Keratin filament binding protein of Clonorchis sinensis and its application in preparing universal oral vaccine for veterinary and fish

Abstract

The present invention belongs to the field of biotechnology, discloses a keratin filament binding protein of Clonorchis sinensis and its application in preparing a universal oral vaccine for animals and fish, and specifically discloses a fusion protein. The present invention obtains a fusion protein through extensive and in-depth research, uses Bacillus subtilis as a vaccine carrier, induces it to form spores, and then prepares it into a universal oral vaccine for preventing Clonorchis sinensis infection in animals and fish. The oral vaccine can produce a good immune effect in the grass carp, the second intermediate host model of Clonorchis sinensis, and the rat, the worm-retaining host model. After immunization with the oral vaccine, the grass carp, the second intermediate host of Clonorchis sinensis, and the rat, the worm-retaining host, can resist the infection of cercariae or metacercariae of Clonorchis sinensis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Clonorchis sinensis keratin filament binding protein and an application thereof in preparing a universal oral vaccine for animals and fish. Background Art

[0002] Clonorchis sinensis (C. sinensis), also known as liver fluke, infects humans and causes Clonorchiasis, or liver fluke disease. It is a zoonotic foodborne parasitic disease that is prevalent in China, South Korea, northern Vietnam, and the Russian Far East. It is estimated that 600 million people worldwide are at risk of infection, and about 35 million are infected. It is difficult for residents in endemic areas to change their habit of eating raw or semi-cooked freshwater fish, which can lead to repeated infection. After being cured by drugs, they can be infected again if they ingest freshwater fish containing live metacercariae. More than 40 mammals other than humans can serve as the final hosts (also known as worm-carrying hosts) of liver flukes. Common worm-carrying hosts, cats and dogs, are easily infected. Both have a close relationship with humans, which greatly promotes the completion of the life cycle of liver flukes and accelerates the rate of disease transmission. It is not realistic to establish a demonstration area for the comprehensive prevention and control of liver fluke disease and to continuously and massively invest manpower and financial resources in many endemic counties and cities. Therefore, to achieve long-term and effective control, we must find a new way and find a more efficient and feasible prevention and control strategy. The "One Health" concept provides a new solution for the long-term and effective control of liver fluke disease. Humans can be reinfected and repeatedly infected, but controlling the infection of the worm-carrying hosts and intermediate hosts, and curbing and blocking the transmission pathways of liver flukes will inevitably reduce and eliminate human infection, and vaccination is one of the most economical and effective health investments in controlling and eliminating infectious diseases; the skin mucosa of freshwater fish, the second intermediate host of Clonorchis sinensis, and the intestinal mucosa and hepatobiliary epithelium of the final host are the earliest immune response sites of the host infected with liver flukes. It is more feasible to adopt an immunization method based on inducing adaptive mucosal immunity to enable the worm-carrying hosts and intermediate host fish to produce immune responses against liver fluke infection. Combined with the management of the worm-carrying hosts of liver flukes and the characteristics of the breeding of freshwater fish, the intermediate host, the strategy of oral vaccines is more suitable for the development of "universal vaccines" for animals and fish to block the transmission of liver fluke disease. Bacillus subtilis (B.subtilis) has been used as a functional food and feed additive. From the perspective of agricultural economy and environmental health, B.subtilis is very suitable for preparing oral "universal vaccines" for animals and fish. The liver fluke keratin filament binding protein and its homologous protein are the main components of the cytoskeleton in most eukaryotic cells, and their main function is to maintain the integrity and continuity of epithelial cells.

[0003] At present, there are no patents or journal articles at home and abroad regarding a universal vaccine for veterinary and fish that can prevent Clonorchis sinensis infection. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a fusion protein.

[0005] The purpose of the second aspect of the present invention is to provide a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention.

[0006] The third aspect of the present invention aims to provide a biological material containing the nucleic acid molecule of the second aspect of the present invention.

[0007] The fourth aspect of the present invention aims to provide a recombinant engineered bacterium of Bacillus subtilis.

