CN114196691A - Gene, protein and vaccine for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep and application - Google Patents
Gene, protein and vaccine for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep and application Download PDFInfo
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Abstract
The invention provides a gene, a protein, a vaccine and an application for preparing a multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, belonging to the technical field of preparation of animal vaccines, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1. The multi-epitope recombinant vaccine prepared by the gene provided by the invention has good immunogenicity and safety, high expression level of target protein, high antigen purity, simple preparation process, no use of urea, safety and environmental protection, improves production efficiency, and has important industrial and research values.
Description
Technical Field
The invention belongs to the technical field of animal vaccine preparation, and particularly relates to a gene, a protein, a vaccine and application for preparing a multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep.
Background
Echinococcosis, commonly known as echinococcosis (Hydatidosis), is a serious zoonosis caused by the parasite of Echinococcus Granulosus (EG) larvae in humans and various animals such as cattle and sheep. The disease is prevalent worldwide, and is more prevalent in western pasturing areas and farming and pasturing areas in China. The focus of echinococcosis is mainly concentrated in the liver and the lung, which causes great damage to human health, not only leads the patients to lose labor capacity, but also brings great economic burden to families and society of the patients. The intercarriage disease is more serious in epidemic areas, the number of livestock suffering from the intercarriage disease is more than 5000 ten thousand every year in China, the direct economic loss caused by the death of the livestock and the waste of viscera (mainly cattle and sheep) is more than 30 hundred million yuan, the serious economic loss is caused to the animal husbandry, and the echinococcosis immunity is incorporated into the mandatory immunity in China.
The prevention and treatment of echinococcosis is mainly based on immune prevention, at present, only aiming at the commercial echinococcosis vaccine of sheep, EG95 protein is taken as an immune antigen for producing the vaccine, the vaccine is expressed in insoluble inclusion bodies in thalli such as escherichia coli, yeast and the like, the vaccine can be prepared through a plurality of steps such as denaturation and renaturation, the production process is very complex, the antigen loss in the middle process is serious, a large amount of ammonia nitrogen wastewater is generated through urea denaturation, and the difficulty in environment-friendly treatment is increased.
Disclosure of Invention
In view of the above, the invention aims to provide a gene, a protein, a vaccine and an application for preparing a multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a gene for preparing a polyepitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1.
The invention also provides the protein encoded by the gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention also provides a recombinant plasmid, and the gene in the technical scheme is connected to a vector to obtain the recombinant plasmid.
Preferably, both ends of the gene contain EcoR I and Sac I enzyme cutting sites and are connected with a vector.
Preferably, the support comprises a PET-32a support.
The invention also provides a preparation method of the protein in the technical scheme, which comprises the following steps:
1) transforming the recombinant plasmid to an expression strain and then culturing to obtain a fermentation strain;
2) fermenting the fermentation strain obtained in the step 1) when the OD of the fermentation liquid is450When the value is 0.4, adding IPTG (isopropyl thiogalactoside) for induction expression, and collecting fermentation thalli;
3) crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain supernatant, and treating the supernatant with Ni2+-purifying with NTA affinity chromatography system to obtain protein.
Preferably, the final concentration of the IPTG in the fermentation liquor in the step 2) is 0.5 mM;
the conditions for inducing expression include: the speed of oscillation was 150rpm, the temperature was 25 ℃ and the time was 10 h.
The invention also provides a multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, and the components of the vaccine comprise the protein, the immunologic adjuvant and the freeze-drying protective agent in the technical scheme.
Preferably, the ratio of the mass of the protein, the mass of the immunoadjuvant and the volume of the lyoprotectant is 2500ug:25mg:0.517 ml.
The invention also provides the application of the vaccine, the protein or the recombinant plasmid in the technical scheme in the preparation of the multi-epitope recombinant vaccine for preventing and treating the echinococcosis of cattle and sheep.
The invention has the beneficial effects that:
the multi-epitope recombinant vaccine prepared by the gene provided by the invention has good immunogenicity and safety, high expression level of target protein, high antigen purity, simple preparation process, no use of urea, safety and environmental protection, improves production efficiency, and has important industrial and research values.
The protein induced by the commercial vaccine is an inclusion body, and is purified after urea denaturation treatment; the protein induced by the invention is soluble protein, has good immunogenicity, and can be directly prepared into vaccine after purification.
