Rabbit hemorrhagic disease virus VP60 recombinant antigen with double-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof
阅读说明：本技术 双位点嵌合巴氏杆菌PlpE抗原表位的兔出血症病毒VP60重组抗原及其制备和应用 (Rabbit hemorrhagic disease virus VP60 recombinant antigen with double-site chimeric Pasteurella pasteurella PlpE epitope and preparation and application thereof ) 是由 朱伟峰 王芳 范志宇 胡波 魏后军 于 2021-05-20 设计创作，主要内容包括：本发明属生物医学技术领域,提供了一种双位点嵌合了巴氏杆菌PlpE抗原表位的兔出血症病毒VP60重组抗原,所述重组抗原通过在分析巴氏杆菌保护性抗原PlpE表位组成的基础上,将PlpE表位集中分布的一段区域(长度合计70个氨基酸)分两段嵌合展示到兔出血症病毒VP60蛋白N端和C端而获得。免疫保护力实验表明该嵌合重组抗原对兔出血症病毒和巴氏杆菌均具有良好的免疫保护力。本发明仅耗费一份人工就可以制备出针对两种病原的抗原,简化了工艺,缩短了制备时间,显著降低了生产成本,为后期生产更低成本的兔病毒性出血症、兔巴氏杆菌病疫苗提供了基础。(The invention belongs to the technical field of biomedicine, and provides a rabbit hemorrhagic disease virus VP60 recombinant antigen with pasteurella PlpE epitope embedded at double sites, which is obtained by embedding and displaying a section of region (total length of 70 amino acids) with the PlpE epitope intensively distributed to the N end and the C end of rabbit hemorrhagic disease virus VP60 protein in two sections on the basis of analyzing the composition of pasteurella protective antigen PlpE epitope. Immune protection experiment shows that the chimeric recombinant antigen has good immune protection to rabbit hemorrhagic disease virus and pasteurella. The invention can prepare the antigens aiming at two pathogens only by consuming one part of manpower, simplifies the process, shortens the preparation time, obviously reduces the production cost and provides a foundation for later-stage production of lower-cost rabbit viral hemorrhagic disease and rabbit pasteurellosis vaccines.)
1. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded at the double sites is characterized in that the recombinant antigen comprises rabbit hemorrhagic disease virus VP60 protein, and the N end and the C end of the recombinant antigen are respectively connected with a PlpE up polypeptide fragment and a PlpE down polypeptide fragment; wherein the PlpE up polypeptide fragment and the PlpE down polypeptide fragment are derived from Pasteurella PlpE protein epitope.
2. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the double-site chimeric Pasteurella pasteurella PlpE epitope as claimed in claim 1, wherein the amino acid sequences of the PlpE epitope embedded at the N-terminal and C-terminal of the VP60 protein are SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
3. The recombinant antigen of rabbit hemorrhagic disease virus VP60 chimeric with Pasteurella pasteurella PlpE epitope at two sites of claim 1, wherein the nucleotide sequences of the PlpE epitope embedded at the N-terminal and the C-terminal of the VP60 protein are SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
4. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the Pasteurella multocida PlpE epitope embedded in the double sites of claim 1, wherein the amino acid sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the Pasteurella multocida PlpE epitope embedded in the double sites is SEQ ID NO: 5.
5. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the double-site chimeric Pasteurella multocida PlpE epitope as claimed in claim 1, wherein the nucleotide sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen encoding the double-site chimeric Pasteurella multocida PlpE epitope is SEQ ID NO 6.
6. An expression vector for recombinant antigen, which is used for expressing rabbit hemorrhagic disease virus VP60 recombinant antigen with a double-site chimeric Pasteurella PlpE epitope and comprises the nucleotide sequence of claim 5.
7. A method for preparing the rabbit hemorrhagic disease virus VP60 recombinant antigen chimeric with Pasteurella pasteurella PlpE epitope at two sites as described in claim 1, which comprises the following steps:
(1) amplifying DNA sequences corresponding to 26 th to 50 th amino acids and 51 st to 95 th amino acids of Pasteurella PlpE by PCR;
(2) sequentially and respectively connecting the PCR products obtained in the step (1) to the 5 'end and the 3' end of the VP60 gene, and connecting the recombinant gene to a pFastBac1 cloning site to obtain a recombinant shuttle plasmid vector;
(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac host bacteria, and obtaining baculovirus plasmids through the transposition of the bacteria;
(4) after the recombinant baculovirus plasmid is transferred into sf9 cells, rabbit hemorrhagic disease virus VP60 recombinant protein antigen embedded with Pasteurella PlpE epitope is obtained.
8. The use of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded in the double sites of claim 1 in the preparation of vaccines for preventing rabbit viral hemorrhagic disease and rabbit pasteurellosis.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a rabbit hemorrhagic disease virus VP60 recombinant antigen embedded with pasteurella epitope, and a preparation method and application thereof.
Background
Rabbit hemorrhagic disease, commonly known as Rabbit plague, is a highly contagious, high-morbidity and highly lethal disease caused by Rabbit Hemorrhagic Disease Virus (RHDV), and mainly occurs in adult rabbits with the age of more than 2 months. The RHDV capsid protein VP60 consists of 579 amino acids, which is the most basic unit of viral capsid composition. The capsid protein VP60 is an immunoprotective antigen of the virus, is directly related to the induction of anti-infection immunity of the organism, and independently expresses the VP60 protein to be capable of self-polymerizing into virus-like particles, so that the vaccine prepared from the virus-like particles can be used for preventing rabbit viral hemorrhagic disease.
