A kind of preparation method and application of Salmonella equine abortus flagellin flic

A Salmonella and flagellin technology, applied in the fields of bioengineering and immunization, can solve the problems of FliC protein research and utilization of rare reports, etc., achieve good application and development application prospects, simple preparation method, and good immune protection effect.

Active Publication Date: 2021-04-30
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In view of the fact that the current research on FliC protein is mostly focused on Salmonella typhimurium and Salmonella typhimurium, and the research and utilization of FliC protein on the immunity of Salmonella equine abortus is rarely reported in China

Method used

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  • A kind of preparation method and application of Salmonella equine abortus flagellin flic
  • A kind of preparation method and application of Salmonella equine abortus flagellin flic
  • A kind of preparation method and application of Salmonella equine abortus flagellin flic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: PCR amplification of FliC gene

[0041] Using the extracted genomic DNA of Salmonella equine abortus as a template, the total reaction volume was 25 μL, and the PCR conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 32 cycles, and final extension at 72°C 10min. After the reaction, the amplified products were electrophoresed on a 1% agarose gel.

Embodiment 2

[0042] Example 2: Construction of recombinant prokaryotic expression plasmid pGEX-4T-FliC

[0043] Primers were designed according to the cloned and sequenced FliC gene sequence: upstream primer: 5'-GTA GGATCC ACCAACTCCCGGTCTGACCTCGACTCCATCC-3'; downstream primer: 5'-TAA CTCGAG TATTGTAGGTTTTTACCGTCGATAGAAACAAC-3', the amplified fragment size is 840bp. The template was the genome of Salmonella equine abortus strain MS97, and the PCR conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 32 cycles, and final extension at 72°C for 10 min. The FliC gene fragment amplified by PCR was digested with BamHI and XhoⅠ respectively, then connected into pGEX-4T-2 vector, and transformed into E.coli BL21 (DE3) competent cells. The positive clones were extracted and identified by enzyme digestion, and the constructed FliC prokaryotic expression vector was identified by enzyme digestion. The re...

Embodiment 3

[0044] Example 3: Expression and purification of FliC protein

[0045] Pick the correct sequenced monoclonal colonies and culture them overnight in LB (Amp+, 100 μg / mL) medium, transfer to fresh LB (Amp+, 100 μg / mL) medium the next day at 1.5%, and culture at 37°C to OD600 Approximately equal to 0.5, add IPTG to a final concentration of 0.1mmol / L, induce for 4.5h at 29°C, collect the bacteria by centrifugation, break the bacteria by ultrasonication, centrifuge at low temperature to get the supernatant, and analyze the expression of the target protein in the supernatant by SDS-PAGE. Purify the sonicated supernatant containing the GST-tagged protein through a purification chromatography column, add 3mL elution buffer to the column to elute the protein, and elute the target protein.

[0046] SDS-PAGE electrophoresis was performed on the induced bacterial samples and the purified protein samples. The results of electrophoresis showed that compared with the control before inductio...

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Abstract

The invention discloses a preparation method of the flagellin FliC derived from Salmonella abortus equine and its possible application in immunization of Salmonella abortus equine disease. By cloning the flagellin FliC gene derived from Salmonella equi abortus and connecting it to the prokaryotic expression vector pGEX‑4T‑2, the prokaryotic recombinant of the protein was obtained, transformed into engineering bacteria BL‑21, and the protein was induced, expressed and purified , the protein has the amino acid sequence in Sequence Table 1, and the purified FliC recombinant protein has good immunogenicity after immunizing experimental animals, and the FliC recombinant protein can overcome the shortcomings of low antibody levels and poor safety produced by inactivated vaccines It can be used to prepare subunit vaccines for preventing and treating equine paratyphoid infection and can be used as an adjuvant for equine animal vaccines, and has good development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and immunization, specifically relates to the flagellin FliC of Salmonella equine abortus and its coded amino acid sequence, and relates to a preparation method of the FliC protein derived from Salmonella equine abortus and its possible application in immunization. Background technique [0002] Equine abortive salmonellosis, also known as equine paratyphoid, is an equine infectious disease caused by Salmonella abortusequ and characterized by abortion in pregnant horses. Primiparous mares and foals are most susceptible to this disease. Young foals usually showed sepsis or local inflammation after infection, while male animals showed orchitis and beard fistula (Xing Tao, 2008). The disease is one of the serious infectious diseases that harm horses. It is difficult to purify among horses. As the number of susceptible horses increases, it will cause the next outbreak and cause serious economic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/255C12N15/70C12N1/21C12R1/19
CPCC07K14/255C12N15/70C12N2800/101
Inventor 苏艳杨康苏玲玲
Owner XINJIANG AGRI UNIV
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