Drawings
FIG. 1 is a gray scale analysis of a purified protein of interest, wherein M: marker; 1.2, 3: purifying the protein; 4: analyzing the content gray level of the target protein;
FIG. 2 is a two-immune 2 week lamb antibody test;
FIG. 3 shows antibody detection in 2-week-hyperimmunized calves.
Detailed Description
The invention provides a gene for preparing a polyepitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, wherein the nucleotide sequence of the gene is shown as SEQ ID No.1, and the gene specifically comprises the following components:
SEQ ID No.1：
the invention screens B cell epitopes of echinococcus EG95 full-length gene (Genebank: AY421719.1) and AgB8/1 full-length gene (Genebank: AF143813.1) by bioinformatics software, and connects 4 screened fragments with strong antigenicity (the thickened part is the 4 screened fragments with strong antigenicity) in series by using a flexible linker to obtain the gene for preparing the polyepitope recombinant vaccine for preventing and treating the bovine and sheep echinococcosis.
The invention also provides a protein encoded by the gene in the technical scheme, wherein the amino acid sequence of the protein is shown as SEQ ID No.2, and specifically comprises the following steps:
SEQ ID No.2：
KGMGIETRTTETPLRKHFNLTPVGSGGGGSGGGGSGGGGSVNPSDPLVYKRQTAKFSDEAAAKEAAAKEAAAKIETPRAGKKESTVMTSGSAGGGGSGGGGSGGGGSDLLKELEEVFQLLRKKLRMA。
the invention also provides a recombinant plasmid, and the gene in the technical scheme is connected to a vector to obtain the recombinant plasmid. In the present invention, the gene preferably contains EcoR I and Sac I cleavage sites, and is ligated into a vector. In the invention, the carrier preferably comprises a PET-32a carrier, the source of the PET-32a carrier is not particularly limited, and the PET-32a carrier can be prepared by a conventional preparation method or a commercial product. The conditions for enzyme digestion and the connection method are not particularly limited, and the ordinary operation can be adopted by the technical personnel in the field.
The invention also provides a preparation method of the protein in the technical scheme, which comprises the following steps:
1) transforming the recombinant plasmid to an expression strain and then culturing to obtain a fermentation strain;
2) fermenting the fermentation strain obtained in the step 1) when the OD of the fermentation liquid is450When the value is 0.4, adding IPTG (isopropyl thiogalactoside) for induction expression, and collecting fermentation thalli;
3) crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain supernatant, and treating the supernatant with Ni2+-purifying with NTA affinity chromatography system to obtain protein.
The recombinant plasmid is transformed into an expression strain and then cultured to obtain a fermentation strain. In the invention, the expression strain is preferably a Rossarta (DE3) expression strain, has chemically transformed competent cells prepared on the basis of a common Escherichia coli BL21 strain, supplements tRNA corresponding to 6 rare codons (AUA, AGG, AGA, CUA, CCC, GGA) lacking in Escherichia coli, and provides the expression level of the exogenous gene in a prokaryotic system. In the present invention, the medium used for the culture preferably includes an LB liquid medium. In the present invention, the conditions for the culture preferably include: the temperature of the culture was 37 ℃ and the time of the culture was 16 hours. The method for transforming the recombinant plasmid into the expression strain is not particularly limited, and those skilled in the art can perform routine operations.
The invention ferments the obtained fermentation strain, when OD of the fermentation liquid is450When the value is 0.4, IPTG is added for induction expression, and the fermentation thalli are collected. In the present invention, the inoculation amount of the fermentation strain is preferably 1%. In the present invention, the conditions of the fermentation preferably include: the speed of shaking was 150rpm and the temperature was 37 ℃. In the present invention, the final concentration of the IPTG in the fermentation broth is preferably 0.5 mM; the conditions for inducing expression preferably include: the speed of oscillation was 150rpm, the temperature was 25 ℃ and the time was 10 h.
