.
  • Cyclophilin is a cytolytic protein widely present in prokaryotic and eukaryotic organisms, and is highly conserved in structure. At present, CyP has been found in Taenia echinococcus, Filaria malayi, Toxoplasma gondii, Plasmondium falciparum, Neospora canis caninum), Schistosomiasis japonica, Entamoeba histolytica, and Eimeria tenella.
  • the present invention provides a recombinant subunit inactivated vaccine for preventing Toxoplasma gondii infection and application thereof.
  • the gene engineering technology is used to clone and transform the cyclophilin gene of Toxoplasma gondii, and the transformed cyclophilin gene is transferred to an engineered bacterium. After induction, the recombinant antigen was highly expressed after induction, and the specificity and immunogenicity of the recombinant antigen were physiologically studied, showing good immunogenicity.
  • the first object of the present invention is to provide a protein having the immunogenicity of Toxoplasma gondii, which is composed of the amino acid sequence described in SEQ ID 2.
  • the source of this sequence is as follows: We modified the spatial structure and hydrophobicity of the protein, and modified the amino acid sequence of the cyclophilin protein.
  • codon optimization website http://www.encorbio.com/protocols/Codon.htm was used to modify the modified
  • the amino acid sequence is codon optimized to facilitate more efficient expression in E. coli.
  • a protein having Toxoplasma immunogenicity is a cyclophilin mutant protein, and is composed of the amino acid sequence described in SEQ ID ID 2.
  • the protein having immunogenicity of Toxoplasma gondii is substituted, deleted, or replaced with the amino acid sequence described in SEQ ID 2 to obtain a protein mutant.
  • the protein having immunogenicity of Toxoplasma gondii is a protein composed of an amino acid sequence having more than 90% homology with the amino acid sequence described in SEQ ID ID 2.
  • the protein having immunogenicity of Toxoplasma gondii is a protein consisting of an amino acid sequence having more than 80% homology with the amino acid sequence described in SEQ ID 2.
  • a nucleic acid corresponding to the SEQ ID 1 or a mutant thereof is digested and ligated into a vector, and then transformed or transfected into a prokaryotic or eukaryotic cell for expression.
  • a method for preparing the aforementioned cyclophilin mutant protein which is characterized by:
  • the vector in step (2) is PET-28a, and E. coli is BL21 (DE3).
  • the recombinant genetically engineered bacterium in step (2) is DH5 ⁇ / pET28a-18C.
  • the genetically engineered bacteria expressing the cyclophilin mutant protein of Toxoplasma gondii in step (3) is constitutively expressed.
  • the recombinant toxoplasma cyclophilin mutant protein described in step (3) is any one of the following three polypeptides:
  • Toxoplasma cyclophilin mutant protein in preparing a subunit inactivated vaccine for preventing Toxoplasma gondii infection.
  • Toxoplasma cyclophilin mutant protein in the preparation of human Toxoplasma vaccine, and in the preparation of Toxoplasma gondii vaccine.
  • a nucleic acid capable of encoding a protein having the immunogenicity of Toxoplasma gondii a nucleic acid encoding the amino acid sequence described in SEQ ID 2 and the nucleic acid sequence described in SEQ ID 1.
  • the invention provides a toxoplasma subunit inactivated vaccine, which comprises the amino acid sequence and the mutant thereof according to claim 1, or the nucleotide sequence and the optimized sequence according to claim 4, and a medically acceptable vector. .
  • toxoplasma subunit inactivated vaccine in the preparation of a medicament for preventing Toxoplasma gondii infection, the medicament comprising the amino acid sequence and the mutant thereof according to claim 1, or the nucleotide sequence and the optimization thereof according to claim 4. Sequence, and a medically acceptable carrier.
  • a method for preparing the inactivated subunit vaccine linking the antigen of Toxoplasma gondii to the expression vector of claim 12, transforming into a prokaryotic expression engineering strain, inducing its high-efficiency expression, and obtaining a soluble fusion protein after purification, and adding a vaccine Made from adjuvant.
  • the vaccine adjuvant is prepared by using MF59 adjuvant or Sybeck 206 adjuvant.
  • the concentration of the protein is at least 10 ⁇ g / ml to 300 ⁇ g / ml.
  • the above vaccine for preventing toxoplasmosis in dogs has a concentration of 100 ⁇ g / ml.
  • the above-mentioned prokaryotic expression engineering bacterium is characterized in that the prokaryotic expression engineering bacterium is BL21 (DE3), BLR (DE3) pLysS, OrigamiB (DE3), C41 (DE3) pLysS, C41 (DE3), BL21, AD494, BL21 -SI, BL21 trxB (DE3) pLysS, BL21 trxB (DE3), Origami 2 (DE3) pLysS, B834 (DE3) pLysS, Rosetta-gami, B (DE3), Rosetta-gami, B (DE3) pLysS, Rosetta-gami ( DE3) pLysS, NovaBlue T1, Tuner (DE3) pLacI, Tuner (DE3) pLysS, Tuner (DE3), Tuner, RosettaBlue (DE3) pLysS, RosettaBlue (DE3) pLacI, Ros
  • a host cell includes the nucleic acid corresponding to SEQ ID 1 or a mutant thereof, and a biological vector.
  • the second object of the present invention is to provide a vector including the nucleotide sequence SEQ ID 1; the vector is pET-28a, pMAL-p5x, pET-42b (+), pCold-GST, pTrcHis A, pGEX-KG, pET-28b (+), pBAD102 / D-TOPO, pAmCyan, ptdTomato, pCS105, pET101 / D-TOPO, pET-24a (+), pET-24c (+), pET-24d (+), pET-27b (+), pET-25b (+), pET-28c (+), pET-29a (+), pET-29b (+), pET-29c (+), pET-30b (+), pET -30c (+), pET-30, Xa / LIC, pET-30, EK / LIC, pET-31b (+), pET-32b (+), pET
  • the third object of the present invention is to provide a soluble fusion protein whose amino acid sequence comprises SEQ ID NO. 2 and a mutant thereof.
  • the soluble fusion protein comprises a Toxoplasma cyclophilin mutant protein and a histidine purification tag; wherein the DNA sequence encoding the Toxoplasma cyclophilin mutant protein is selected from SEQ ID NO. 1; The amino acid sequence of the purification tag is SEQ ID NO.3.
  • amino acid sequence of the soluble fusion protein is SEQ ID NO. 2.
  • a third object of the present invention is to provide a host cell comprising the above-mentioned vector, or which has been transformed or transfected with the nucleotide sequence according to claim 1.