Pasteurellosis (Pasteurellosis) in rabbits is mainly caused by pasteurella multocida ((R))Pasteurella multocida) Causing a major bacterial blight which is harmful to rabbit industry. Rabbits of various ages and varieties are susceptible to pasteurella multocida, and rabbits of 2-6 months are particularly susceptible to the disease, which is one of the main epidemic diseases causing death of rabbits of 9 weeks to 6 months. At present, protective antigens such as PlpE and the like are reported, and animal experiments show good immunoprotection efficacy.
At present, the bivalent inactivated vaccine for the rabbit viral hemorrhagic disease and the rabbit pasteurellosis is used for simultaneously preventing and controlling the rabbit viral hemorrhagic disease and the rabbit pasteurellosis. However, the vaccine has the problems of high production cost and the like, and the immune protection effect on the pasteurellosis is not ideal. The preparation process of the traditional bivalent inactivated vaccine for the rabbit hemorrhagic disease and the rabbit pasteurellosis comprises the steps of preparing the rabbit hemorrhagic disease and the rabbit pasteurellosis inactivated vaccine respectively, and then carrying out physical mixing to prepare the combined vaccine. The production process of the bivalent vaccine needs to go through the production process of two vaccines, two workers, materials, water and electricity and the like are consumed, and the production cost is increased. Although there is a trend of using genetic engineering subunit vaccines (using VP60 and PlpE as antigens) to replace traditional bacterial or viral inactivated vaccines in recent years, the current production process of genetic engineering bivalent subunit vaccines still needs to go through the process of preparing single vaccine separately and then mixing physically, and the production cost is not reduced significantly. Therefore, the scheme of forming a new antigen by chimeric connection of different antigens and epitopes thereof becomes a new combined vaccine development direction, which simplifies the production process of the combined vaccine and effectively reduces the production cost.
The invention aims to display the epitope of the pasteurella protective antigen PlpE on rabbit hemorrhagic disease virus VP60 in a chimeric way, thereby obtaining a new antigen required by the rabbit viral hemorrhagic disease and rabbit pasteurella disease bigeminal subunit vaccine.
Disclosure of Invention
The technical problem solved by the invention is as follows: the recombinant protein antigen VP60 of rabbit hemorrhagic disease virus embedded with the pasteurella protective antigen PlpE epitope is constructed to obtain the recombinant protein antigen which can produce the rabbit hemorrhagic disease virus and pasteurella with immunoprotection only by consuming one part of manual work, thereby being applied to the preparation of the bigeminal vaccine of rabbit viral hemorrhagic disease and rabbit pasteurella disease
In order to solve the technical problems, the invention provides a rabbit hemorrhagic disease virus VP60 recombinant antigen with a pasteurella PlpE epitope embedded at a double site, wherein the recombinant antigen comprises rabbit hemorrhagic disease virus VP60 protein, and the N end and the C end of the recombinant antigen are respectively connected with a PlpE up polypeptide fragment and a PlpE down polypeptide fragment; wherein the PlpE up polypeptide fragment and the PlpE down polypeptide fragment are derived from Pasteurella PlpE protein epitope.
The amino acid sequences of the PlpE epitope embedded at the N end and the C end of the VP60 protein are SEQ ID NO. 1 and SEQ ID NO. 2 respectively.
The nucleotide sequences of the PlpE epitope embedded at the N end and the C end of the VP60 protein are SEQ ID NO. 3 and SEQ ID NO. 4 respectively.
The amino acid sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded at the double sites is SEQ ID NO. 5; the nucleotide sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen for coding the double-site chimeric Pasteurella pneumophila PlpE epitope is SEQ ID NO. 6.
In addition, an expression vector for a recombinant antigen for expressing a rabbit hemorrhagic disease virus VP60 recombinant antigen having an epitope of Pasteurella pasteurella PlpE antigen chimeric at two sites, comprising the nucleotide sequence of claim 7 is provided.
The invention relates to a preparation method of a rabbit hemorrhagic disease virus VP60 recombinant antigen with a Pasteurella pasteurella PlpE epitope embedded at double sites, which comprises the following steps:
(1) amplifying DNA sequences corresponding to 26 th to 50 th amino acids and 51 st to 95 th amino acids of Pasteurella PlpE by PCR;
(2) sequentially and respectively connecting the PCR products obtained in the step (1) to the 5 'end and the 3' end of the VP60 gene, and connecting the recombinant gene to a pFastBac1 cloning site to obtain a recombinant shuttle plasmid vector;
(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac host bacteria, and obtaining baculovirus plasmids through the transposition of the bacteria;
(4) after the recombinant baculovirus plasmid is transferred into sf9 cells, rabbit hemorrhagic disease virus VP60 recombinant protein antigen embedded with Pasteurella PlpE epitope is obtained.
The rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded in the double sites is applied to the preparation of vaccines for preventing rabbit viral hemorrhagic disease and rabbit pasteurellosis.