The invention crushes and centrifuges the obtained fermentation thalli to obtain supernatant fluid, and the supernatant fluid is processed by Ni2+-purifying with NTA affinity chromatography system to obtain protein. In the invention, the fermentation thalli is preferably washed by Buffer A, then resuspended by Buffer A according to the mass volume ratio of 1g to 10ml, and then ultrasonically crushed. In the present invention, the conditions for the ultrasonication preferably include: the power is 300W, the work time is 3s, the pause time is 3s, and the ultrasound is 120 min. In the present invention, the conditions of the centrifugation preferably include: the centrifugation temperature was 4 ℃, the centrifugation force was 12000g, and the centrifugation time was 20 min.
In the present invention, the Ni2+The conditions for purification by the NTA affinity chromatography system preferably comprise: after balancing the nickel column by using a balance Buffer solution Buffer A with the volume 5 times that of the nickel column, circularly loading the supernatant to ensure that the total loading volume is 3 times of the volume of the protein sample; after the sample loading is finished, eluting the hybrid protein by using 10 times of nickel column volume Buffer B, and finally, eluting by using eluent BufferAnd C, eluting the target protein. In the invention, the formula of Buffer A is as follows: taking 29.22g of NaCl and 20ml of 1M Tris-HCL with pH8.0, and adding water to fix the volume to 1000 ml. The formula of Buffer B is as follows: taking 29.22g of NaCl, 20ml of 1M Tris-HCL with pH8.0 and 1.7g of imidazole, and adding water to a constant volume of 1000 ml. The formula of Buffer C is as follows: taking 29.22g of NaCl, 20ml of 1M Tris-HCL with pH8.0, and 17g of imidazole, and adding water to fix the volume to 1000 ml.
The invention also provides a multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, and the components of the vaccine comprise the protein, the immunologic adjuvant and the freeze-drying protective agent in the technical scheme.
In the invention, the ratio of the mass of the protein, the mass of the immunoadjuvant and the volume of the lyoprotectant is preferably 2500ug:25mg:0.517 ml. In the present invention, the immunological adjuvant preferably comprises the immunological adjuvant Quil A (saponin Sigma aldrich-S7900). In the present invention, the lyoprotectant preferably includes a sucrose solution and a mannitol solution, wherein the sucrose solution preferably has a mass percentage of 15%, the mannitol solution preferably has a mass percentage of 6.25%, and the volume ratio of the sucrose solution to the mannitol solution is 0.417: 0.1. The preparation method of the vaccine is not particularly limited, and the vaccine is obtained by mixing the components, freezing the mixture for 24 hours at the temperature of minus 80 ℃ and freeze-drying the mixture by a freeze dryer.
In the present invention, the method of use of the vaccine preferably comprises: when the animal is cattle, the dosage is 500 ug/head; when the animal is sheep, the dosage is 100 ug/head.
The invention also provides the application of the gene, the protein or the recombinant plasmid in the technical scheme in the preparation of the multi-epitope recombinant vaccine for preventing and treating the echinococcosis of cattle and sheep. The kind and preparation method of the vaccine are not particularly limited in the present invention, and those skilled in the art can select the vaccine according to the routine practice.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of multi-epitope recombinant plasmid, protein purification and vaccine preparation
1. Construction of a Multi-epitope recombinant plasmid
The method comprises the steps of screening the B cell epitope of echinococcus EG95 full-length gene (Genebank: AY421719.1) and AgB1 full-length gene (Genebank: AF143813.1) by bioinformatics software, connecting 4 screened fragments with strong antigenicity in series by using a flexible linker, connecting enzyme cutting sites at two ends of the fragments in series by using EcoR I and Sac I, connecting a carrier by using PET-32a, and sending the fragments to a biological company for artificially synthesizing multi-epitope recombinant plasmids (the DNA sequence and the amino acid sequence after the fragments are connected in series by using the flexible linker are respectively SEQ ID No.1 and SEQ ID No. 2).