  • a fourth object of the present invention is to provide a method for preparing a soluble fusion protein, which is characterized by including expressing the soluble fusion protein by a host cell and performing isolation.
  • a fifth object of the present invention is to provide a vaccine, which is characterized in that the vaccine includes a nucleotide sequence in SEQ ID 2 and a medically acceptable carrier.
  • the vaccine is double-digested by Toxoplasma gondii and ligated into pET28a expression vector, and transformed into BL21 (DE3) engineered bacteria to induce efficient expression.
  • the purified protein is a soluble fusion protein, and MF59 adjuvant or Saibike 206 adjuvant is prepared. Its vaccine maintains its unique immunogenicity and is suitable for industrial production.
  • the invention has the advantages that the subunit inactivated vaccine prepared by the antigen of Toxoplasma gondii effective antigen is selected, and the subunit inactivated vaccine prepared by the antigen has strong cellular and humoral immune effects, and can induce the body's innate immunity In response, it secretes various cellular inflammatory factors, thereby effectively preventing canine toxoplasmosis.
  • the invention optimizes the sequence of the cyclophilin protein, and the expression amount in the prokaryotic cell E. coli is twice the expression amount before the optimization.
  • the vaccine provided by the present invention can be prepared from engineering strains and added with a vaccine adjuvant. The vaccine maintains its unique immunogenicity and is suitable for industrial production.
  • M DL2000marker
  • C18 the gene fragment of interest.
  • M protein marker
  • r1 soluble expression of Toxoplasma cyclophilin protein
  • r2 soluble expression of Toxoplasma cyclophilin mutant protein.
  • M protein marker
  • r recombinant cyclophilin mutant protein
  • Figure 4 Western-blot-specific results of in vitro expression of recombinant proteins.
  • M protein marker
  • r recombinant cyclophilin mutant protein
  • Toxoplasma cyclophilin recombinant mutant protein stimulates RAW264.7 cells to produce TNF- ⁇ levels in mice.
  • Toxoplasma cyclophilin recombinant mutant protein stimulates mouse dendritic cells to produce IL-12 levels.
  • Figure 8 Protective effect of toxoplasma cyclophilin recombinant mutant protein vaccine against toxoplasma infection in mice.
  • Figure 9 Figure of antibody titer produced by Toxoplasma cyclophilin recombinant mutant protein vaccine in dogs.
  • Example 1 Construction of a prokaryotic expression vector of recombinant cyclophilin mutant protein of Toxoplasma gondii
  • the gene was artificially synthesized based on the nucleic acid sequence SEQ ID NO.1, and the primers were designed based on the physical map of the prokaryotic expression vector pET28a and the restriction sites were introduced.
  • Upstream primer ATGGAATTC ATGGAAAACGCGGGCGTGCGCAAA
  • Downstream primer AAGCTT, TTCTTTTTTGCCAATATCGGTAA
  • the positive plasmids are EcoRI and Hind III.
  • the purified target fragment after double digestion was ligated to the prokaryotic expression vector pET28a.
  • the ligation product was transformed into E. coli DH5 ⁇ competent cells and the recombinant plasmid was selected.
  • the double digestion reaction was identified with EcoRI and Hind III to obtain the prokaryotic expression plasmid pET28a- C18.
  • PET28a-C18 was transformed into BL21 (DE3) engineering bacteria, and a single colony was picked and inoculated into 5 ml of LB liquid medium (containing 100 ⁇ g / ml kanamycin), and cultured at 37 ° C with shaking at 220 rpm overnight.
  • LB liquid medium containing 100 ⁇ g / ml kanamycin
  • a single colony of E. coli BL21 (DE3) transformed with the recombinant plasmid pET-28a (+)-C18 was inoculated into 5 mL of LB liquid medium, and cultured at 37 ° C with shaking overnight. The next day, the seed liquid was transferred to 800 mL of LB liquid medium and propagated at 37 ° C. When the OD600 of the bacterial liquid was monitored to 0.6 to 0.7, expression was induced under optimal conditions. The bacterial cells were collected by centrifugation, and the cells were suspended in 20 mL of PBS, repeatedly frozen and thawed 5 times (-80 ° C, 1h / 37 ° C, 10min), and then sonicated.
  • the supernatant was centrifuged to obtain soluble protein (active protein).
  • the pellet was suspended in 10 mL of denaturing buffer, shaken at room temperature for 1 h, and the supernatant was centrifuged to obtain non-soluble protein (denatured protein).
  • the above two proteins were separately tested by SDS-PAGE to verify the solubility of the target protein. If the target protein is mainly located in the soluble protein supernatant, it means soluble expression; if the target protein is mostly located in the insoluble protein supernatant, then It is expressed as inclusion body.
  • the concentration of solution B (PBS, containing 1M imidazole) is 10%, that is, the concentration of imidazole is 100 mM, and the non-specific adsorbed impurities are washed away for 30 minutes. Keep the flow rate unchanged and set the elution conditions.
  • the concentration of solution B is 30%, that is, the concentration of imidazole is 300 mM, and the time is 120 minutes.
  • the target protein is eluted.
  • the collected target protein is subjected to SDS-PAGE to observe the purity. The results are shown in Figure 3. See Figure 4 for specific results of Western-blot identification of recombinant mutant protein expressed in vitro.
  • RAW264.7 cells were inoculated into 24-well culture plates at 0.5 ⁇ 106 / mL, 0.5mL / well, 5% CO2, and cultured at 37 ° C for 24h. After aspiration of the supernatant, the prepared recombinant cyclophilin mutant protein of Toxoplasma gondii was dissolved in RAW264.7 cell culture medium, and added to 24 at a concentration of 1 ⁇ g / mL, 0.1 ⁇ g / mL, 0.01 ⁇ g / mL, 0.5 mL / well. In the well plate, an equal volume of PBS was used as a negative control.
  • the cell culture plate was cultured in a 5% CO2 incubator at 37 ° C for 48 hours, and the supernatant was collected, and the TNF-a level was detected by an ELISA experiment.
  • the results showed that the production of TNF-a increased with increasing protein concentration, ranging from 40 pg to 310 pg.
  • the results are shown in Figure 5 of the accompanying drawings. These results indicate that the recombinant cyclophilin mutant protein of Toxoplasma gondii can stimulate the production of TNF-a in RAW264.7 cells.
  • Isolate mouse dendritic cells adjust the cell concentration to 0.5 ⁇ 106 cells / mL, inoculate them into a 24-well tissue cell culture plate, and inoculate 0.5 mL per well.
