Application of using streptococcus suis type-2 hy0245 gene encoded proteins as protective antigens

A protective antigen, Streptococcus suis technology, applied in the field of veterinary microbiology and animal infectious diseases, can solve the problems of strong virulence and biological safety hazards, and achieve the effects of good immune protection, production safety, and strong immunogenicity

Inactive Publication Date: 2012-05-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because inactivated vaccines, attenuated vaccines and gene-deleted vaccines have biological safety hazards, subunit vaccines designed based on conserved proteins are considered to be a hot spot in the field of Streptococcus suis vaccine research

Method used

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  • Application of using streptococcus suis type-2 hy0245 gene encoded proteins as protective antigens
  • Application of using streptococcus suis type-2 hy0245 gene encoded proteins as protective antigens
  • Application of using streptococcus suis type-2 hy0245 gene encoded proteins as protective antigens

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 recombinant protein HP0245 EC Cloning and expression of

[0019] In the present invention, the SS2 local isolate SC-19 is used as the source strain of DNA clones encoding extracellular peptides in hp0245. This strain is isolated from a pig with streptococcosis in Sichuan Province in 2005. It is a highly virulent strain with MRP + EF + SLY + Phenotype (see literature Li W, Liu L, Qiu D, ChenH and Zhou R. Identification of Streptococcus suis serotype 2 genes preferential expressed in the natural host. IntJ Med Microb, 2010, 300: 482-488).

[0020] 1. Cloning of DNA encoding extracellular peptides in hp0245

[0021] Using the genome of Streptococcus suis type 2 SC-19 as a template, the applicant designed the hp0245 gene sequence based on the S. suis 05ZYH33 genome sequence published on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) Upstream primer P1: 5'-GC GAATTC GGTGCTAGTCGAACGTTG-3', where the underlined part is the EcoR I restriction site; downstrea...

Embodiment 2

[0027] Embodiment 2 recombinant protein HP0245 EC Detection of Immunogenicity

[0028] In order to prove that the recombinant protein HP0245 EC It has the characteristics of immunogenicity of the natural protein HP0245 of Streptococcus suis type 2, and can be used as an antigen in the application of a diagnostic test kit for Streptococcus suis type 2 infection, the applicant has passed Western blot method (see Sam Brook and La Searle, Molecular Cloning Experiment Guide, Third Edition, Jin Dongyan et al. (Translation), Science Press, Beijing, 2001) the purified recombinant protein HP0245 EC After SDS-PAGE running, transfer to PVDF membrane, and hybridize it with pig serum infected with Streptococcus suis type 2, the specific steps are as follows: 5 μg recombinant protein HP0245 EC After SDS-PAGE electrophoresis, it was electrotransferred to PVDF membrane (purchased from U.S. Invitrogen Corporation) by transfer membrane instrument (purchased from U.S. Bio-Rad Co.), and PVDF me...

Embodiment 3

[0029] Embodiment 3 recombinant protein HP0245 EC Detection of protection against Streptococcus suis type 2 infection

[0030] 1. For recombinant protein HP0245 EC Evaluation of antibody levels in mice after immunization

[0031] For recombinant protein HP0245 ECTo evaluate the antibody level after immunization, the applicant will use the recombinant protein HP0245 EC , inactivated vaccine and adjuvant control were used to evaluate the antibody level of mice after immunization. The method is: Divide 4-week-old female Balb / c mice into 3 groups, 20 in each group. Group 1 was intraperitoneally injected with 50 μg HP0245 EC . Group 2 was injected with homemade 1 x 10 9 The inactivated vaccine of Streptococcus suis type 2 of CFU, the preparation process of inactivated vaccine is as follows: cultivate Streptococcus suis type 2 overnight with tryptic soybean medium (TSB, purchased from U.S. BD company), then add 0.5% polymer The formaldehyde solution was left to culture for 2...

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Abstract

The invention belongs to the technical fields of veterinary microbiology and zoonotic diseases, and in particular relates to application of immunogenic protein genes of streptococcus suis type-2 for separating, cloning and expressing exocellular peptides and recombinant proteins of the encoded proteins in vaccine and diagnosis. In the invention, a new immunogenic protein gene hp0245 is separated from SC-19 bacterial strains of streptococcus suis type-2 virulent strains, wherein DNA (hp0245EC) for encoding the exocellular peptides has nucleotide sequences represented in the sequence table SEQ ID No:1 and is encoded with 261 amino acids. The recombinant protein HP0245EC remains the immunogenic feature of the primitive protein, can provide effective immunological protection to the SC-19 bacterial strain of the streptococcus suis type-2 infected in mice and has potential application value of vaccine. The invention further comprises a composition and preparation method for a streptococcus suis type-2 HP0245-ELISA diagnostic reagent kit, and a preparation of hp0245EC for cloning hp0245EC escherichia coli; the recombinant escherichia coli has been preserved in the China Center for Type Culture Collection (CCTCC) and the preservation number is CCTCC No: M2010258.

Description

technical field [0001] The invention belongs to the technical fields of veterinary microbiology and animal infectious diseases. It specifically relates to the isolation and cloning of a protective antigen gene of Streptococcus suis type 2 and the application of its encoded protein in vaccines and ELISA diagnostic kits. Background technique [0002] Streptococcus suis is the pathogenic bacterium that causes streptococcus suis, which can cause meningitis, sepsis, pneumonia, endocarditis, arthritis, and even death in pigs, and has caused huge economic losses to the pig industry in the world. Among the 33 known serotypes (1-31, 33, 1 / 2), Streptococcus suis type 2 is the most widespread and most pathogenic serotype, and can cause infection in specific populations. Since Danish scholars first reported human infection with S. suis type 2 in 1968, there have been more than 550 cases of human infection with S. suis type 2 in the world. Among them, in 1998 and 2005, large-scale outb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315C12N15/31C12N1/21A61K39/09A61P31/04G01N33/569C12R1/19
Inventor 周锐李薇刘磊
Owner HUAZHONG AGRI UNIV
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