2. Recombinant protein purification
Transforming the recombinant plasmid into a Rosseta (DE3) expression strain, culturing for 16h at 37 ℃ in an incubator, selecting a single colony to 10ml LB liquid culture medium containing Amp, and performing shaking culture at 150r/min at 37 ℃ to obtain a fermentation strain; inoculating the fermentation strain into 1L LB liquid culture medium according to the proportion of 1%, and performing shaking culture at 150 r/min; bacteria solution OD450When the value reaches 0.4, IPTG with the final concentration of 0.5mM is added for induction expression, shaking culture is carried out at 150r/min, the induction temperature is 25 ℃, and induction is carried out for 10 hours. Washing the fermentation bacteria once with Buffer A, adding 10 times volume of Buffer A to the precipitate according to the weight-volume ratio, and then carrying out ultrasonic crushing; the ultrasonic power is 300W, the work time is 3s, the intermittence time is 3s, and the time is 120 min. Centrifuging at 4 deg.C for 20min at 12000g after ultrasonication to obtain supernatant, and treating with Ni2+-NTA affinity chromatography system purification. After balancing the nickel column by using a balance Buffer solution Buffer A with the volume 5 times that of the nickel column, circularly loading the supernatant to ensure that the total loading volume is 3 times of the volume of the protein sample; after the sample loading is finished, eluting the hybrid protein by using Buffer B with 10 times of the volume of the nickel column, and finally eluting the target protein by using eluent Buffer C. (the formula of Buffer A is that NaCl 29.22g, 1M pH8.0 Tris-HCl 20ml are taken, and water is added to fix the volume to 1000 ml; the formula of Buffer B is that NaCl 29.22g, 1M pH8.0 Tris-HCl 20ml, imidazole 1.7g are taken, and water is added to fix the volume to 1000 ml; the formula of Buffer C is that NaCl 29.22g, 1M pH8.0 Tris-HCl 20ml, imidazole 17g are taken, and water is added to fix the volume to 1000 ml).
The purified recombinant protein was assayed for total protein content by the Bradford method. Based on the total protein content determined, each sample was diluted to 1mg/mL, mixed with 5 × Loading Buffer at a ratio of 4:1 and boiled for 5 minutes. 10 μ L of the prepared sample was subjected to SDS-PAGE gel electrophoresis, each sample was set to 3 replicates, and the results were subjected to grayscale analysis by Bandscan software and the content of the target protein was determined. As a result: the concentration of the purified recombinant protein was 2.1mg/ml, and the content of the target protein was 80.09% (as shown in FIG. 1).
3. Vaccine preparation
The purified recombinant protein, an immunologic adjuvant Quil A and a freeze-drying protective agent (15% of cane sugar and 6.25% of mannitol) are taken to prepare the vaccine according to the component requirements of table 1 (the immunologic dose of cattle is 5 times of that of sheep, so that the immunologic dose of sheep is 100 mu g/head part and 25 head parts/bottle, the immunologic dose of cattle is 500 mu g/head part and 5 head parts/bottle), each bottle is subpackaged with 2.5mL, the freezing is carried out for 24 hours at minus 80 ℃, and then the freeze-drying vaccine is prepared by a freeze-drying machine.
TABLE 1 vaccine formulation
| Purification of proteins | Quil A | 15% sucrose | 6.25% mannitol | |
| Vaccine | 2500μg | 25mg | 0.417mL | 0.1mL |
The result of the embodiment 1 shows that the multi-epitope recombinant plasmid is successfully constructed, the expression quantity of the induced protein is good, the purity of the purified protein is high, and the freeze-dried vaccine is prepared by using the multi-epitope recombinant plasmid.
Example 2
Immunogenicity test of echinococcosis (echinococcosis) disease multi-epitope recombinant vaccine for cattle and sheep
1. Immunogenicity testing of Multi-epitope recombinant vaccines (sheep)
Labeling ear labels of 10 healthy lambs of 2-4 months of age which are negative in echinococcosis serum antibody detection result, randomly dividing the lambs into 2 groups, injecting 1 part (1ml) of prepared vaccine (prepared in example 1) subcutaneously into the neck of each sheep of an immunization group, and injecting diluent with the same volume into a control group; boosting once 28 days after the first immunization in the same manner with the same dose; serum was collected for the second immunization for 14 days and antibody detection was performed.
As a result: as shown in Table 2, the control group showed negative results in all the sheep sera (antibody OD mean 0.197). The immunized group of sheep all showed antibody reaction, the serum detection results were all positive (antibody OD value >0.3 is positive), the average antibody OD value of the immunized group was 1.694, and the differences were significant compared with the control group, as shown in FIG. 2.