  • Treated toxoplasma recombinant cyclophilin mutant proteins were added to cell culture plates at a concentration of 100 ⁇ g / mL, 50 ⁇ g / mL, 10 ⁇ g / mL, 1 ⁇ g / mL, 0.1 ⁇ g / mL, and 0.01 ⁇ g / mL per well, respectively.
  • 0.5mL The cell culture plate was placed in 5% CO2 and cultured in a 37 ° C incubator for 24 hours.
  • the culture supernatant was collected, and the IL-12 level in the supernatant was detected by an ELISA experiment.
  • the results show that the IL-12 content of each group is protein concentration dependent, that is, increases with increasing protein concentration.
  • the results are shown in FIG. 6.
  • the results indicate that the recombinant cyclophilin mutant protein of Toxoplasma gondii can stimulate the dendritic immune cells in mice to produce a large amount of IL-12.
  • Example 5 Toxoplasma gondii recombinant subunit vaccine mice immune challenge protection experiment
  • mice 125 female SPF BALB / c mice aged 8 to 10 weeks old were divided into 5 groups of 25 each, of which the first group was cyclophilin experimental group I (adjuvant MF59, 30 ⁇ g / one, subcutaneous injection), and The second group was cyclophilin experimental group II (adjuvant 206, 30 ⁇ g / head, subcutaneous injection), the third group was negative control group I (equal volume of PBS, with MF59 adjuvant, subcutaneous injection), and the fourth group was negative control Group II (equal volume of PBS, with 206 adjuvant, subcutaneous injection), and the fifth group was the blank control group (equal volume of PBS, subcutaneous injection). Each group received three injections at two-week intervals. The antibody titer is measured weekly. One week after the third immunization injection, 103 purified Toxoplasma gondii trophozoites were injected intraperitoneally, and the condition of the mice was observed and recorded daily.
  • the antibody results showed that the antibody level increased rapidly three weeks after one immunization, and reached its peak at 6 weeks.
  • the antibody titer of the vaccine is shown in Figure 7 of the accompanying drawings.
  • Example 6 Experimental study on the protection rate of Toxoplasma gondii recombinant subunit vaccine in dogs
  • the experimental animals were divided into 2 groups, of which the first group was the cyclophilin experimental group (adjuvant MF59, 300 ⁇ g / head, subcutaneous injection), a total of 15 dogs.
  • the second group was a blank control group (equal volume of PBS, injected subcutaneously) with a total of 5 dogs. Each group received three injections at two-week intervals. The antibody titer is measured weekly.
  • Toxoplasma gondii trophozoites were injected intraperitoneally. After 30 days, each dog was dissected and the brain, heart, liver, spleen, lung, kidney, lymph nodes, masseter muscle, and tongue muscle were detected by PCR. 2. The infection of Toxoplasma gondii in abdominal muscle tissue. If any of the tissues is positive for PCR, the dog is determined to be infected by Toxoplasma. If the PCR results of all tissues are negative, it is determined to be protected by Toxoplasma gondii.
  • the antibody results showed that the antibody level in the immune group continued to increase after the first immunization. See Figure 9 for the antibody titer of the vaccine.
  • the toxoplasma gondii subunit inactivated vaccine provided by the present invention has a high immune protection rate against toxoplasma gondii, and can be used as a vaccine candidate for prevention and treatment of toxoplasmosis in canines.

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Abstract