[0008] The purpose of the fifth aspect of the present invention is to provide a method for constructing the recombinant engineered Bacillus subtilis bacteria of the fourth aspect of the present invention.

[0009] The purpose of the sixth aspect of the present invention is to provide the use of the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the biological material of the third aspect of the present invention or the recombinant engineered bacteria of the fourth aspect of the present invention in the preparation of a product for preventing or treating Clonorchiasis sinensis.

[0010] The seventh aspect of the present invention aims to provide a vaccine.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a fusion protein, wherein the fusion protein is any one of a1) to a3):

[0013] a1) a fusion protein with an amino acid sequence as shown in SEQ ID NO: 5;

[0014] a2) a fusion protein in which one or more amino acids of SEQ ID NO:5 are substituted and / or deleted and / or added and which has the same or similar function as SEQ ID NO:5;

[0015] a3) a fusion protein that has at least 99%, 98%, 97%, 96%, or 95% homology to SEQ ID NO: 5 and has the same or similar function as SEQ ID NO: 5.

[0016] The second aspect of the present invention provides a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the nucleic acid molecule is as shown in SEQ ID NO:6.

[0018] The third aspect of the present invention provides a biological material related to the nucleic acid molecule of the second aspect of the present invention, wherein the biological material comprises at least one of b1) to b8):

[0019] b1) the nucleic acid molecule according to the second aspect of the present invention;

[0020] b2) an expression cassette containing the nucleic acid molecule described in b1);

[0021] b3) a recombinant vector containing the nucleic acid molecule described in b1);

[0022] b4) a recombinant vector containing the expression cassette described in b2);

[0023] b5) a recombinant cell containing the nucleic acid molecule described in b1);

[0024] b6) a recombinant cell containing the expression cassette described in b2);

[0025] b7) a recombinant cell containing the recombinant vector described in b3);

[0026] b8) A recombinant cell containing the recombinant vector described in b4)

[0027] In some embodiments of the present invention, the recombinant vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, a F cosmid, or an artificial chromosome.

[0028] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cell vector does not include propagation materials.

[0029] The fourth aspect of the present invention provides a recombinant engineered bacterium of Bacillus subtilis, wherein the recombinant engineered bacterium expresses the fusion protein of the first aspect of the present invention.

[0030] The recombinant Bacillus subtilis engineered bacteria expressing the keratin filament binding protein of Clonorchis sinensis uses the keratin filament binding protein of Clonorchis sinensis as a candidate antigen molecule, constructs a recombinant plasmid to fuse the keratin filament binding protein of Clonorchis sinensis with the spore capsid protein CotC and express it on the outer capsid of Bacillus subtilis, induces an immune response in the skin mucosa of the second intermediate host or the intestinal mucosa of the worm-retaining host through oral immunization, produces specific antibodies against the keratin filament binding protein of Clonorchis sinensis, hinders the cercariae from attacking the infected fish body to form a cyst wall, prevents the metacercariae from forming cysts or prevents the metacercariae from excysting and developing into adults in the worm-retaining host, thereby blocking the infection of Clonorchis sinensis. The recombinant engineered bacteria that induce the formation of spores can be used to prepare a universal oral vaccine for animals and fish to prevent infection of Clonorchis sinensis.

[0031] The fifth aspect of the present invention provides a method for constructing the Bacillus subtilis recombinant engineered bacteria of the fourth aspect of the present invention, comprising the following steps:

[0032] (1) Obtain the target gene;

[0033] (2) constructing a recombinant plasmid: linking the target gene obtained in step (1) with the vector plasmid using homologous recombination to obtain a recombinant plasmid;

[0034] (3) Transforming the recombinant plasmid obtained in step (2) into Bacillus subtilis competent cells to obtain the Bacillus subtilis recombinant engineered bacteria.

[0035] In some embodiments of the present invention, the target gene described in (1) contains a nucleotide sequence encoding a keratin filament binding protein of Clonorchis sinensis.