The invention has the beneficial effects that: compared with the prior art, although the rabbit viral hemorrhagic disease and the rabbit pasteurella combined inactivated vaccine or the genetic engineering subunit vaccine are adopted to simultaneously prevent and control the rabbit viral hemorrhagic disease and the rabbit pasteurella disease in the prior art, the traditional preparation process of the combined vaccine is to prepare two vaccines respectively and then physically mix the two vaccines, the production process of the combined vaccine needs to go through the production process of the two vaccines, two manual work, materials, water and electricity and the like are consumed, the production cost is increased, and the risk of mutual interference of different components is brought. Although the rabbit hemorrhagic disease virus structural protein VP60 theoretically has the capability of displaying foreign epitopes at the N-terminal and the C-terminal, great difficulty is faced in displaying foreign epitopes (particularly bacterial epitopes with relatively far relativity) as much as possible and ensuring that the self-epitopes are not influenced (the VP60 protein can only display foreign animal virus epitopes of about 40 amino acids at the longest at the N-terminal and the C-terminal under the current technical conditions). On the basis of analyzing the composition of the Pasteurella multocida protective antigen PlpE epitope, a section of region (total length of 70 amino acids) in which the PlpE epitope is intensively distributed is displayed at the N end and the C end of the rabbit hemorrhagic disease virus VP60 protein in a two-section chimeric way, and the recombinant antigen is unexpectedly found to still have hemagglutination activity (namely, virus-like particles can be formed, and the neutralizing epitope of the rabbit hemorrhagic disease virus is not influenced). The immune protective force experiment result shows that the recombinant antigen has immune protective effect on pasteurella and rabbit hemorrhagic disease virus.
In a word, the invention obtains the recombinant antigen with immunoprotection effect on rabbit hemorrhagic disease virus and pasteurellosis by constructing the rabbit hemorrhagic disease virus Vp60 recombinant protein embedded with the pasteurellosis protective antigen PlpE epitope, and provides a foundation for later-stage production of lower-cost rabbit viral hemorrhagic disease and rabbit pasteurellosis vaccines.
Drawings
FIG. 1 shows the prediction of the ability of the amino acid residues of PlpE to form B cell epitopes (using the online software Bepipred-2.0 (http:// tools. immuneepitope. org/bcell /), the prediction score is higher than 0.5 and the probability of forming B cell epitopes is higher);
FIG. 2 is a schematic diagram of the construction process of a recombinant shuttle plasmid vector of PlpE epitope double-site chimeric VP60 recombinant antigen;
FIG. 3 shows the expression of the PlpE epitope chimeric VP60 recombinant protein VP60-PlpE, wherein M represents a molecular size marker; VP60-PlpE is a VP60-PlpE recombinant protein (68 kDa) expressed by a baculovirus-insect cell expression system; VP60 represents the rabbit hemorrhagic disease virus VP60 protein (60 kDa) expressed by the baculovirus-insect cell expression system;
FIG. 4 shows the hemagglutination properties of VP60-PlpE after the PlpE epitope is embedded in VP60 recombinant protein, and the left picture shows the agglutination of VP60-PlpE to human type O red blood cells; right panel is control, i.e. agglutination of human O-type erythrocytes by wild-type baculovirus cultures;
FIG. 5 is a diagram showing the reaction of PlpE epitope chimeric VP60 recombinant protein VP60-PlpE with PlpE hyperimmune serum, wherein M represents molecular size markers; VP60-PlpE is VP60-PlpE recombinant protein expressed by baculovirus-insect cell expression system; VP60 represents rabbit hemorrhagic disease virus VP60 protein expressed by baculovirus-insect cell expression system;
FIG. 6 shows the specific antibody production (A) and the survival of the mouse after challenge with Pasteurella in the immunoprotection test (B)
FIG. 7 survival of rabbits after challenge with rabbit hemorrhagic disease virus (A) and Pasteurella virus (B) in immunoprotection experiments.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1: preparation of PlpE epitope chimeric VP60 recombinant protein
(1) Plasmids, strains and culture vectors, namely plasmids pFastBac1 and pFastBac1-VP60 are constructed and stored in the laboratory;E．coli DH5 α was purchased from tokyo kezam biotechnology limited;E．coliDH10Bac is obtained from Shanghai Weidi Biotechnology GmbH, and Pasteurella multocida C51-17 is provided by laboratory preservation; sf9 insect cells were maintained and cultured in this laboratory.
Protein amino acid sequence analysis was performed using the sequence of the PlpE gene in the whole genome sequence of Pasteurella multocida strain C51-17 published by Genbank as a reference sequence. B cell epitope distribution of PlpE was determined using Bepipred-2.0 (http:// tools. immuneepitope. org/bcell /).
The B cell epitope prediction of PlpE is shown in FIG. 1, where the primary structure of PlpE comprises a total of 336 amino acid residues. The default 0.5 of the Bepipred-2.0 software was chosen as the threshold for the epitope composed of amino acid residues, and the prediction showed that the epitope for PlpE is mainly distributed at the N-terminus, especially the amino acid residue sequence from position 26 to 105. Therefore, the invention selects the N-end 26-95 amino acid to be connected to the N-end and the C-end of the rabbit hemorrhagic disease virus VP60 in a segmented manner to obtain the epitope chimeric VP60, namely the recombinant protein antigen prepared by the rabbit viral hemorrhagic disease and rabbit pasteurellosis vaccine has the amino acid sequence of SEQ ID NO. 5.