本发明提供一种具有弓形虫免疫原性的蛋白，该蛋白为cyclophilin突变体蛋白，由SEQ ID 2所述氨基酸序列组成。本发明还提供一种可编码具有弓形虫免疫原性的蛋白的核酸，具有SEQ ID 1所述的核酸序列。本发明提供一种疫苗，是将弓形虫抗原基因双酶切后连接pET28a等原核表达载体，转化入BL21(DE3)等原核表达工程菌，诱导其高效表达，纯化后得到的蛋白为可溶性蛋白，并且保持其特有的免疫原性。

Description

一种用于预防犬弓形虫感染的疫苗及其制备方法 技术领域
本发明涉及免疫学和生物学领域，涉及一种用于预防犬弓形虫感染的疫苗和制备方法，尤其涉及疫苗在犬弓形虫病感染预防中的应用。
背景技术
弓形虫病(Toxoplasmosis)是由孢子虫纲、弓形虫属的龚地弓形虫(Toxoplasma gondii)引起的一种寄生于动物或人的细胞内的多宿主原虫病，是一种呈世界性分布的人畜共患寄生虫病，猫科动物是终末宿主，人和多种动物是中间宿主。弓形虫病已成为我国亟待解决的重要公共卫生问题之一，其原因主要体现在以下几点：(1)人类弓形虫感染率极高，一般为20％～50％，尤其是妇女和儿童，感染率更高，全球约有1/3的人感染弓形虫，我国人群弓形虫感染率为5％～20％。孕妇感染后不管是否有临床症状，大约50％可发生垂直传播，造成早产、流产、胎儿发育畸形或死胎等症状；有6％～10％的艾滋病患者并发弓形虫病，而且艾滋病病人所患脑炎中有50％是由弓形虫感染所引起的。(2)猫、犬、猪、羊、牛、兔等家畜感染弓形虫非常普遍，其感染率高达10％～50％，猪、牛、羊的流产率达到30-40.7％；猪感染弓形虫还可引起“无名高热”，病死率达60％，因此弓形虫病是造成家畜流产的重要因素之一，影响畜牧业生产。(3)随着家庭饲养宠物犬的数量不断增加，犬猫作为伴侣动物更是掀起一阵"犬猫宠物热"，犬作为弓形虫的重要中间宿主，猫作为唯一终末宿主，与人接触亲密，成为人类弓形虫病的主要传染源。(4)弓形虫可以感染所有的有核细胞，(猪牛羊鸡鸭鹅等)动物感染率平均为15.4％，感染后弓形虫以缓殖子形式遍布全身各个组织。这就造成弓形虫很容易经肉、乳、蛋类大量流入食品市场，成为人类的传染源，严重威胁着动物性食品的公共卫生安全。
然而，迄今为止国内外仍然没有理想的商品化防治犬弓形虫病的疫苗和药物，弓形虫感染后可引起宿主保护性免疫应答，因此研制安全、有效的疫苗应是弓形虫病良好的预防措施。弓形虫疫苗的研制从60年代至今一直围绕全虫疫苗。全虫疫苗包括灭活疫苗和减毒活疫苗，但灭活疫苗对小鼠缺乏免疫保护性，因此无实际应用价值；弓形虫速殖子经紫外线、放射线及化学试剂等处理后毒力减弱，能诱导较强的免疫应答，如Ts-4、T-263和S48，但减毒活疫苗存在减毒不充分和毒力返祖等危险，因此弱毒疫苗不能广泛使用；本发明的基因工程疫苗是将弓形虫抗原基因在高效表达载体中表达，从而得到大量纯化的单一抗原，其优点是免疫原性好、生物安全性高、免疫刺激性小。综合以上论述可以发现，基因工程疫苗是弓形虫疫苗发展的希望，更具有开发应用价值。
弓形虫的发育可分成五个阶段，即滋养体、包囊、裂殖体、配子体和卵囊，其中前两个阶段是在中间宿主内进行，而后三个阶段只在终末宿主也就是猫科动物的肠道和体表上。虫体发育过程是猫科动物食入发育成熟的卵囊或者包含弓形虫包囊的动物组织，卵囊内的子孢子或者包囊内的滋养体就会侵入机体消化道，且逐渐移动到肠上皮细胞，并在细胞内呈现球虫型的发育和繁殖。
犬患弓形虫病多因食入了孢子化的卵囊或吞食含有包囊及滋养体的肉、内脏等而引起感染，除此之外还可经受损的皮肤、呼吸道、眼及胎盘等途径感染。患犬多是1岁以内的幼犬或青年犬，怀孕母犬可感染弓形虫病引起流产、早产，成年犬多呈隐性感染或一过性，但也有发生死亡的病例的报道。另外，该病的发生还与犬具有的食粪恶习或者捕食鼠类等相关。当犬食入猫排出的卵囊后，里面的子孢子会通过淋巴液和血液循环侵入肠道组织以外的细胞中，并进行双芽方式繁殖，生成很多速殖子，此时为急性感染期。当机体产生免疫力时，速殖子会变成缓殖子，并形成包囊，且会长时间生存在脑、眼、骨骼肌以及心中，此时为慢性感染期。如果犬没有形成免疫力和抵抗力，加之弓形虫具有很强毒性，就可能导致机体出现急性发病。反之，如果弓形虫繁殖收到阻碍，就会出现轻微的发病或者没有表现出任何临床症状。
亲环蛋白(cyclophilin,CyP)是广泛存在于原核和真核生物体内的胞溶性蛋白质，在结构上高度保守。目前，已经发现，CyP存在于细粒棘球绦虫(Taenia echinococcus)、马来丝虫(Filaria malayi)、刚地弓形虫(Toxoplasma gondii)、恶性疟原虫(Plasmondium falciparum)、犬新孢子虫(Neospora caninum)、日本血吸虫(Schistosomiasis japonica)、痢疾变形虫(Entamoeba histolytica)及柔嫩艾美耳球虫(Eimeria tenella)等寄生虫中。
发明内容
针对目前问题，本发明提供一种用于预防犬弓形虫感染的重组亚单位灭活疫苗和应用，利用基因工程技术，克隆并改造弓形虫cyclophilin基因，将改造后的cyclophilin基因转入工程菌进行表达，经诱导后重组抗原获得高效表达，并对重组抗原的特异性和免疫原性进行生理性研究，表现良好的免疫原性。
本发明解决上述技术问题所采用的技术方案是：
本发明第一个目的是提供一种具有弓形虫免疫原性的蛋白，由SEQ ID 2所述氨基酸序列组成。该序列的来源如下：我们对蛋白的空间结构及疏水性，针对cyclophilin蛋白的氨基酸序列进行改造，同时利用密码子优化网站http://www.encorbio.com/protocols/Codon.htm对改造后的氨基酸序列进行密码子优化，以 利于其在大肠杆菌中更高效的表达。
本发明解决上述技术问题所采用的技术方案是：
一种具有弓形虫免疫原性的蛋白，该蛋白为cyclophilin突变体蛋白，由SEQ ID 2所述氨基酸序列组成。
所述的具有弓形虫免疫原性的蛋白，该蛋白由SEQ ID 2所述氨基酸序列取代、删除、或置换得到蛋白突变体。
所述的具有弓形虫免疫原性的蛋白，该蛋白是与SEQ ID 2所述氨基酸序列有90％以上同源性的氨基酸序列组成的蛋白。
所述的具有弓形虫免疫原性的蛋白，该蛋白是与SEQ ID 2所述氨基酸序列有80％以上同源性的氨基酸序列组成的蛋白。
制备以上蛋白的方法，将所述SEQ ID 1或其突变体对应的核酸酶切后连接到载体中，转化或转染到原核或真核细胞中进行表达。
一种制备上述cyclophilin突变体蛋白的方法，其特征在于：
(1)克隆相关基因到表达载体质粒中，获得重组表达载体；
(2)所述重组表达载体转化大肠杆菌，获得基因工程菌；
(3)所述基因工程菌进行发酵培养，表达cyclophilin突变体蛋白；
(4)回收基因工程菌菌体破碎后的上清液，分离纯化弓形虫cyclophilin突变体蛋白。
所述的制备cyclophilin突变体蛋白的方法方法中，步骤(2)所述载体为PET-28a，大肠杆菌为BL21(DE3)。
所述的制备cyclophilin突变体蛋白的方法方法中，步骤(2)所述重组基因工程菌为DH5α/pET28a-18C。
所述的制备cyclophilin突变体蛋白的方法方法中，步骤(3)所述基因工程菌表达弓形虫cyclophilin突变体蛋白为组成型表达。
所述的制备cyclophilin突变体蛋白的方法方法中，步骤(3)中所述的重组弓形虫cyclophilin突变体蛋白为如下三种多肽的任意一种：
1)含有序列表中的SEQ ID NO2序列的多肽；
2)与1)所述多肽至少80％同源的多肽；
弓形虫cyclophilin突变体蛋白在制备预防犬弓形虫感染的亚单位灭活疫苗中的应用。
弓形虫cyclophilin突变体蛋白在制备预防在制备人弓形虫疫苗中的应用，在制备猫弓形虫疫苗中的应用。
一种可编码具有弓形虫免疫原性的蛋白的核酸，编码SEQ ID 2所述氨基酸序列的核酸，具有SEQ ID 1所述的核酸序列。
本发明提供一种弓形虫亚单位灭活疫苗，该疫苗包括权利要求1所述氨基酸序列及其突变体，或权利要求4所述核苷酸序列及其优化序列，以及医学上可接受的载体。
所述弓形虫亚单位灭活疫苗在制备预防犬弓形虫感染的药物中的应用，该药物包括权利要求1所述氨基酸序列及其突变体，或权利要求4所述核苷酸序列及其优化序列，以及医学上可接受的载体。
一种制备所述亚单位灭活疫苗的方法，将弓形虫抗原连接到权利要求12所述的表达载体上，转化入原核表达工程菌，诱导其高效表达，纯化后得到可溶性融合蛋白，添加疫苗佐剂制备而成。优选疫苗佐剂为MF59佐剂或赛比克206佐剂制备而成。
所述的用于预防犬弓形虫病的疫苗中，所述蛋白的浓度至少为10μg/ml～300μg/ml.