[0036] In some embodiments of the present invention, the encoding nucleotide sequence of the Clonorchis sinensis keratin filament binding protein is shown in SEQ ID NO:1.

[0037] In some embodiments of the present invention, the vector plasmid is pBE03-CotC plasmid.

[0038] The pBE0-CotC plasmid is an existing plasmid in the prior art. Reference may be made to the document “Production of N-Acetyl-d-Neuraminic Acid by Use of an Efficient Spore Surface Display System. The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine ​​protease”.

[0039] In some embodiments of the present invention, the restriction endonucleases in (2) are HindⅢ and SacⅠ.

[0040] In some embodiments of the present invention, the Bacillus subtilis competent cells are Bacillus subtilis WB600 competent cells.

[0041] The sixth aspect of the present invention provides the use of the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the biological material of the third aspect of the present invention or the recombinant engineered bacteria of the fourth aspect of the present invention in the preparation of a product for preventing or treating Clonorchiasis sinensis.

[0042] In some embodiments of the present invention, the products include but are not limited to reagents, kits and vaccines.

[0043] In some embodiments of the invention, the product is a vaccine.

[0044] The seventh aspect of the present invention provides a vaccine, wherein the vaccine comprises the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the biological material of the third aspect of the present invention, or the recombinant engineered bacteria of the fourth aspect of the present invention.

[0045] In some embodiments of the invention, the vaccine is an oral vaccine.

[0046] In some embodiments of the present invention, the vaccine is a vaccine universal for animals and fish.

[0047] In some embodiments of the present invention, the vaccine further comprises an adjuvant, such as cod liver oil.

[0048] The beneficial effects of the present invention are:

[0049] The present invention obtains a fusion protein through extensive and in-depth research, uses Bacillus subtilis as a vaccine carrier, induces the fusion protein to form spores, and then prepares a universal oral vaccine for preventing infection of Clonorchis sinensis in animals and fish. The oral vaccine can produce a good immune effect in grass carp, a second intermediate host model of Clonorchis sinensis, and rats, a worm-retaining host model. Through immunization with the oral vaccine, grass carp, a second intermediate host of Clonorchis sinensis, and rats, a worm-retaining host, can resist infection by cercariae or metacercariae of Clonorchis sinensis.

[0050] The vaccine (the universal oral vaccine for animals and fish of Clonorchis sinensis) provided by the invention can cut off the transmission route of Clonorchis sinensis and achieve the effect of controlling Clonorchiasis sinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the result of colony PCR identification after the keratin filament binding protein of Clonorchis sinensis was constructed into plasmid pBE03-CotC. In the figure, M is the DNA molecular weight marker, and 1 to 10 are the results of PCR identification of 10 monoclonal colonies respectively.

[0052] Figure 2 This is the SDS-PAGE verification of the spore surface expression of the keratin filament binding protein of Clonorchis sinensis. In the figure, M is the protein molecular weight marker, lane 1 is Bacillus subtilis WB600 without plasmid, lane 2 is Bacillus subtilis WB600 containing plasmid pBE03-CotC, and lane 3 is Bacillus subtilis WB600 expressing the keratin filament binding protein of Clonorchis sinensis.

[0053] Figure 3The IgM levels in serum, surface mucus, intestinal mucus and bile, and the IgZ levels in surface mucus and intestinal mucus of grass carp after immunization with spore vaccine; wherein, A is serum IgM level, B is surface mucus IgM level, C is intestinal mucus IgM level, D is bile IgM level, E is surface mucus IgZ level, and F is intestinal mucus IgZ level; in the figure, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.005; the experimental group in the figure refers to the high-dose group.

[0054] Figure 4 The serum IgG, IgG1, and IgG2a antibody levels as well as the IgA antibody levels in the jejunum and ileum tissues of rats after immunization with the spore vaccine; A is the serum IgG level, B is the serum IgG1 level, C is the IgG2a level, D is the jejunal tissue mucus IgA level, and E is the ileal tissue mucus IgA level; in the figure, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.005, and **** represents p < 0.001.