(3) Construction of recombinant bacmid the sequence of the PlpE gene of Pasteurella multocida strain C51-17 was used as a reference sequence, and the primer Plpeupf was designed: 5'-aggcatgcggtaccaagcttatgTGTAGCGGTGGTGGCGGT-3' and Plpeupr: 5'-gggctttgccctcATTTGATTGAACCGGTTGTGCT-3' is used to amplify a DNA fragment corresponding to amino acids 26 to 50 of PlpE, Plpedown F: 5'-ggcttttcttatgtcACTCCAATCAAACATCCTATGACTAATAG-3' and PlpE down R: 5'-atcctctagtacttctcgacctaTTTTTCTTGTTCTAGAGGGGCTTG-3' A DNA fragment corresponding to amino acids 51 to 95 of PlpE was amplified. The Pasteurella C51-17 strain genome is extracted and PCR amplification is carried out on the PlpE gene fragment of interest. The VP60 gene fragment was prepared by amplifying pFastBac1-VP60 plasmid vector using primers VP60F 5'-atcaaatGAGGGCAAAGCCCGCACA-3' and VP60R 5 'ggagtGACATAAGAAAAGCCATTGGTTGTG 3'. The pFastBacF 1 plasmid vector was PCR amplified using primers pFastBacF5 '-GTCGAGAAGTACTAGAGGATCATAATCAGCC-3' and pFastBacR5 '-AAGCTTGGTACCGCATGCC-3' to prepare a linearized pFastBac1 linearized vector. Gene fragments corresponding to the 26 th-50 th amino acid and the 51 th-95 th amino acid of PlpE are respectively connected to the 5 'end and the 3' end of the VP60 gene according to the operation in the instruction of a homologous recombinant cloning kit (Novozam, Nanjing), and then the positive recombinant plasmid is transferred into DH5 alpha host bacteria. And selecting positive clone PCR to identify correctly, sending the positive clone PCR to Nanjing subsection sequencing of Beijing Optimalaceae New industry Limited company to identify whether the cloned gene is complete and correctly connected to the carrier, and displaying the result of sequencing that the target gene fragment is correctly connected to the carrier. Extracting positive cloning plasmid according toE． coliProcedures in the DH10Bac competent cell Specification, transformation of recombinant plasmids intoE． coliAnd (3) obtaining positive clones in DH10Bac competent cells through blue-white screening and PCR identification, and extracting recombinant Bacmid-VP 60-PlpE. The recombinant bacmid constructed is shown in FIG. 2.
Transfection, expression and identification Bacmid-VP60-PlpE was passed through Liposomal LipofectaminTM3000 Sf9 insect cells transfected to logarithmic growth phase. After transfection, the cells were observed 1 time every 12 h, and when cytopathic effect was evident, the cells and supernatant were collected as a stock solution of recombinant baculovirus and stored at 4 ℃. And (3) repeatedly freezing and thawing the 1 st generation virus solution for 3 times, then inoculating Sf9 cells with the volume ratio of 1%, and carrying out passage to obtain the 2 nd generation recombinant virus. After 3 generations of virus obtained by repeating the above procedure, the recombinant baculovirus was inoculated into the suspension sf9 cells in logarithmic phase for spinner culture, and the culture was collected after about 100 hours of culture. The recombinant VP60-PlpE protein was obtained after 3 repeated freeze-thawing of the culture, and observed after SDS-PAGE and Coomassie blue staining, a band of about 68kDa was present in the sample (FIG. 3), corresponding to the theoretical predicted size of VP 60-PlpE.
Example 2 hemagglutination assay of two-site chimeric VP60-PlpE recombinant proteins
VP60-PlpE and wild type baculovirus culture were added to 50. mu.L of each well in two wells of a 50-well U-plate. Then 50. mu.L of 1% human O-type erythrocyte suspension is added into each hole, and after 30 min of action at 4 ℃, the hemagglutination condition is observed. The result shows that the VP60-PlpE recombinant protein has obvious hemagglutination (figure 4), namely, the VP60-PlpE recombinant protein is considered to still form rabbit hemorrhagic disease virus-like particles, and effectively displays the rabbit hemorrhagic disease virus neutralizing epitope.
EXAMPLE 3 reaction of the two-site chimeric VP60-PlpE recombinant protein with PlpE hyperimmune serum
The VP60-PlpE recombinant protein and VP60 recombinant protein (control) were subjected to SDS-PAGE gel electrophoresis, and then the proteins were transferred onto NC membranes. After being sealed by 5 percent skim milk, 1/500 diluted mouse anti-PlpE serum preserved in the laboratory is added, and the mixture is incubated for 1 h at 37 ℃; PBST was rinsed 5 times, 1/10000 diluted HRP-conjugated goat anti-mouse IgG was added, and incubation was performed at 37 ℃ for 1 h; PBST was rinsed 5 times and ECL color development was performed. Western blot results showed that there was a band of about 68kDa in lane VP60-PlpE (FIG. 5), but there was no band of expected size in lane VP60 as a control, indicating that the recombinant protein VP60-PlpE was able to specifically react with mouse anti-PlpE serum, i.e., the PlpE partial immunogenic fragment was successfully integrated into VP 60.