为了的更好的技术效果，以上用于预防犬弓形虫病的疫苗，所述蛋白的浓度为100μg/ml。
上述的原核表达工程菌，其特征在于，所述原核表达工程菌为BL21(DE3)，BLR(DE3)pLysS、OrigamiB(DE3)、C41(DE3)pLysS、C41(DE3)、BL21、AD494、BL21-SI、BL21 trxB(DE3)pLysS、BL21 trxB(DE3)、Origami 2(DE3)pLysS、B834(DE3)pLysS、Rosetta-gami B(DE3)、Rosetta-gami B(DE3)pLysS、Rosetta-gami(DE3)pLysS、NovaBlue T1、Tuner(DE3)pLacI、Tuner(DE3)pLysS、Tuner(DE3)、Tuner、RosettaBlue(DE3)pLysS、RosettaBlue(DE3)pLacI、RosettaBlue(DE3)、RosettaBlue、Rosetta-gami B(DE3)pLacI、Rosetta-gami B、Rosetta-gami2(DE3)pLacI、Rosetta-gami 2、Rosetta-gami(DE3)pLacI、Rosetta-gami、Rosetta2(DE3)pLacI、Rosetta2(DE3)、Rosetta 2、Origami 2(DE3)pLacI、Rosetta(DE3)pLacI、Rosetta、OrigamiB(DE3)pLacI、OrigamiB(DE3)pLysS、Origami B、Origami 2、Origami(DE3)pLacI、Origami(DE3)pLysS、Origami、BL21(DE3)pLacI、BLR、B834(DE3)、BLR(DE3)、DH10MultiBac、ER2738、ET12567(pUZ8002)、BL21-AI、BJ5183、Rosetta-gami 2(DE3)pLysS、Rosetta-gami 2(DE3)、Rosetta-gami(DE3)、BL21(DE3)pLySs、Rosetta 2(DE3)pLySs、Rosetta(DE3)pLySs、Rosetta(DE3)、Origami(DE3)、Origami 2(DE3)、BL21-Gold(DE3)、M15[pREP4]、Sure、BL21 Star(DE3)pLySs、BL21 Star(DE3)，及以上述任意工程菌为基础改造而成的工程菌。
一种宿主细胞，包括所述SEQ ID 1或其突变体对应的核酸，以及生物学载体。
本发明第二个目的是提供一种载体，所述载体包括核苷酸序列SEQ ID 1；所述载体为 pET-28a、pMAL-p5x、pET-42b(+)、pCold-GST、pTrcHis A、pGEX-KG、pET-28b(+)、pBAD102/D-TOPO、pAmCyan、ptdTomato、pCS105、pET101/D-TOPO、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-27b(+)、pET-25b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30 Xa/LIC、pET-30 EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32EK/LIC、pET-32Xa/LIC、pET-33b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、pET-43.1c(+)、pET-43.1EK/LIC、pET-43.1EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44 EK/LIC、pET-45b(+)、pET-46 EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pETDuet-1、pET-37b(+)、pET-5b(+)、pET-51b(+)、pET-52b(+)、pBV220、pkk232-8、pET-15b、pQE-16、pCold IV、pQE-70、pSUMO、pET-SUMO、pDsRed-Express2、pColdS-SUMO、pCold TF、pCold III、pCold II、pCold I、pE-SUMO、pCold-ProS2、pBAD202/D-TOPO、pACYC184、pBAD/Thio-TOPO、pBad/Myc-His C、pBad/Myc-His B、pBad/Myc-His A、pBad/His C、pBad/His B、pBad/His A、pBAD-TOPO、pET-23b(+)、pET-23a(+)、pET-23c(+)、pET-23(+)、pET-12b(+)、pET-12c(+)、pET-12a(+)、pET-11b(+)、pET-11a(+)、ET-11c(+)、pBad24、pQE-81L、pQE-32、pQE-9、pQE-31、pQE-60、pQE-40、pET-50b(+)、pET-26b(+)、pET-32a(+)、pET-21b(+)、pET-22b(+)、pET-14b、pET-16b、pET-19b、pET-20b(+)、pET-21d(+)、pET-21c(+)、pET-21b(+)、pET-21a(+)、pET-30a(+)、pGEX-4T-3、pGEX-5X-2、pG-KJE8、pGro7、pCDFDuet-1、pTf16、pEZZ18、pBAD18、pMAL-c5x、pMal-p2E、pMal-p2X、pET-41 EK/LIC、pMal-c4X、pTrcHis B、pET-3b(+)、pGEX-3X、pGEX-4T-2、pGEX-4T-1、pTrc99a、pET-His、pALEX a,b,c、pACYC177、pKD4、pKD20、pMXB10、pKJE7、pRSET B、pGEX-2T、pRSFDuet-1、pCOLADuet-1、pET-3a(+)、pGEX-6P-3、pGEX-6P-2、pGEX-6P-1、pGEX-5X-3、pGEX-5X-1、pGEX-2TK、pRSET A、pMal-c2G、pMal-c2E、pMal-c2X、pRSET C及以上述任意载体为基础改造而成的载体，优选地，表达载体为pET-28a载体。
本发明第三个目的是提供一种可溶性融合蛋白，所述可溶性融合蛋白的氨基酸序列包含SEQ ID NO.2及其突变体。