[0055] Figure 5 The proliferation index of spleen cells (A) and the secretion level of IL-4 (C) and IFN-γ (B) of spleen cells in rats after immunization with spore vaccine; in the figure, * represents p < 0.05, ** represents p < 0.01, and ns represents no significant difference.

[0056] Figure 6 The number of IgA secreting cells in the jejunum and ileum of rats immunized with spore vaccine was analyzed by immunohistochemistry; A is the immunohistochemistry result of IgA in the jejunum of the blank group, B is the immunohistochemistry result of IgA in the jejunum of the control group, C is the immunohistochemistry result of IgA in the jejunum of the experimental group, D is the immunohistochemistry result of IgA in the ileum of the blank group, E is the immunohistochemistry result of IgA in the ileum of the control group, F is the immunohistochemistry result of IgA in the ileum of the experimental group, G is the average optical density value of IgA secreting cells in the jejunum, and H is the average optical density value of IgA secreting cells in the ileum; in the figure, ** represents p < 0.01, and *** represents p < 0.005.

[0057] Figure 7 These are the statistics of the number of worm eggs in feces and the number of adult worms in the bile tract after infection with Clonorchis sinensis metacercariae after immunization with spore vaccine, among which, A is the detection of adult worms in the bile tract of rats in the blank group, B is the detection of adult worms in the bile tract of rats in the control group, C is the detection of adult worms in the bile tract of rats in the experimental group, D is the detection of worm eggs in feces of the three groups of rats, E is the number of worm loads in the bile tract of the three groups of rats, and F is the number of worm eggs per gram of feces of the three groups of rats; in the figure, * represents p < 0.05, and ** represents p < 0.01.

[0058] Figure 8These are Masson staining and Sirius red staining of liver tissues after infection with Clonorchis sinensis metacercariae after immunization with spore vaccine, as well as the transcription levels of Acta2 and Col1a1 genes in liver tissues. A is Masson staining of liver tissues in the blank group, B is Masson staining of liver tissues in the control group, C is Masson staining of liver tissues in the experimental group, D is Sirius red staining of liver tissues in the blank group, E is Sirius red staining of liver tissues in the control group, F is Sirius red staining of liver tissues in the experimental group, G is statistical analysis of Masson staining-positive areas, H is statistical analysis of Sirius red staining-positive areas, I is the transcription level of Acta2 gene in liver tissue, and J is the transcription level of Col1a1 gene in liver tissue. In the figure, * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.001. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below through specific examples.

[0060] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0062] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0063] Reagent preparation

[0064] ①10×Spizizen Salts solution (10×SS solution, 100mL): K2HPO4·3H2O 15g, KH2PO4 6g, (NH4)2SO4 2g, MgSO4 0.2g, and sodium citrate 1g were dissolved in deionized water and made up to 100mL. After high pressure sterilization, store at 4℃.

[0065] ②Spore rupture buffer: 1mM PMSF, 50mM Tris-HCl, 0.5mM EDTA.

[0066] ③SDS-DTT solution: 0.5% SDS, 0.1M DTT, 0.1M NaCl.

[0067] ④GMⅠ solution (10 mL): 10×SS solution 1 mL, 10% yeast powder 0.1 mL, 20% glucose 0.25 mL, 1% casein hydrolysate 0.2 mL, 0.25% tryptophan solution 0.2 mL, deionized water 8.25 mL.

[0068] ⑤GMⅡ(10mL): 10×SS solution 1mL, 10% yeast powder 0.05mL, 20% glucose 0.25mL, 1% casein hydrolyzate 0.04mL, 0.25% tryptophan solution 0.2mL, 100mM CaCl2 0.05mL, 25mM MgCl2 1mL

[0069] Example 1 Preparation of a universal oral vaccine for animals and fish against Clonorchiasis sinensis

[0070] (1) Construction of a recombinant plasmid containing the gene for keratin filament binding protein of Clonorchis sinensis