Example 4 immunoprotection assay
(1) Mouse immunoprotection experiments 20 4-6 week old female ICRs were randomly divided into 2 groups, 1 control, and another 10 mice. Experimental groups immunization of VP60-PlpE recombinant protein: after 20% volume of alumina gel was added to 200. mu.g/. mu.l of recombinant protein, the immunized mice were injected intraperitoneally at 100. mu.l each. The booster immunization after 21 days, the dose, the mode and the like are the same as those of the primary immunization. Negative control group: PBS was used instead of recombinant protein, and the adjuvant used was the same as that of the experimental group. After blood was collected from tail veins of all mice 10 days after the booster immunization, it was confirmed that high levels of specific antibodies had been produced (FIG. 6A), and then the mice were challenged subcutaneously with Pasteurella C51-17 strain at about 50CFU each (about 5 × LD 50). The survival of the mice after challenge is shown in fig. 6B, only 2 mice survived in the control group, and 8 mice survived in the experimental group. This indicates that VP60-PlpE confers immunoprotection against challenge with Pasteurella.
(2) In the rabbit immunoprotection experiment, 60 healthy and susceptible rabbits with the age of 2-3 months are selected and randomly divided into 6 groups, 2 groups of controls and 4 groups of experiments, wherein each group comprises 10 rabbits. Two experimental groups immunized against the VP60-PlpE recombinant protein group: adding 20% volume of alumina gel adjuvant into 200mg/ml recombinant protein, and injecting immunized rabbit subcutaneously, each 1 ml; two further experimental groups immunized rabbits with a physical Mixture of VP60 and PlpE recombinant protein (mix) comprising 100mg/ml each of VP60 recombinant protein and PlpE recombinant protein, after addition of 20% by volume of an alumina gel adjuvant. The remaining two groups served as negative controls: the recombinant protein was replaced with the immunophosphate using the same adjuvant as that of the experimental group. After 3 weeks of immunization, the rabbit hemorrhagic disease virus Wanfan strain was used to challenge the VP60-PlpE chimeric antigen immunization group, the physical Mixture group and a group of control groups, and the survival condition of the rabbits was observed at 100 × LD 50/rabbit for 7 days continuously. After 3 weeks of immunization, the remaining VP60-PlpE chimeric antigen immunization group, the physically mixed texture group and the other group were challenged with 100CFU (10 × MLD)/rabbit survival was observed for 7 consecutive days using Pasteurella C51-17 strain. After the rabbit hemorrhagic disease virus challenge, the survival rate of the experimental group (the immunity VP60-PlpE chimeric antigen recombinant protein group and the immunity two-protein physical mixture group) rabbits is 100%, and the control group is totally dead (the survival rate is 0/10, and the survival rate is shown in figure 7A). The survival rate of rabbits in the experimental group after pasteurella challenge was 100%, while the survival rate of the control group was only 20% (fig. 7B). The results of these experiments show that the immunization of VP60-PlpE recombinant protein can ensure that the rabbit has immunoprotection against rabbit hemorrhagic disease virus and pasteurella simultaneously, and the VP60-PlpE recombinant protein has the same protective power as the vaccine of the physical mixture of the two proteins.
Example 5 Process and time consuming comparison
Although the chimeric antigen and the two protein mixture vaccine provided by the patent have similar immune protection effects, the preparation process has obvious difference. As shown in Table 1, the chimeric antigen preparation process does not need to undergo steps of antigen purification, endotoxin removal, physical mixing and the like, the preparation time is shortened by 50%, material and water and electricity consumption of corresponding steps does not exist, the production cost is obviously reduced, and the application prospect is good.