较佳地，所述一种可溶性融合蛋白包括弓形虫cyclophilin突变体蛋白，以及组氨酸纯化标签；其中编码所述弓形虫cyclophilin突变体蛋白的DNA序列选自SEQ ID NO.1；所述的纯化标签的氨基酸序列为SEQ ID NO.3。
优选，可溶性融合蛋白的氨基酸序列为SEQ ID NO.2。
本发明第三个目的是提供一种宿主细胞，其特征在于，含有上述的载体，或已用权利 要求1所述的核苷酸序列转化或转染。
本发明第四个目的是提供一种制备可溶性融合蛋白的方法，其特征在于，包括通过宿主细胞表达可溶性融合蛋白，并进行分离。
本发明第五个目的是提供一种疫苗，其特征在于，所述疫苗包括SEQ ID 2中核苷酸序列，以及医学上可接受的载体。
优选，所述疫苗是将弓形虫抗原双酶切后连接到pET28a表达载体，转化入BL21(DE3)工程菌，诱导其高效表达，纯化后得到的蛋白为可溶性融合蛋白，再添加MF59佐剂或赛比克206佐剂，制备而成，其疫苗保持其特有的免疫原性，适合于工业化生产。
本发明的优点在于：选用了弓形虫有效地抗原基因进行亚单位灭活疫苗的研制，该抗原制备的亚单位灭活疫苗具有较强的细胞免疫和体液免疫效果，能够诱导机体的先天性免疫应答，分泌各种细胞炎性因子，从而有效的预防犬弓形虫病。本发明对cyclophilin蛋白的序列进行优化，在原核细胞大肠杆菌中的表达量为优化前的表达量的2倍。本发明提供的疫苗可以在工程菌种制备，再添加疫苗佐剂，制备而成，其疫苗保持其特有的免疫原性，适合于工业化生产。
附图说明
图1.SEQ ID 2基因ORF扩增结果。
附图中：M,DL2000marker；C18，目的基因片段。
图2.弓形虫cyclophilin蛋白和弓形虫cyclophilin突变体蛋白的SDS-PAGE结果。
附图中：M,蛋白marker；r1,弓形虫cyclophilin蛋白可溶性表达；r2,弓形虫cyclophilin突变体蛋白可溶性表达。
图3.弓形虫cyclophilin重组突变体表达蛋白纯化SDS-PAGE结果。
附图中：M,蛋白marker；r,重组cyclophilin突变体蛋白。
图4.Western-blot鉴定体外表达重组蛋白特异性结果。
附图中：M,蛋白marker；r,重组cyclophilin突变体蛋白。
图5.弓形虫cyclophilin重组突变体蛋白刺激小鼠RAW264.7细胞产生TNF-α水平。
图6.弓形虫cyclophilin重组突变体蛋白刺激小鼠树突状细胞产生IL-12水平。
图7.弓形虫cyclophilin重组突变蛋白疫苗注射小鼠产生抗体效价图。
图8.弓形虫cyclophilin重组突变体蛋白疫苗对小鼠抗弓形虫感染的保护作用。
图9.弓形虫cyclophilin重组突变体蛋白疫苗注射犬产生抗体效价图。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：弓形虫重组cyclophilin突变体蛋白原核表达载体的构建
根据核酸序列SEQ ID NO.1人工合成基因，同时根据原核表达载体pET28a物理图谱设计引物并引入酶切位点。
上游引物：ATGGAATTC ATGGAAAACGCGGGCGTGCGCAAA
下游引物：AAGCTT TTCTTTTTTGCCAATATCGGTAA
利用人工合成的方法合成SEQ ID NO.1基因序列，利用上述引物大量扩增，并将PCR纯化产物克隆至pMD-18-T经酶切和测序鉴定阳性克隆菌，阳性质粒经EcoRI和Hind III进行双酶切后纯化的目的片段连接到原核表达载体pET28a，将连接产物转化E.coli DH5α感受态细胞后筛选重组质粒，用EcoRI和Hind III进行双酶切反应鉴定，获得原核表达质粒pET28a-C18。将pET28a-C18转化入BL21(DE3)工程菌，挑取单个菌落接种到5ml LB液体培养基中(含100μg/ml卡那霉素)中，37℃，220rpm振荡培养过夜。cyclophilin突变体ORF扩增结果见附图1。
实施例2：表达蛋白的纯化
(1)表达蛋白的提取及可溶性验证
以选取的转化了重组质粒pET-28a(+)-C18的E.coli BL21(DE3)单菌落接种于5mL LB液体培养基中，37℃过夜振荡培养。次日将种子液转接到800mL LB液体培养基中37℃扩繁，监测菌液OD600至0.6～0.7时，在最适条件下诱导表达。离心收集菌体，菌体用20mLPBS悬浮，反复冻融5次(-80℃,1h/37℃,10min)后，经超声处理,离心取上清即为可溶性蛋白(具有活性的蛋白)。沉淀用10mL变性缓冲液悬浮，室温振摇1h，离心取上清即为非可溶性蛋白(变性蛋白)。将上述两种蛋白分别进行SDS-PAGE检测以验证目的蛋白的可溶性，若目的蛋白主要位于可溶性蛋白上清，说明为可溶性表达；若目的蛋白大部分位于非可溶性蛋白性蛋白上清液中，则说明为包涵体形式表达。结果表明，改造后的弓形虫cyclophilin突变体蛋白为可溶性表达，其表达量为120mg/L,未突变改造的cyclophilin蛋白为可溶性表达(制备方法同文献：弓形虫亲环蛋白基因的克隆及原核表达，李运娜，2010年)，其表达量为60mg/L，其结果见附图2。
(2)融合表达蛋白的纯化
将镍柱使用A液(PBS)平衡至稳定，使用A液冲洗系统，流速20mL/min，冲洗2min， 将平衡后的镍柱连接至上样接口，以流速1mL/min流穿样品，使目的蛋白挂柱。流穿结束后，将已挂有目的蛋白的镍柱连接至洗脱接口，首先用A液平衡镍柱，流速1mL/min，洗掉未结合的杂蛋白。保持流速不变，设置梯度洗杂条件，B液(PBS，含有1M咪唑)浓度为10％，即咪唑浓度为100mM,时间30min，洗掉非特异性吸附杂蛋白。保持流速不变，设置洗脱条件，B液浓度为30％，即咪唑浓度为300mM，时间120min，洗脱目的蛋白，将收集的目的蛋白进行SDS-PAGE，观察纯度。其结果见附图3。通过Western-blot鉴定体外表达重组突变体蛋白特异性结果见附图4。
实施例3：表达产物对小鼠巨噬细胞免疫原性检测
将RAW264.7细胞以0.5×106/mL接种到24孔培养板上，每孔0.5mL，5％CO2，37℃培养24h。吸出上清后将制备好的弓形虫重组cyclophilin突变体蛋白溶于RAW264.7细胞培养液中，并按1μg/mL、0.1μg/mL、0.01μg/mL的浓度，0.5mL/孔添加至24孔板中，等体积PBS作为阴性对照。将细胞培养板置于5％CO2，37℃培养箱中培养48h，收集上清液，通过ELISA实验检测TNF-a水平。结果表明TNF-a的产生随蛋白浓度的升高而增加，范围从40pg-310pg不等，结果见附图5。说明弓形虫重组cyclophilin突变体蛋白可以刺激RAW264.7细胞产生TNF-a。
