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CN-122005770-A - Canine anti-cyst type borrelia double antigen recombinant protein subunit vaccine and preparation method thereof

CN122005770ACN 122005770 ACN122005770 ACN 122005770ACN-122005770-A

Abstract

The invention discloses a canine anti-cyst type borrelia antigen recombinant protein subunit vaccine and a preparation method thereof, and relates to the technical field of biological medicines. The canine anti-cyst type bag worm double antigen recombinant protein subunit vaccine uses recombinant proteins rEg 05752 and rEg 09809 as core double antigens, both of which have no transmembrane region and signal peptide and contain a plurality of B cell antigen epitopes. After the beagle is immunized, the organism can be efficiently induced to generate specific IgG antibodies, the antibody titer reaches a peak value after 35 days of immunization and still maintains a higher level after attack, and simultaneously, th1/Th2 type mixed immune response is obviously activated, so that the cytokine levels of IL-2, IFN-gamma, IL-4, IL-5 and the like are greatly improved. The invention has obvious immune protection effect and provides a safe and efficient novel technical means for preventing and controlling the source of the echinococcosis.

Inventors

  • WANG ZHENGRONG
  • BO XINWEN
  • ZHAO JIAXIN
  • ZHANG YANYAN
  • SUN YAN

Assignees

  • 新疆农垦科学院

Dates

Publication Date
20260512
Application Date
20260228

Claims (10)

  1. 1. An application of a recombinant protein composition in preparing a canine anti-cyst type borrelia antigen recombinant protein subunit vaccine, which is characterized by comprising rEg 05752 protein and rEg 09809 protein; the amino acid sequence of rEg 05752 protein is shown as SEQ ID NO.2, and the amino acid sequence of rEg 09809 protein is shown as SEQ ID NO.4.
  2. 2. The use according to claim 1, wherein the nucleotide sequence of the rEg 05752 protein-encoding gene is shown in SEQ ID NO.1, and the nucleotide sequence of the rEg 09809 protein-encoding gene is shown in SEQ ID NO. 3.
  3. 3. The use according to claim 2, wherein the method for preparing rEg 05752 protein comprises: Transforming recombinant plasmid containing coding genes with nucleotide sequences shown as SEQ ID NO.1 into escherichia coli, and then carrying out induced expression and purification treatment to obtain rEg 05752 protein; the preparation method of rEg 09809,09809 protein comprises the following steps: And (3) transforming the recombinant plasmid containing the coding gene with the nucleotide sequence shown as SEQ ID NO.3 into escherichia coli, and then carrying out induced expression and purification treatment to obtain the rEg 09809 protein.
  4. 4. The recombinant protein subunit vaccine of the anti-cyst type borrelia canis double antigen of the dog is characterized in that the active ingredients comprise rEg 05752 protein and rEg 09809 protein; the amino acid sequence of rEg 05752 protein is shown as SEQ ID NO.2, and the amino acid sequence of rEg 09809 protein is shown as SEQ ID NO.4.
  5. 5. The recombinant protein subunit vaccine of anti-cyst type bordeaux of claim 4, the canine anti-cyst type bordeaux double antigen recombinant protein subunit vaccine also comprises a vaccine adjuvant.
  6. 6. The canine anti-cyst recombinant protein subunit vaccine of claim 5, wherein the vaccine adjuvant is a Quil a adjuvant.
  7. 7. The recombinant protein subunit vaccine of anti-canine cyst type borrelia antigen of claim 6 wherein the mass ratio of rEg 05752 protein, rEg 09809 protein and Quil a adjuvant is 1:1:1.
  8. 8. The recombinant protein subunit vaccine of anti-canine cyst type borrelia antigen of claim 4 wherein the method for preparing rEg 05752 protein comprises: Transforming recombinant plasmid containing coding genes with nucleotide sequences shown as SEQ ID NO.1 into escherichia coli, and then carrying out induced expression and purification treatment to obtain rEg 05752 protein; the preparation method of rEg 09809,09809 protein comprises the following steps: And (3) transforming the recombinant plasmid containing the coding gene with the nucleotide sequence shown as SEQ ID NO.3 into escherichia coli, and then carrying out induced expression and purification treatment to obtain the rEg 09809 protein.
  9. 9. A method for preparing the recombinant protein subunit vaccine of the anti-cyst type bordeaux of dog as claimed in any one of claims 4 to 8, which comprises the step of uniformly mixing the rEg 05752 protein, the rEg 09809 protein and a vaccine adjuvant to prepare the recombinant protein subunit vaccine of the anti-cyst type bordeaux of dog.
  10. 10. The method of claim 9, wherein the rEg 05752 protein is prepared by a process comprising: Transforming recombinant plasmid containing coding genes with nucleotide sequences shown as SEQ ID NO.1 into escherichia coli, and then carrying out induced expression and purification treatment to obtain rEg 05752 protein; the preparation method of rEg 09809,09809 protein comprises the following steps: And (3) transforming the recombinant plasmid containing the coding gene with the nucleotide sequence shown as SEQ ID NO.3 into escherichia coli, and then carrying out induced expression and purification treatment to obtain the rEg 09809 protein.

