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A fusion protein antigen for diagnosis of canine brucellosis

A technology of Brucella canis and fusion protein, which is applied in the field of fusion protein antigen for diagnosis of canine brucellosis, can solve the problems of lack of safety and achieve the effects of ensuring accuracy, overcoming poor safety and high sensitivity

Active Publication Date: 2022-04-19
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the deficiencies of the prior art and the shortcomings of low safety, the present invention provides a brucellosis-specific fusion protein antigen for detecting brucellosis caused by Brucella canis

Method used

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  • A fusion protein antigen for diagnosis of canine brucellosis
  • A fusion protein antigen for diagnosis of canine brucellosis
  • A fusion protein antigen for diagnosis of canine brucellosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Prediction and Screening of Brucella Major Membrane Protein B Cell Antigen Polypeptides

[0021] 1. Obtain the amino acid sequences of Brucella's main outer membrane proteins BP26, Omp2b, Omp16, Omp25 and Omp31 from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, using Bepipred Linear EpitopePrediction, ABCpred The prediction server software predicts the B cell epitope of the outer membrane protein, and selects the sequence with α helix, less β sheet, good hydrophilicity and antigenicity as the dominant epitope (see Table 1).

[0022] Table 1 Prediction information table of main outer membrane protein B cell antigen polypeptide

[0023]

[0024]

[0025] 2. Use the protein sequence comparison function of the NCBI website to compare the above-mentioned predicted peptides with the protein sequences in the database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and eliminate those with other bacteria. A polypeptide with a protein sequence identity of 90%-...

Embodiment 2

[0039] The design of embodiment 2 Brucella specific fusion protein and the construction of prokaryotic expression plasmid

[0040] The above-mentioned predicted antigen epitopes were connected in series, and the flexible polypeptide linker "GGGGS" was used to link each epitope to finally form a protein with a sequence of 366 amino acids, the sequence of which is shown in SEQ ID NO.1. Using online software to analyze its solubility (https: / / www.novopro.cn / tools / prot-sol.html), the predicted solubility (after normalization) is 0.659, indicating that the protein has a high solubility (>0.45 Indicates that the solubility may be higher than the average value of the data set). Hydrophobicity analysis of the protein showed that the protein had good hydrophilicity ( figure 1 ). Good solubility and hydrophilicity indicate that the fusion protein can be used as an ideal antigen for antibody detection.

[0041] The codon optimization tool (ExpOptimizer) was used to optimize the codon ...

Embodiment 3

[0042] Embodiment 3: the expression and purification of Brucella fusion protein

[0043] Transform the constructed pET28a(+) plasmid containing the fusion protein sequence (SEP ID NO.2) into Escherichia coli BL21(DE3) expression bacteria by heat shock method, spread on LB plates (containing 30 μg / mL kanamycin), and culture at 37°C overnight. Pick a single colony from the plate and inoculate it in LB liquid medium (containing 30 μg / mL kanamycin), at 37° C., 200 r / min overnight. Then, take the overnight culture bacterial liquid and freshly prepared LB liquid medium (containing 30 μg / mL kanamycin) to expand the culture at a volume of 1:100, when the OD of the bacterial liquid 600 When 0.6 was reached, IPTG was added at a final concentration of 0.3 mmol / L. After optimization, the best induction condition is 25°C, 150r / min induction for 8h, at this time the expression of the fusion protein in the supernatant is the highest.

[0044] After resuspending the collected cells with ex...

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Abstract

The present invention provides a brucellosis-specific fusion protein antigen, the amino acid sequence of which is shown in SEQ ID No:1. The fusion protein antigen provided by the invention contains multiple Brucella dominant outer membrane antigen epitopes, and has good extensibility, stability and biological activity. The canine brucellosis ELISA antibody detection kit established by using the fusion protein of the invention has high sensitivity and specificity. Specificity cross experiments showed that the OD450 ratios of the fusion protein with Escherichia coli O157 serum, Escherichia coli H7 serum, Salmonella O antigen multivalent serum, Yersinia enterica serum and Listeria monocytogenes serum and negative serum were all less than 1.5, The OD450 ratio of Brucella canis positive serum to negative serum is greater than 5, indicating that the fusion protein provided by the invention does not cross-react with serum infected with other bacteria, thereby ensuring the accuracy of detection.

Description

technical field [0001] The invention belongs to the technical field of animal epidemic detection, and in particular relates to a fusion protein antigen for diagnosing canine brucellosis. Background technique [0002] Brucellosis (hereinafter referred to as brucellosis) is an important zoonotic infectious disease caused by Brucella. It is an animal disease that must be notified by the International Organization for Animal Health (OIE). The Prevention and Control Law stipulates that it is a Class B infectious disease, which poses a major threat to my country's aquaculture industry, food safety and human health. At present, the genus Brucella mainly includes 6 species, namely ovine species, bovine species, porcine species, ovine epididymis species, shilling rat species and canine brucellosis. In my country, the sheep, cattle, porcine and dog Brucellas are mainly prevalent, all of which are pathogenic to humans. Humans can be infected through the digestive tract and respiratory...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/569G01N33/68G01N33/58G01N33/558G01N33/543C12R1/19
CPCC07K14/23C12N15/70G01N33/56911G01N33/6854G01N33/587G01N33/558G01N33/54393C07K2319/31C12N2800/22G01N2333/23G01N2469/20
Inventor 孙明军孙翔翔殷德辉刘蒙达樊晓旭田莉莉孙淑芳邵卫星范伟兴
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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