实施例4:表达产物对小鼠树突状细胞免疫原性检测
分离小鼠树突状细胞，调整细胞浓度为0.5×106个/mL，接种至24孔组织细胞培养板中，每孔接种0.5mL。将处理的弓形虫重组cyclophilin突变体蛋白分别以每孔100μg/mL、50μg/mL、10μg/mL、1μg/mL、0.1μg/mL、0.01μg/mL的浓度添加至细胞培养板中，每孔0.5mL。将细胞培养板置于5％CO2，37℃培养箱中培养24h，收集培养上清液，通过ELISA实验检测上清液中IL-12水平。结果表明各组产生IL-12含量呈现蛋白浓度依赖性，即随着蛋白浓度升高而增加，其结果见附图6。结果说明弓形虫重组cyclophilin突变体蛋白可以刺激小鼠树突状免疫细胞产生大量的IL-12。
实施例5:弓形虫重组亚单位疫苗小鼠免疫攻毒保护实验
将125只8～10周龄左右，雌SPF级BALB/c小鼠分成5组，每组25只，其中第一组cyclophilin实验组I(佐剂为MF59，30μg/只，皮下注射)，第二组为cyclophilin实验组II(佐剂为206，30μg/只，皮下注射)，第三组为阴性对照组I(相等体积量PBS，配以MF59佐剂，皮下注射)，第四组阴性对照组II(相等体积量的PBS，配以206佐剂，皮下注射)，第五组为空白对照组(相等体积量的PBS，皮下注射)。各组注射三次，间隔两周。每周检测抗体滴度。第三次免疫注射一周后腹腔注射纯化的弓形虫滋养体103个/只，每天观察记录小鼠状况。
抗体结果显示一免后三周抗体水平迅速提高，并在6周时达到顶点，疫苗抗体效价见附图7。
免疫保护性试验结果显示实验组免疫存活率明显优于阴性对照组和空白对照组，且MF59佐剂疫苗组效果优于206佐剂疫苗组对照组(见附图8)。
实施例6:弓形虫重组亚单位疫苗犬免疫攻毒保护率实验
选择无病原(包括无寄生虫病、无病毒病、无细菌性传染病)的实验动物犬(比格犬，性成熟的母犬)20只，尤其是无弓形虫、新孢子虫、犬瘟热、犬传染性肝炎、细小病毒等传染性病原。将实验动物分为2组，其中第一组为cyclophilin实验组(佐剂为MF59，300μg/只，皮下注射)，共15只犬。第二组为空白对照组(相等体积量的PBS，皮下注射)，共5只犬。各组注射三次，间隔两周。每周检测抗体滴度。第三次免疫注射一周后腹腔注射纯化的弓形虫滋养体104个/只，30天后，将各犬解剖，通过PCR检测脑、心、肝、脾、肺、肾、淋巴结、咬肌、舌肌、腹肌组织感染弓形虫情况，若其中任意组织PCR结果为阳性，则该犬判定为弓形虫感染，若全部组织PCR结果为阴性，则判定为弓形虫保护。
抗体结果显示免疫组一免后抗体水平持续升高，疫苗抗体效价见附图9。
攻毒后结果显示对照组犬全部判定为弓形虫感染，免疫组15只犬中13只犬判定为弓形虫保护，2只犬判定为弓形虫感染，保护率为86％。
综合上述实验结果，本发明提供的一种弓形虫亚单位灭活疫苗，对犬弓形虫免疫保护率较高，可以用来作为犬弓形虫病的预防和治疗性候选疫苗。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (21)

  1. 一种具有弓形虫免疫原性的蛋白，其特征在于：该蛋白为cyclophilin突变体蛋白，由SEQ ID 2所述氨基酸序列组成。
  2. 根据权利要求1所述的具有弓形虫免疫原性的蛋白，其特征在于：该蛋白由SEQ ID 2所述氨基酸序列取代、删除、或置换得到蛋白突变体。
  3. 根据权利要求1所述的具有弓形虫免疫原性的蛋白，其特征在于：该蛋白是与SEQ ID 2所述氨基酸序列有90％以上同源性的氨基酸序列组成的蛋白。
  4. 一种可编码具有弓形虫免疫原性的蛋白的核酸，其特征在于：编码SEQ ID 2所述氨基酸序列的核酸，具有SEQ ID 1所述的核酸序列。
  5. 一种制备权利要求1-3中任一项cyclophilin突变体蛋白的方法，其特征在于：
    (1)克隆相关基因到表达载体质粒中，获得重组表达载体；
    (2)所述重组表达载体转化大肠杆菌，获得基因工程菌；
    (3)所述基因工程菌进行发酵培养，表达cyclophilin突变体蛋白；
    (4)回收基因工程菌菌体破碎后的上清液，分离纯化弓形虫cyclophilin突变体蛋白。
  6. 根据权利要求5所述的制备cyclophilin突变体蛋白的方法方法，其特征在于，步骤(2)所述载体为PET-28a，大肠杆菌为BL21(DE3)。
  7. 根据权利要求5所述的制备cyclophilin突变体蛋白的方法方法，其特征在于，步骤(2)所述重组基因工程菌为DH5α/pET28a-18C。
  8. 根据权利要求5所述的制备cyclophilin突变体蛋白的方法方法，其特征在于，步骤(3)所述基因工程菌表达弓形虫cyclophilin突变体蛋白为组成型表达。
  9. 根据权利要求5所述的方法，其特征在于，步骤(3)中所述的重组弓形虫cyclophilin突变体蛋白为如下三种多肽的任意一种：
    1)含有序列表中的SEQ ID NO2序列的多肽；
    2)与1)所述多肽至少80％同源的多肽。
  10. 根据权利要求5所述的方法，其特征在于，步骤(1)中所述的表达载体质粒还包括pMAL-p5x、pET-42b(+)、pCold-GST、pTrcHis A、pGEX-KG、pET-28b(+)、pBAD102/D-TOPO、pAmCyan、ptdTomato、pCS105、pET101/D-TOPO、pET-24a(+)、pET-24c(+)、pET-24d(+)、pET-27b(+)、pET-25b(+)、pET-28c(+)、pET-29a(+)、pET-29b(+)、pET-29c(+)、pET-30b(+)、pET-30c(+)、pET-30Xa/LIC、pET-30EK/LIC、pET-31b(+)、pET-32b(+)、pET-32c(+)、pET-32EK/LIC、pET-32Xa/LIC、pET-33b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42c(+)、pET-43.1a(+)、pET-43.1b(+)、 