[0071] According to the multiple cloning site of the vector plasmid pBE03-CotC and the coding sequence of the keratin filament binding protein of Clonorchis sinensis (nucleotide sequence such as SEQ ID NO: 1), specific gene amplification primers were designed, and the coding sequence of the keratin filament binding protein of Clonorchis sinensis was connected to the pBE03-CotC after enzyme digestion to obtain a recombinant plasmid containing the keratin filament binding protein of Clonorchis sinensis. The restriction endonucleases used were HindⅢ and SacⅠ, and the primer sequences used were: upstream primer P1: 5'-ATAAAAACACTAC AAGCTT ATGGAGGCTTTTGAGGTCCAGA-3' (SEQ ID NO: 3), the underline is the HindⅢ restriction site, ATAAAAACACTAC is the vector plasmid homologous sequence; downstream primer P2: 5'-CAAAGTGCTA GAGCTC CCATATATTACCCCCGGGCTTA-3' (SEQ ID NO: 4), the underline is the SacⅠ restriction site, and CAAAGTGCTA is the vector plasmid homologous sequence. The genetic engineering technology was operated in Escherichia coli DH5α, and the recombinant plasmid was sequenced and identified as correct and stored routinely.

[0072] (2) Chemical transformation of recombinant plasmid of Clonorchis sinensis keratin filament binding protein into Bacillus subtilis WB600

[0073] Pick a freshly cultured single colony (WB600), inoculate it into 10mL GMⅠ medium, culture it at 30℃ overnight (180rpm / min), take the above bacterial solution at a ratio of 10% (100μL) and inoculate it into 10mL GMⅠ medium, culture it on a shaker for 3.5h (37℃, 250rpm / min); take the above bacterial solution at a ratio of 10% (100μL) and inoculate it into 10mL GMⅡ medium, culture it on a shaker for 60 minutes (37℃, 250rpm / min). The above bacterial solution was divided into 1mL per tube, centrifuged at 5000rpm / min for 5 minutes, removed about 900μL of the supernatant, and resuspended the bacterial precipitate with the remaining 100μL of supernatant. Take about 5 μg of the recombinant plasmid of the keratin filament binding protein of Clonorchis sinensis and add it to the above 100 μL bacterial solution, let it stand in a 37°C water bath for 30 minutes, and culture it at 180 rpm / min at 37°C for 2 hours. Evenly spread the bacterial solution on an LB plate (spectinomycin, 10 μg / mL), and culture it at 37°C overnight for about 16 hours. The next day, pick a single clone and add it to LB medium (spectinomycin, 10 μg / mL), and culture it at 37°C, 250 rpm / min on a shaker for about 16 hours to obtain the recombinant Bacillus subtilis engineered bacteria containing the keratin filament binding protein of Clonorchis sinensis. The positive clones were identified by PCR of the recombinant engineered bacteria solution, and the identification results were as follows: Figure 1 At the same time, the recombinant engineering bacteria were shaken to extract plasmids and the nucleic acid sequence of the keratin filament binding protein of Clonorchis sinensis was sequenced and analyzed, and the results were consistent with the nucleotide sequence of the target gene fragment. The nucleotide sequence of the target gene fragment of the keratin filament binding protein of Clonorchis sinensis is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0074] (3) Detection of Clonorchis sinensis keratin filament binding protein expressed by recombinant engineered bacteria outer capsid fusion

[0075] A single clone of the recombinant engineered bacteria was picked and inoculated into 5 mL of LB medium (spectinomycin, 10 μg / mL), cultured overnight at 37°C in a shaking incubator, and then transferred into 100 mL of DSM medium at a ratio of 1:100, and cultured at 37°C, 250 rpm / min in a shaking incubator for 24 h to induce spore formation. The spore solution was centrifuged at 4°C, 10,000×g for 15 min, the supernatant was discarded, and the precipitate was collected; the precipitate was resuspended with 10 mL SDS-DTT solution, incubated in a 37°C water bath for 2 h, centrifuged to remove the supernatant, and the precipitate was resuspended with 1 M Tris-HCl solution, centrifuged at 10,000×g for 10 min, and repeated 6 times; the precipitate was resuspended with 5 mL spore breaking buffer, ultrasonically broken for 15 min at low temperature by an ultrasonic disruptor, centrifuged at 4°C, 10,000×g for 10 min, and the supernatant and precipitate were collected respectively; the supernatant and precipitate were subjected to 10% SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue R250 for 2 h, and then decolorized to observe the expression of the fusion protein of Clonorchis sinensis keratin filament binding protein and capsid protein CotC and its localization in spores. The results are shown in Figure 2 As shown, it indicates that the fusion protein is successfully expressed in Bacillus subtilis. The amino acid sequence of the fusion protein is shown in SEQ OD NO:5, and the nucleotide sequence encoding the fusion protein is shown in SEQ OD NO:6.