Table 1 comparison of preparation processes
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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Ala Cys Ala Ala Cys Cys Gly Gly Thr Thr Cys Ala Ala Cys Thr Ala
35 40 45
Cys Cys Cys Cys Ala Ala Ala Thr Thr Gly Thr Thr Ala Thr Cys Cys
50 55 60
Cys Ala Cys Cys Thr Cys Ala Ala Ala Ala Ala Ala Cys Gly Gly Ala
65 70 75 80
Gly Gly Gly Cys Ala Ala Ala Gly Cys Cys Cys Gly Cys Ala Cys Ala
85 90 95
Gly Cys Gly Cys Cys Gly Cys Ala Ala Gly Gly Cys Gly Ala Ala Gly
100 105 110
Cys Ala Gly Cys Ala Gly Gly Cys Ala Cys Thr Gly Cys Thr Ala Cys
115 120 125
Cys Ala Cys Ala Gly Cys Ala Thr Cys Ala Gly Thr Thr Cys Cys Cys
130 135 140
Gly Gly Ala Ala Cys Cys Ala Cys Gly Ala Cys Cys Gly Ala Cys Gly
145 150 155 160
Gly Cys Ala Thr Gly Gly Ala Thr Cys Cys Thr Gly Gly Thr Gly Thr
165 170 175
Ala Gly Thr Gly Gly Cys Cys Gly Cys Ala Ala Cys Thr Ala Gly Thr
180 185 190
Gly Thr Gly Gly Thr Cys Ala Cys Thr Gly Cys Ala Gly Ala Ala Ala
195 200 205
Ala Thr Thr Cys Ala Thr Cys Cys Gly Cys Ala Thr Cys Gly Gly Thr
210 215 220
Thr Gly Cys Ala Ala Cys Gly Gly Cys Gly Gly Gly Gly Ala Thr Thr
225 230 235 240
Gly Gly Cys Gly Gly Thr Cys Cys Ala Cys Cys Cys Cys Ala Ala Cys
245 250 255
Ala Gly Gly Thr Gly Gly Ala Cys Cys Ala Ala Cys Ala Ala Gly Ala
260 265 270
Ala Ala Cys Ala Thr Gly Gly Ala Gly Gly Ala Cys Ala Ala Ala Cys
275 280 285
Thr Thr Cys Thr Ala Cys Thr Ala Cys Ala Ala Thr Gly Ala Thr Gly
290 295 300
Thr Thr Thr Thr Cys Ala Cys Thr Thr Gly Gly Thr Cys Cys Gly Thr
305 310 315 320
Cys Gly Cys Gly Gly Ala Cys Gly Cys Ala Cys Cys Cys Gly Gly Cys
325 330 335
Ala Gly Cys Ala Thr Thr Cys Thr Cys Thr Ala Cys Ala Cys Thr Gly
340 345 350
Thr Cys Cys Ala Ala Cys Ala Cys Thr Cys Thr Cys Cys Ala Cys Ala
355 360 365
Gly Ala Ala Cys Ala Ala Cys Cys Cys Ala Thr Thr Cys Ala Cys Ala
370 375 380
Gly Cys Thr Gly Thr Ala Cys Thr Gly Ala Gly Cys Cys Ala Gly Ala
385 390 395 400
Thr Gly Thr Ala Cys Gly Cys Thr Gly Gly Cys Thr Gly Gly Gly Cys
405 410 415
Thr Gly Gly Thr Gly Gly Cys Ala Thr Gly Cys Ala Gly Thr Thr Cys
420 425 430
Cys Gly Cys Thr Thr Cys Ala Thr Ala Gly Thr Thr Gly Cys Thr Gly
435 440 445
Gly Ala Thr Cys Ala Gly Gly Thr Gly Thr Gly Thr Thr Thr Gly Gly
450 455 460
Thr Gly Gly Gly Cys Gly Ala Cys Thr Gly Gly Thr Cys Gly Cys Gly
465 470 475 480
Gly Cys Thr Gly Thr Gly Ala Thr Ala Cys Cys Ala Cys Cys Ala Gly
485 490 495
Gly Cys Ala Thr Cys Gly Ala Gly Ala Thr Thr Gly Gly Gly Cys Cys
500 505 510
Ala Gly Gly Gly Thr Thr Gly Gly Ala Gly Gly Thr Cys Ala Gly Gly
515 520 525
Cys Ala Ala Thr Thr Thr Cys Cys Thr Cys Ala Thr Gly Thr Thr Gly
530 535 540
Thr Thr Ala Thr Cys Gly Ala Cys Gly Cys Cys Cys Gly Thr Thr Cys
545 550 555 560
Ala Cys Thr Cys Gly Ala Ala Cys Cys Thr Gly Thr Thr Ala Cys Cys
565 570 575
Ala Thr Cys Ala Cys Cys Ala Thr Gly Cys Cys Ala Gly Ala Cys Thr
580 585 590
Thr Gly Cys Gly Thr Cys Cys Cys Ala Ala Cys Ala Thr Gly Thr Ala
595 600 605
Cys Cys Ala Thr Cys Cys Ala Ala Cys Thr Gly Gly Thr Gly Ala Cys
610 615 620
Cys Cys Thr Gly Gly Cys Cys Thr Thr Gly Thr Cys Cys Cys Cys Ala
625 630 635 640
Cys Ala Cys Thr Ala Gly Thr Cys Cys Thr Thr Ala Gly Thr Gly Thr
645 650 655
Thr Thr Ala Cys Ala Ala Cys Ala Ala Cys Cys Thr Cys Ala Thr Cys
