Neospora caninum NcMIC26 antigen and application thereof

A technology for Neospora and Neospora, applied in the biological field, can solve the problems of false negative, result error, low sensitivity of detection method, etc., and achieve the effects of simple operation, good repeatability and good specificity

Inactive Publication Date: 2019-08-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cows with Neospora-positive fetuses were found to be serologically negative in some studies, which may be due to fluctuations in antibody levels in the cows or due to insensitive assays false negative
The reported detection antigens still have limitations in the actual diagnosis of neosporosis in cattle, causing some errors in the results of epidemiological investigations

Method used

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  • Neospora caninum NcMIC26 antigen and application thereof
  • Neospora caninum NcMIC26 antigen and application thereof
  • Neospora caninum NcMIC26 antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Screening of Neospora diagnostic antigen

[0059] 1) Collection of Neospora secreted proteins

[0060] The Neospora Nc1 was mass-cultured in Vero cells, and when the Neospora multiplied for about 72 hours to the state of being released, the worms were separated, purified and collected from the Vero cells. Count up to 5×10 7 Individuals / mL or more, resuspend the pellet with 200 μL serum-free DMEM, and incubate in a 37°C incubator for 3-5 hours; centrifuge after the incubation to obtain Neospora secreted proteins, namely extracellular tachyzoite excretion and secretion antigen (esa) and Tachyzoite holozoite antigen (pellet); SDS-PAGE electrophoresis was performed on the collected secreted protein, and silver staining was performed according to the instructions of the silver staining kit. The results were as follows: figure 1 .

[0061] 2) Western Blot identification of Neospora secreted protein

[0062] The antigenicity and specificity of the secreted prot...

Embodiment 2

[0065] Example 2: Prokaryotic induced expression and purification of recombinant protein r NcMIC26

[0066] 1) Cloning of NcMIC26 gene

[0067] According to the Neospora dominant antigen NcMIC26 known in Example 1, find the coding gene sequence of Neospora NcMIC26 antigen in ToxoDB online database (http: / / www.toxodb.org), remove its signal peptide, select with Toxoplasma gondii The lower part (271bp-1014bp) of homologous gene similarity, design primer F and R, take the cDNA of Neospora as template, amplify the gene fragment of Neospora MIC26, the nucleotide sequence of described gene fragment is as follows Shown in SEQ ID NO.2, as the result image 3 As shown, the sequence size is 744bp;

[0068] Wherein, the cDNA of Neospora extracts Neospora RNA and reverse transcription by the Trizol-chloroform method, specifically including the following steps:

[0069] Collect and purify Neospora tachyzoites up to 3~5×10 7 each, use 1mL Resuspend the RNA isolation reagent evenly, an...

Embodiment 3

[0084] Embodiment 3: the establishment of bovine serum Neospora antibody indirect ELISA detection method

[0085] 1) Reaction conditions

[0086] The bovine serum Neospora antibody indirect ELISA reaction conditions are shown in Table 2:

[0087] Table 2 bovine serum Neospora antibody indirect ELISA reaction conditions

[0088]

[0089] The method steps are as follows:

[0090] a) Antigen coating: the recombinant protein rNcMIC26 was diluted to 10 μg / mL with 0.05 M carbonate buffer solution of pH=9.6, 100 μL per well in a 96-well enzyme-labeled microreaction plate, and the coating condition was 37° C. for 1 hour, Overnight at 4°C (14~16h);

[0091] b) Washing: Discard the liquid in the well, add 300 μL of 0.5% PBST to wash 4 times, with an interval of 5 minutes between each time;

[0092] c) Blocking: use 5% horse serum-PBST as the blocking solution, 100 μL per well, block at 37° C. for 1 h;

[0093] d) washing: the operation steps are the same as b);

[0094] e) Prim...

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Abstract

The invention discloses a neospora caninum NcMIC26 antigen and an application thereof. Firstly, the invention discloses neospora caninum NcMIC26 antigen. The amino acid sequence of the antigen is as shown in SEQID NO.1. The invention further discloses an application of recombinant protein of the antigen to construction of a neospora caninum indirect ELISA diagnosis reagent kit. Through screening,a new diagnosis antigen suitable for neospora caninum is obtained, an indirect ELISA reagent kit is established by using the recombinant protein of the antigen as a coating antigen, the operation is simple, quick and efficient, the specificity is good, the sensibility is high, the repeatability is good, the neospora caninum NcMIC26 antigen can be used for detecting neospora caninum infection situation of cattle and can detect positive samples which cannot be detected by common NcSRS2 protein, more accurate detection data can be obtained, and the neospora caninum NcMIC26 antigen has important significance in preventing and treating the neospora caninum.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Neospora NcMIC26 antigen and its application. Background technique [0002] Neosporosis (neosporosis) is a protozoan disease caused by Neospora caninum that can infect a variety of animals. The disease caused by Neospora has a global distribution trend. The disease is the most serious harm to cattle and dogs. It is one of the main causes of cattle abortion, weak fetus, stillbirth, mummified fetus and dog's nervous system disorder, and it brings serious economic losses to animal husbandry. The infection intensity of bovine neosporosis reported by different countries and regions is different, and the positive rate of serum antibody is 13.5%-82%. Currently, there are no effective drugs or vaccines against Neospora. [0003] The main detection methods for neosporosis include etiological diagnosis, histopathological diagnosis, molecular biological diagnosis and serological diagnosis. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/44C12N15/30C12N15/70A61P33/02G01N33/531G01N33/569
CPCA61P33/02C07K14/44C12N15/70G01N33/531G01N33/56905
Inventor 刘晶王先梅刘群许建海
Owner CHINA AGRI UNIV
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