[0076] (4) Mass cultivation of recombinant Bacillus subtilis

[0077] The recombinant engineering bacteria of Clonorchis sinensis keratin filament binding protein were inoculated into 40 mL LB medium (spectinomycin, 10 μg / mL) at a ratio of 1:100, and cultured overnight at 37°C, 250 rpm / min in a shaking incubator for about 16 h. The next day, the culture was transferred to 4LDSM spore medium and cultured at 37°C, 250 rpm / min in a shaking incubator for 24 h. The spore solution was centrifuged at 4°C, 10,000 × g for 15 min, the supernatant was discarded, and the precipitate was collected. The lysozyme solution containing PMSF was used to The precipitate was resuspended in 1M NaCl solution and centrifuged, and the supernatant was discarded. The precipitate was resuspended in 1M KCl solution and centrifuged, and the supernatant was discarded. The precipitate was resuspended in distilled water, centrifuged, and the supernatant was discarded. The precipitate was resuspended in 40mL distilled water, centrifuged, and the supernatant was discarded. This was repeated three times. Finally, the precipitate was resuspended in 40mL distilled water and incubated at 69℃ for 1 hour to inactivate the remaining spore propagules. The spores were counted and stored at -80℃.

[0078] The spore vaccine induced by mass cultivation of Bacillus subtilis in this part is a spore containing the fusion protein of Clonorchis sinensis keratin filament binding protein-capsid protein, that is, a universal oral vaccine for animals and fish against Clonorchis sinensis.

[0079] Example 2 Feeding of a Universal Oral Vaccine for Animals and Fishes for Clonorchiasis to the Second Intermediate Host Grass Carp

[0080] Feeding plan and sampling plan:

[0081] 240 grass carps were randomly divided into 4 groups, 60 in each group, and divided into blank group, control group and experimental group. After the grass carps were purchased, they were temporarily kept in the laboratory fish tank for 2 weeks without eating during this period. After the grass carps were in a stable state, they were fed with grass carp feed without spores and fish liver oil for 1 week (grass carp feed was provided by Guangdong Taifeng Extruded Feed Co., Ltd., ingredients: crude protein ≥30%, crude fat ≥3%, crude ash powder ≤13%, crude fiber ≤12%, lysine ≥1.3%, total phosphorus ≥0.7%), and fed twice a day to train the grass carp eating time. Blank group: grass carp ate grass carp feed coated with fish liver oil (5g feed / 1mL fish liver oil); control group: grass carp ate fish liver oil and Bacillus subtilis containing the vector plasmid pBE03-CotC (1×10 10 cfu / g) coated grass carp feed; experimental group: the Bacillus subtilis spore vaccine containing the keratin filament binding protein of Clonorchis sinensis obtained in Example 1 was added at a rate of 1×10 8 cfu / g (low dose group), 1×10 10 cfu / g (high dose group) was mixed with grass carp feed, and after being placed at room temperature for 30 minutes, cod liver oil was used to coat the spores and feed at a ratio of 5g feed / 1mL cod liver oil to prevent the spores from spreading on the water surface. The fish were fed for 6 consecutive weeks, twice a day, with about 0.2g feed / fish each time. After 6 weeks, feed was fed without spores and cod liver oil, and 1 / 3 of the water was replaced with fresh water every day to remove impurities such as feces in the fish tank.