660 665 670
Ala Ala Cys Cys Cys Gly Thr Thr Thr Gly Gly Thr Gly Gly Ala Thr
675 680 685
Cys Cys Ala Cys Cys Ala Ala Cys Gly Cys Ala Ala Thr Cys Cys Ala
690 695 700
Gly Gly Thr Gly Ala Cys Ala Gly Thr Gly Gly Ala Ala Ala Cys Gly
705 710 715 720
Ala Gly Gly Cys Cys Gly Ala Gly Thr Gly Ala Thr Gly Ala Cys Thr
725 730 735
Thr Thr Gly Ala Gly Thr Thr Cys Gly Thr Gly Ala Thr Gly Ala Thr
740 745 750
Thr Ala Gly Ala Gly Cys Cys Cys Cys Cys Thr Cys Cys Ala Gly Cys
755 760 765
Ala Ala Ala Ala Cys Thr Gly Thr Thr Gly Ala Cys Thr Cys Ala Ala
770 775 780
Thr Cys Thr Cys Ala Cys Cys Cys Gly Cys Ala Gly Gly Cys Cys Thr
785 790 795 800
Thr Cys Thr Cys Ala Cys Gly Ala Cys Cys Cys Cys Ala Gly Thr Cys
805 810 815
Cys Thr Cys Ala Cys Thr Gly Gly Thr Gly Thr Thr Gly Gly Cys Ala
820 825 830
Ala Thr Gly Ala Cys Ala Ala Cys Ala Gly Gly Thr Gly Gly Ala Ala
835 840 845
Cys Gly Gly Cys Cys Ala Ala Ala Thr Ala Gly Thr Gly Gly Gly Ala
850 855 860
Cys Thr Gly Cys Ala Ala Cys Cys Ala Gly Thr Ala Cys Cys Thr Gly
865 870 875 880
Gly Gly Gly Gly Gly Thr Thr Thr Thr Cys Cys Ala Cys Gly Thr Gly
885 890 895
Cys Ala Ala Cys Ala Gly Gly Cys Ala Cys Thr Gly Gly Ala Ala Cys
900 905 910
Cys Thr Gly Ala Ala Cys Gly Gly Cys Ala Gly Cys Ala Cys Ala Thr
915 920 925
Ala Thr Gly Gly Cys Thr Gly Gly Thr Cys Ala Ala Gly Cys Cys Cys
930 935 940
Thr Cys Gly Gly Thr Thr Thr Gly Cys Cys Gly Ala Cys Ala Thr Thr
945 950 955 960
Gly Ala Cys Cys Ala Thr Cys Gly Ala Ala Gly Ala Gly Gly Cys Ala
965 970 975
Gly Thr Gly Cys Ala Ala Gly Thr Thr Ala Thr Thr Cys Thr Gly Gly
980 985 990
Gly Ala Ala Cys Ala Ala Cys Thr Cys Cys Ala Cys Cys Ala Ala Cys
995 1000 1005
Gly Thr Gly Cys Thr Thr Cys Ala Gly Thr Thr Thr Thr Gly Gly Thr
1010 1015 1020
Ala Cys Gly Cys Thr Ala Ala Thr Gly Cys Thr Gly Gly Gly Thr Cys
1025 1030 1035 1040
Thr Gly Cys Gly Ala Thr Thr Gly Ala Cys Ala Ala Cys Cys Cys Thr
1045 1050 1055
Ala Thr Cys Thr Cys Cys Cys Ala Gly Gly Thr Thr Gly Cys Ala Cys
1060 1065 1070
Cys Ala Gly Ala Cys Gly Gly Cys Thr Thr Cys Cys Cys Thr Gly Ala
1075 1080 1085
Cys Ala Thr Gly Thr Cys Ala Thr Thr Cys Gly Thr Gly Cys Cys Cys
1090 1095 1100
Thr Thr Thr Ala Ala Cys Ala Gly Cys Cys Cys Cys Ala Ala Cys Ala
1105 1110 1115 1120
Thr Thr Cys Cys Gly Ala Cys Cys Gly Cys Gly Gly Gly Gly Thr Gly
1125 1130 1135
Gly Gly Thr Cys Gly Gly Gly Thr Thr Thr Gly Gly Thr Gly Gly Thr
1140 1145 1150
Ala Thr Thr Thr Gly Gly Ala Ala Cys Ala Gly Thr Ala Ala Cys Ala
1155 1160 1165
Ala Cys Gly Gly Thr Gly Cys Cys Cys Cys Cys Gly Cys Thr Gly Cys
1170 1175 1180
Thr Ala Cys Ala Ala Cys Thr Gly Thr Gly Cys Ala Gly Gly Cys Cys
1185 1190 1195 1200
Thr Ala Thr Gly Ala Gly Thr Thr Ala Gly Gly Thr Thr Thr Thr Gly
1205 1210 1215
Cys Cys Ala Cys Thr Gly Gly Gly Gly Cys Ala Cys Cys Ala Ala Ala
1220 1225 1230
Cys Ala Gly Cys Cys Thr Cys Cys Ala Gly Cys Cys Cys Ala Cys Cys
1235 1240 1245
Ala Cys Cys Ala Ala Cys Ala Cys Thr Thr Cys Ala Gly Gly Thr Gly
1250 1255 1260
Cys Ala Cys Ala Gly Ala Cys Thr Gly Thr Cys Gly Cys Thr Ala Ala
1265 1270 1275 1280
Gly Thr Cys Cys Ala Thr Thr Thr Ala Thr Gly Cys Cys Gly Thr Gly
1285 1290 1295
Gly Thr Ala Ala Cys Cys Gly Gly Cys Ala Cys Ala Ala Ala Cys Cys
1300 1305 1310