pET-43.1c(+)、pET-43.1EK/LIC、pET-43.1EK/LIC、pET-44a(+)、pET-44b(+)、pET-44c(+)、pET-44EK/LIC、pET-45b(+)、pET-46EK/LIC、pET-47b(+)、pET-48b(+)、pET-49b(+)、pETDuet-1、pET-37b(+)、pET-5b(+)、pET-51b(+)、pET-52b(+)、pBV220、pkk232-8、pET-15b、pQE-16、pCold IV、pQE-70、pSUMO、pET-SUMO、pDsRed-Express2、pColdS-SUMO、pCold TF、pCold III、pCold II、pCold I、pE-SUMO、pCold-ProS2、pBAD202/D-TOPO、pACYC184、pBAD/Thio-TOPO、pBad/Myc-His C、pBad/Myc-His B、pBad/Myc-His A、pBad/His C、pBad/His B、pBad/His A、pBAD-TOPO、pET-23b(+)、pET-23a(+)、pET-23c(+)、pET-23(+)、pET-12b(+)、pET-12c(+)、pET-12a(+)、pET-11b(+)、pET-11a(+)、ET-11c(+)、pBad24、pQE-81L、pQE-32、pQE-9、pQE-31、pQE-60、pQE-40、pET-50b(+)、pET-26b(+)、pET-32a(+)、pET-21b(+)、pET-22b(+)、pET-14b、pET-16b、pET-19b、pET-20b(+)、pET-21d(+)、pET-21c(+)、pET-21b(+)、pET-21a(+)、pET-30a(+)、pGEX-4T-3、pGEX-5X-2、pG-KJE8、pGro7、pCDFDuet-1、pTf16、pEZZ18、pBAD18、pMAL-c5x、pMal-p2E、pMal-p2X、pET-41EK/LIC、pMal-c4X、pTrcHis B、pET-3b(+)、pGEX-3X、pGEX-4T-2、pGEX-4T-1、pTrc99a、pET-His、pALEX a,b,c、pACYC177、pKD4、pKD20、pMXB10、pKJE7、pRSET B、pGEX-2T、pRSFDuet-1、pCOLADuet-1、pET-3a(+)、pGEX-6P-3、pGEX-6P-2、pGEX-6P-1、pGEX-5X-3、pGEX-5X-1、pGEX-2TK、pRSET A、pMal-c2G、pMal-c2E、pMal-c2X、pRSET C，及以上述任意载体为基础改造而成的载体。
  11. 一种可溶性融合蛋白，其特征在于，包括权利要求1或2所述弓形虫cyclophilin突变体蛋白，以及纯化标签。
  12. 根据权利要求11所述可溶性蛋白，其特征在于：所述的纯化标签的氨基酸序列为SEQ ID NO.3。
  13. 权利要求1或2，或11所述蛋白在制备预防犬弓形虫感染的亚单位灭活疫苗中的应用。
  14. 权利要求1或2，或11所述蛋白在制备预防人弓形虫感染的亚单位灭活疫苗中的应用。
  15. 权利要求1或2，或11所述蛋白在制备预防猫弓形虫感染的亚单位灭活疫苗中的应用。
  16. 一种弓形虫亚单位灭活疫苗，其特征在于，该疫苗包括权利要求1所述氨基酸序列及其突变体，或权利要求4所述核苷酸序列及其优化序列，以及医学上可接受的载体。
  17. 权利要求16所述亚单位灭活疫苗在制备预防犬弓形虫感染的药物中的应用，其特 征在于，该药物包括权利要求1所述氨基酸序列及其突变体，或权利要求4所述核苷酸序列及其优化序列，以及医学上可接受的载体。
  18. 一种制备权利要求16所述的亚单位灭活疫苗的方法，其特征在于，所述亚单位灭活疫苗将弓形虫抗原连接到权利要求12所述的表达载体上，转化入原核表达工程菌，诱导其高效表达，纯化后得到可溶性融合蛋白，添加疫苗佐剂制备而成。
  19. 根据权利要求18任一项所述的用于预防犬弓形虫病的疫苗，其特征在于：所述蛋白的浓度至少为10μg/ml～300μg/ml.
  20. 根据权利要求18所述的原核表达工程菌，其特征在于，所述原核表达工程菌为BL21(DE3)，BLR(DE3)pLysS、OrigamiB(DE3)、C41(DE3)pLysS、C41(DE3)、BL21、AD494、BL21-SI、BL21 trxB(DE3)pLysS、BL21 trxB(DE3)、Origami 2(DE3)pLysS、B834(DE3)pLysS、Rosetta-gami B(DE3)、Rosetta-gami B(DE3)pLysS、Rosetta-gami(DE3)pLysS、NovaBlue T1、Tuner(DE3)pLacI、Tuner(DE3)pLysS、Tuner(DE3)、Tuner、RosettaBlue(DE3)pLysS、RosettaBlue(DE3)pLacI、RosettaBlue(DE3)、RosettaBlue、Rosetta-gami B(DE3)pLacI、Rosetta-gami B、Rosetta-gami2(DE3)pLacI、Rosetta-gami 2、Rosetta-gami(DE3)pLacI、Rosetta-gami、Rosetta2(DE3)pLacI、Rosetta2(DE3)、Rosetta 2、Origami 2(DE3)pLacI、Rosetta(DE3)pLacI、Rosetta、OrigamiB(DE3)pLacI、OrigamiB(DE3)pLysS、Origami B、Origami 2、Origami(DE3)pLacI、Origami(DE3)pLysS、Origami、BL21(DE3)pLacI、BLR、B834(DE3)、BLR(DE3)、DH10MultiBac、ER2738、ET12567(pUZ8002)、BL21-AI、BJ5183、Rosetta-gami 2(DE3)pLysS、Rosetta-gami 2(DE3)、Rosetta-gami(DE3)、BL21(DE3)pLySs、Rosetta 2(DE3)pLySs、Rosetta(DE3)pLySs、Rosetta(DE3)、Origami(DE3)、Origami 2(DE3)、BL21-Gold(DE3)、M15[pREP4]、Sure、BL21 Star(DE3)pLySs、BL21 Star(DE3)，及以上述任意工程菌为基础改造而成的工程菌。
  21. 一种宿主细胞，其特征在于：包括所述SEQ ID 1或其突变体对应的核酸，以及生物学载体。
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