[0082] Serum, bile, body mucus and intestinal mucus were collected 2, 4, 6 and 8 weeks after immunization. 1 cm of hindgut tissue and liver tissue of grass carp were collected and fixed at 8 weeks and preserved in Bouin fixative. The contents of hindgut tissue of fish were collected at 8 weeks and spread on LB plates (spectinomycin, 10 μg / mL) to identify spore colonization.

[0083] ELISA was used to detect the levels of IgM and IgZ in serum, bile, body surface mucus and intestinal mucus at different time points. The test results were as follows: Figure 3 The test results show that the spore vaccine can increase the levels of fish serum, bile, body surface mucus and intestinal mucus specific IgM and body surface mucus and intestinal mucus IgZ, and induce systemic and local mucosal immunity and systemic immune response of grass carp (the experimental group refers to the high-dose group).

[0084] Two weeks after the last immunization, the cercariae (1×10 4 Tail / m 3) was added to the grass carp in the control group, low-dose group, and high-dose group, and the metacercariae were challenged and infected once a day for 7 days. After the challenge infection, the grass carp was fed with a spore-free and cod liver oil-free feed for 30 days. The back muscle and the mung bean-sized chicken of the tail were taken and the metacercariae infection was observed under a microscope by the compression method. Then the whole grass carp was digested by the protease digestion method and the metacercariae were counted. The results are shown in Table 1. The test results show that the use of 1×10 10 CFU spore vaccine mixed feed can effectively resist the attack infection of cercariae.

[0085] Table 1 Metacercariae infection in grass carp after immunization with spore vaccine

[0086]

[0087] Example 3 Feeding of a universal oral vaccine for animals and fish against Clonorchiasis sinensis in worm-carrying host rats

[0088] Feeding and sampling scheme: 24 SD rats aged 6-7 weeks were fed in the animal room for one week and randomly divided into an experimental group (gavage with Bacillus subtilis recombinant engineered bacteria containing Clonorchis sinensis keratin filament binding protein), a control group (gavage with WB600-pBE03-CotC) and a blank group (gavage with PBS). Rats were orally immunized by gavage, and each rat was gavaged with 1×10 10 cfu spores were gavaged for three consecutive days as one immunization, and immunized once every two weeks for a total of three times; serum was collected at the 2nd, 4th and 6th week; jejunum and ileum tissues were collected at the 6th week and fixed in 4% paraformaldehyde, and spleen tissues were collected to prepare spleen cell suspension.

[0089] ELISA was used to detect the levels of IgG, IgG1, and IgG2a antibodies in serum and the levels of IgA antibodies in intestinal mucus in jejunal and ileal tissues. The test results are shown in Figure 4 ELISA test results showed that the spore vaccine could significantly increase the level of specific antibodies in rat serum and local mucosal immune response.

[0090] At the sixth week of the experiment, spleen tissues of rats in each group were collected and rat spleen cell suspensions of 1×10 5 / mL, add 70μL of cells to each well of a 96-well plate, use concanavalin (purchased from Guangzhou Jiayan Biotechnology Co., Ltd., catalog number BS206) and recombinant Clonorchis sinensis keratin filament binding protein (prepared by this laboratory) to stimulate spleen cells, use cell culture medium to prepare the stimulator concentration to 50μg / mL, add 20μL of stimulator to each well to make the final concentration 10μg / mL, stimulate and culture for 48 hours, and detect the proliferation ability of spleen cells by CCK-8 method. Adjust the cell concentration to 5×10 5 / mL, add 1mL of cells to each well of a 2-well plate, use cell culture medium to prepare concanavalin and recombinant Clonorchis sinensis keratin filament binding protein at a concentration of 1mg / mL, add 10μL of stimulant to each well to make the final concentration of 10μg / mL, stimulate and culture for 48 hours, and use the microplate method to detect the ability of spleen cells to secrete cytokines IL-4 and IFN-γ. The results are as follows Figure 5 As shown in the results, oral spore vaccine can significantly increase the proliferation ability of rat spleen cells and the secretion levels of IL-4 and IFN-γ.