Ala Ala Ala Ala Thr Cys Cys Ala Ala Cys Cys Gly Gly Ala Cys Thr
1315 1320 1325
Gly Thr Thr Thr Gly Thr Gly Ala Thr Gly Gly Cys Cys Thr Cys Gly
1330 1335 1340
Gly Gly Thr Gly Thr Thr Ala Thr Cys Thr Cys Cys Ala Cys Gly Cys
1345 1350 1355 1360
Cys Ala Ala Ala Cys Gly Cys Cys Ala Gly Cys Gly Cys Cys Gly Thr
1365 1370 1375
Cys Ala Cys Ala Thr Ala Cys Ala Cys Gly Cys Cys Cys Cys Ala Ala
1380 1385 1390
Cys Cys Ala Gly Ala Cys Ala Gly Ala Ala Thr Thr Gly Thr Gly Ala
1395 1400 1405
Cys Thr Ala Cys Ala Cys Cys Cys Gly Gly Cys Ala Cys Thr Cys Cys
1410 1415 1420
Thr Gly Cys Cys Gly Cys Thr Gly Cys Ala Cys Cys Thr Gly Thr Ala
1425 1430 1435 1440
Gly Gly Thr Ala Ala Gly Ala Ala Cys Ala Cys Ala Cys Cys Cys Ala
1445 1450 1455
Thr Cys Ala Thr Gly Thr Thr Cys Gly Cys Gly Thr Cys Thr Gly Thr
1460 1465 1470
Thr Gly Thr Cys Ala Gly Gly Cys Gly Cys Ala Cys Cys Gly Gly Thr
1475 1480 1485
Gly Ala Cys Gly Thr Cys Ala Ala Cys Gly Cys Cys Gly Cys Ala Gly
1490 1495 1500
Cys Cys Gly Gly Gly Thr Cys Ala Ala Cys Cys Ala Ala Cys Gly Gly
1505 1510 1515 1520
Gly Ala Cys Cys Cys Ala Gly Thr Ala Thr Gly Gly Cys Ala Cys Gly
1525 1530 1535
Gly Gly Cys Thr Cys Cys Cys Ala Ala Cys Cys Ala Cys Thr Gly Cys
1540 1545 1550
Cys Ala Gly Thr Gly Ala Cys Ala Ala Thr Thr Gly Gly Ala Cys Thr
1555 1560 1565
Thr Thr Cys Gly Cys Thr Cys Ala Ala Cys Ala Ala Cys Thr Ala Cys
1570 1575 1580
Thr Cys Gly Thr Cys Ala Gly Cys Ala Cys Thr Cys Gly Thr Gly Cys
1585 1590 1595 1600
Cys Thr Gly Gly Gly Cys Ala Gly Thr Thr Cys Thr Thr Cys Gly Thr
1605 1610 1615
Thr Thr Gly Gly Cys Ala Gly Thr Thr Ala Ala Cys Cys Thr Thr Thr
1620 1625 1630
Gly Cys Ala Thr Cys Thr Gly Gly Thr Thr Thr Cys Ala Thr Gly Gly
1635 1640 1645
Ala Gly Ala Thr Cys Gly Gly Cys Cys Thr Ala Ala Gly Thr Gly Thr
1650 1655 1660
Gly Gly Ala Cys Gly Gly Gly Thr Ala Cys Thr Thr Thr Thr Ala Thr
1665 1670 1675 1680
Gly Cys Ala Gly Gly Ala Ala Cys Ala Gly Gly Ala Gly Cys Cys Thr
1685 1690 1695
Cys Ala Ala Cys Cys Ala Cys Gly Cys Thr Cys Ala Thr Thr Gly Ala
1700 1705 1710
Cys Thr Thr Gly Ala Cys Thr Gly Ala Ala Cys Thr Cys Ala Thr Thr
1715 1720 1725
Gly Ala Cys Gly Thr Ala Cys Gly Cys Cys Cys Cys Gly Thr Gly Gly
1730 1735 1740
Gly Ala Cys Cys Cys Ala Gly Gly Cys Cys Gly Thr Cys Cys Ala Ala
1745 1750 1755 1760
Ala Ala Gly Cys Ala Cys Ala Cys Thr Cys Gly Thr Gly Thr Thr Cys
1765 1770 1775
Ala Ala Cys Cys Thr Gly Gly Gly Gly Gly Gly Cys Ala Cys Ala Ala
1780 1785 1790
Cys Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Thr Cys Thr Thr Ala
1795 1800 1805
Thr Gly Thr Cys Thr Cys Ala Cys Thr Ala Gly Cys Ala Ala Ala Thr
1810 1815 1820
Ala Ala Ala Cys Ala Ala Cys Ala Ala Ala Thr Thr Cys Ala Ala Ala
1825 1830 1835 1840
Thr Ala Cys Cys Thr Ala Cys Ala Ala Cys Thr Cys Ala Ala Ala Ala
1845 1850 1855
Thr Thr Cys Thr Thr Thr Thr Ala Ala Ala Cys Ala Thr Ala Thr Cys
1860 1865 1870
Gly Ala Ala Gly Ala Ala Gly Ala Gly Ala Ala Ala Ala Thr Gly Ala
1875 1880 1885
Thr Ala Ala Ala Gly Gly Ala Thr Ala Cys Cys Ala Cys Ala Ala Ala
1890 1895 1900
Cys Gly Ala Thr Thr Gly Gly Thr Thr Thr Ala Ala Ala Ala Gly Thr
1905 1910 1915 1920
Thr Gly Cys Gly Thr Thr Thr Cys Cys Thr Cys Thr Thr Ala Thr Cys
1925 1930 1935
Ala Gly Ala Ala Thr Ala Cys Thr Thr Gly Thr Thr Gly Ala
1940 1945 1950