Description

Canine anti-cyst type borrelia double antigen recombinant protein subunit vaccine and preparation method thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a canine anti-cyst type borrelia antigen recombinant protein subunit vaccine and a preparation method thereof. Background Echinococcosis (CE) is caused by infection with echinococcus granulosus (e. granulosus) one of the helminths of humans and animals. Infected canines are the primary source of infection for CE transmission, and after eggs in their faeces pollute the environment, they can be used to infect intermediate hosts (cattle, sheep, humans) via mouth feel, completing the life cycle and causing disease. At present, praziquantel insect repellent is a main means for controlling echinococcus granulosus infection of dogs, but the method has obvious limitations that, on one hand, the reinfection rate of dogs is high, and the propagation chain is difficult to block from the source, and on the other hand, the dogs have discharged eggs in a large amount into the environment before insect repellent, and the continuous propagation risk still exists. Therefore, developing a novel prevention and control technology (such as a canine vaccine) to realize accurate prevention and control of infection of a final host becomes an urgent need for comprehensive prevention and control of CE. The screening and functional verification of vaccine candidate antigens are the key links of vaccine research and development, and the scientificity and effectiveness of the vaccine candidate antigens directly determine the immunity protection capability and clinical application prospect of the vaccine. The recombinant protein antigen has the advantages of capability of accurately simulating natural antigen epitope, strong specificity, no risk of pathogenic microorganism pollution, high safety, capability of large-scale preparation through a genetic engineering technology and the like, and becomes a research hot spot in the field of parasite vaccines. At present, animal experiments prove that the echinococcus granulosus candidate antigen with good insect-reducing effect on dogs has advanced to a certain extent, mainly comprises EgM, eg14-3-3, egG1Y162 and the like, and lays a foundation for the subsequent development of novel vaccines. It is noted that most echinococcus granulosus recombinant protein antigens have weak autoimmune property, and individual immunization is difficult to induce a host to generate specific immune response with sufficient intensity, and proper adjuvant is needed to strengthen the immune effect. The adjuvant can remarkably improve the immunogenicity of the recombinant protein antigen by prolonging the residence time of the antigen in the body, activating the innate immune cells, regulating and controlling immune response types and other mechanisms, and is an indispensable component in vaccine research and development. Disclosure of Invention The invention aims to provide a canine anti-cyst type borrelia antigen recombinant protein subunit vaccine and a preparation method thereof, so as to solve the problems in the prior art. The invention has obvious immune protection effect and provides a safe and efficient novel technical means for preventing and controlling the source of the echinococcosis. In order to achieve the above object, the present invention provides the following solutions: The invention provides an application of a recombinant protein composition in preparing a canine anti-cyst type bag worm double antigen recombinant protein subunit vaccine, wherein the recombinant protein composition comprises rEg 05752 protein and rEg 09809 protein; the amino acid sequence of rEg 05752 protein is shown as SEQ ID NO.2, and the amino acid sequence of rEg 09809 protein is shown as SEQ ID NO.4. Further, the nucleotide sequence of the coding gene of rEg 05752 protein is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene of rEg 09809 protein is shown as SEQ ID NO. 3. Further, the preparation method of rEg 05752 protein comprises the following steps: Transforming recombinant plasmid containing coding genes with nucleotide sequences shown as SEQ ID NO.1 into escherichia coli, and then carrying out induced expression and purification treatment to obtain rEg 05752 protein; the preparation method of rEg 09809,09809 protein comprises the following steps: And (3) transforming the recombinant plasmid containing the coding gene with the nucleotide sequence shown as SEQ ID NO.3 into escherichia coli, and then carrying out induced expression and purification treatment to obtain the rEg 09809 protein. The invention also provides a canine anti-cyst type borrelia antigen recombinant protein subunit vaccine, and the active ingredients comprise rEg 05752 protein and rEg 09809 protein; the amino acid sequence of rEg 05752 protein is shown as SEQ ID NO.2, and the amino acid sequence of rEg 09809 protein i