[0091] At the 6th week of the experiment, immunohistochemistry was used to analyze the number of IgA secreting cells in the jejunum and ileum tissues of rats in each group. Figure 6 Compared with the control group, oral administration of spore vaccine significantly increased the number of IgA cells in the jejunum and ileum of rats, indicating that oral spore vaccine can significantly enhance the immune response of intestinal mucosa, which corresponds to the antibody level detection in intestinal mucus.

[0092] In the sixth week of the experiment, each rat was gavaged with 50 metacercariae of Clonorchis sinensis; after four weeks of infection, the number of eggs in the rat feces was counted; after eight weeks of infection, the number of adult worms in the bile duct was counted. Masson staining and Sirius red staining were used to analyze the collagen deposition in liver tissue, and real-time fluorescence quantitative PCR was used to analyze the transcription levels of Acta2 and Col1a1 in liver tissue. The results are shown in Table 2. Figure 7 as well as Figure 8 The results showed that oral spore vaccine could significantly reduce the number of worm eggs in feces and the number of worms in the bile duct, with the worm and egg reduction rates being 54.69% and 52.73%, respectively, and reduce the collagen deposition in liver tissue, and the expression of Acta2 and Col1a1 was significantly downregulated.

[0093] Table 2 Calculation of worm reduction rate and egg reduction rate after infection with Clonorchis sinensis metacercariae in rats immunized with spore vaccine

[0094]

[0095]

[0096] Note: ** represents p < 0.01.

[0097] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A fusion protein, wherein the fusion protein is a fusion protein having an amino acid sequence as shown in SEQ ID NO:

5.

2. A nucleic acid molecule encoding the fusion protein of claim 1.

3. A biological material related to the nucleic acid molecule according to claim 2, characterized in that: The biological material comprises at least one of b1) to b8): b1) The nucleic acid molecule according to claim 2; b2) an expression cassette containing the nucleic acid molecule described in b1); b3) a recombinant vector containing the nucleic acid molecule described in b1); b4) a recombinant vector containing the expression cassette described in b2); b5) a recombinant cell containing the nucleic acid molecule described in b1); b6) a recombinant cell containing the expression cassette described in b2); b7) a recombinant cell containing the recombinant vector described in b3); b8) A recombinant cell containing the recombinant vector described in b4).

4. A recombinant engineered bacterium of Bacillus subtilis, characterized in that: The recombinant engineered bacteria express the fusion protein according to claim 1.

5. The method for constructing the recombinant engineered bacteria according to claim 4, comprising the following steps: (1) Obtain the target gene; (2) constructing a recombinant plasmid: linking the target gene obtained in step (1) with the vector plasmid using homologous recombination to obtain a recombinant plasmid; (3) The recombinant plasmid obtained in step (2) is transformed into Bacillus subtilis competent cells to obtain the recombinant engineered bacteria.

6. The construction method according to claim 5, characterized in that: The target gene described in (1) contains the nucleotide sequence encoding the keratin filament binding protein of Clonorchis sinensis.

7. The construction method according to claim 6, characterized in that: The encoding nucleotide sequence of the Clonorchis sinensis keratin filament binding protein is shown in SEQ ID NO:

1.

8. The construction method according to claim 6, characterized in that: The vector plasmid is pBE03-CotC plasmid.

9. Use of the fusion protein according to claim 1, the nucleic acid molecule according to claim 2, the biological material according to claim 3 or the recombinant engineered bacteria according to claim 4 in the preparation of a product for preventing Clonorchiasis sinensis.

10. The use according to claim 9, characterized in that: The products include reagents, kits and vaccines.

11. A vaccine comprising the fusion protein of claim 1, the nucleic acid molecule of claim 2, the biological material of claim 3 or the recombinant engineered bacteria of claim 4.

12. The vaccine according to claim 11, characterized in that The vaccine is an oral vaccine; and / or, the vaccine is a vaccine for animals and fish.

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