TABLE 2 antibody test results (lamb)
2. Immunogenicity test of Multi-epitope recombinant vaccines (cattle)
Labeling 10 healthy calves of 3-6 months old who are negative in echinococcosis serum antibody detection result, randomly dividing the calves into 2 groups, injecting 1 part (2ml) of prepared vaccine (prepared in example 1) subcutaneously into the neck of each sheep of an immunization group, and injecting diluent with the same volume into a control group; boosting once 28 days after the first immunization in the same manner with the same dose; serum was collected for the second immunization for 14 days and antibody detection was performed.
As a result: as shown in Table 3, the bovine serum samples of the control group were all negative (antibody OD mean 0.188). All the cattle in the immune group showed antibody reaction, the serum detection results were positive (antibody OD value >0.3 is positive), the average antibody OD value in the immune group was 1.245, and the differences were significant compared with the control group, as shown in FIG. 3.
TABLE 3 results of antibody detection (Calf)
The results of example 2 show that the prepared freeze-dried vaccine has good immunogenicity, and cattle and sheep after immunization generate higher antibody levels, so that the freeze-dried vaccine can provide a good protection effect against echinococcosis.
Example 3
Safety test of polyepitope recombinant vaccine (prepared in example 1) for echinococcosis (hydatid) disease in cattle and sheep
1. Safety test of polyepitope recombinant vaccine (sheep)
After inoculation, the animals are observed for 7 days, so the observation indexes of the experimental sheep comprise local and systemic reactions such as body temperature, spirit, appetite and the like, and the results are shown in a table 4. As can be seen from table 4: therefore, no abnormality is found in the spirit, body temperature, appetite and the like of the tested sheep. Abnormal reaction caused by vaccination is not seen in the vaccinated lambs; the vaccine is inoculated to the pregnant sow to ensure normal pregnancy without abortion. The multi-epitope recombinant vaccine is proved to be safe to sheep immunity.
TABLE 4 sheep vaccine safety test results
2. Safety test of polyepitope recombinant vaccine (ox)
After inoculation, the animals were observed for 7 days, so the observation indexes of the test sheep include local and systemic reactions such as body temperature, spirit and appetite, and the results are shown in Table 5. As can be seen from table 5: therefore, no abnormality is found in the spirit, body temperature, appetite and the like of the tested cattle. Abnormal reaction caused by vaccination is not seen in the vaccinated calves; the vaccine inoculated pregnant cow is normal in pregnancy and no abortion occurs. The multi-epitope recombinant vaccine is proved to be safe for the immunity of cattle.
TABLE 5 bovine vaccine safety test results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Chongqing Aolong biological products Co., Ltd
<120> gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep
<141> 2021-12-28
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Leu Glu Glu Val Phe Gln Leu Leu Arg Lys Lys Leu Arg Met Ala
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Claims (10)
1. A gene for preparing a polyepitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep is characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 1.
2. The protein encoded by the gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID No. 2.
3. A recombinant plasmid obtained by ligating the gene of claim 1 to a vector.
4. The recombinant plasmid of claim 3, wherein the gene contains EcoR I and Sac I cleavage sites at both ends and is linked to a vector.
5. The recombinant plasmid of claim 3 or 4, wherein the vector comprises a PET-32a vector.
6. A method for producing the protein of claim 2, comprising the steps of:
1) transforming the recombinant plasmid according to any one of claims 3 to 5 into an expression strain, and culturing to obtain a fermentation strain;
2) fermenting the fermentation strain obtained in the step 1) when the OD of the fermentation liquid is450When the value is 0.4, adding IPTG (isopropyl thiogalactoside) for induction expression, and collecting fermentation thalli;
3) crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain supernatant, and treating the supernatant with Ni2+-purifying with NTA affinity chromatography system to obtain protein.
7. The method of claim 6, wherein the final concentration of IPTG in step 2) is 0.5mM in the fermentation broth;
the conditions for inducing expression include: the speed of oscillation was 150rpm, the temperature was 25 ℃ and the time was 10 h.
8. A polyepitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, wherein the components of the vaccine comprise the protein of claim 2, an immunoadjuvant and a lyoprotectant.
9. The vaccine according to claim 8, wherein the ratio of the mass of the protein, the mass of the immunoadjuvant and the volume of the lyoprotectant is 2500ug:25mg:0.517 ml.
10. Use of the gene of claim 1, the protein of claim 2 or the recombinant plasmid of claim 3 in the preparation of a polyepitopic recombinant vaccine for the prevention and treatment of echinococcosis in cattle and sheep.
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