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Peste des petits ruminants virus recombinant protein antigen and rapid test strip for peste des petits ruminants virus antibody

A kind of PPR, recombinant protein technology, applied in the direction of virus/phage, antisense single-stranded RNA virus, virus, etc., can solve the problems of economic loss of animal breeding industry in Tibet, achieve easy storage and transportation, small amount of testing samples, Detect the effect of low cost

Inactive Publication Date: 2015-02-11
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2007, the disease first broke out in the Ngari area of ​​Tibet, my country, and in the following years, the disease still occurred in some areas of Tibet, causing huge economic losses to the animal breeding industry in Tibet
At present, colloidal gold immunochromatographic test strips or related products for the rapid detection of PPR virus at home and abroad are used for rapid detection of PPR virus antibodies, on-site detection and import and export animal quarantine

Method used

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  • Peste des petits ruminants virus recombinant protein antigen and rapid test strip for peste des petits ruminants virus antibody
  • Peste des petits ruminants virus recombinant protein antigen and rapid test strip for peste des petits ruminants virus antibody
  • Peste des petits ruminants virus recombinant protein antigen and rapid test strip for peste des petits ruminants virus antibody

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Expression of truncated PPRVN protein

[0030] 1. PPRVN protein antigenic epitope analysis and codon optimization: by searching the PPRVN gene sequence and corresponding amino acid sequence (GenBank: AFR66760.1) in the NCBI database, using online software to analyze its main antigenic epitope, determine the selected antigenic epitope Located at the 130th to 491st amino acid in the N protein, its amino acid sequence is SEQ ID No.1 in the sequence table, and its corresponding nucleotide sequence (GenBank: JX217850.1) is the 495th to 1580th nucleotide, see sequence List SEQ ID No.5.

[0031] 2. PPRVN gene codon optimization and artificial synthesis: codon optimization, that is, redesign the gene sequence according to the preference of the expression system for amino acid codons, and modify the codons with low utilization or rare codons in the gene sequence to the expression system Frequent use of codons ensures that the amino acid sequence of the expressed prot...

Embodiment 2

[0046] Embodiment 2: the preparation of colloidal gold label test strip

[0047] 1. Preparation of colloidal gold and labeling of streptococcal protein G (SPG) by colloidal gold: Colloidal gold solution was prepared by trisodium citrate reduction method. Add 1mL of 1% chloroauric acid solution to 99mL of three-distilled water, heat to boiling, quickly add 2.5mL of freshly prepared 1% (m / v) trisodium citrate solution, and mix well, continue heating and boiling for 5min, the color of the solution changes from Blue turns to red. After cooling to room temperature, the pH of the colloidal gold solution was adjusted to 6.5 with 1% sodium carbonate solution. Slowly add 20 μL of SPG solution with a concentration of 1 mg / mL to 50 mL of colloidal gold solution, stir at room temperature for 45 min, slowly add ovalbumin (OVA) with a concentration of 10 g / L to a final concentration of 10 mg / mL, and continue stirring for 30 min. Centrifuge at 4,000r / min for 10min, transfer the supernatant...

Embodiment 3

[0053] Embodiment 3: specificity, sensitivity and coincidence rate test of test strip

[0054] 1. Specificity of test strips: Use test strips to detect PPRV standard positive serum samples, other related viruses including Akabane disease virus (AKV), O-type foot-and-mouth disease virus (FMDVO), bluetongue virus (BTV), deer epidemic Hemorrhagic disease virus (EHDV), bovine viral diarrhea virus (BVDV) positive serum samples and negative control samples. The results showed that the test strip was positive when detecting PPRV standard positive serum samples, and was negative when detecting other virus positive serum and negative control samples, indicating that the test strip had good specificity.

[0055] 2. Sensitivity of the test strip: Detect 5 serially diluted PPRV positive serum samples (numbered 1 to 5) with the test strip, and use the ELISA kit as a control at the same time. The degrees are 1:64, 1:64, 1:128, 1:128 and 1:64 respectively, and the ELISA detection limit is 1...

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Abstract

The invention discloses a peste des petits ruminants virus recombinant protein antigen and a rapid test strip for detecting a peste des petits ruminants virus antibody by using the recombinant protein antigen as a detection line reagent. The peste des petits ruminants virus recombinant protein antigen can be obtained by the following steps: selecting main antigenic epitope-containing amino acid segment SEQ IDNo.1 through analyzing the N protein antigenic epitope of the peste des petits ruminants virus, optimizing a codon and artificially synthesizing the codon-optimized gene sequence SEQIDNo.2, constructing a recombinant expression carrier and transforming Escherichia coli for protein expression. The purified peste des petits ruminants virus recombinant protein antigen can be used for detecting the peste des petits ruminants virus antibody. The rapid test strip established based on the peste des petits ruminants virus recombinant protein antigen has the advantages of being convenient to operate, fast to detect, free from special laboratories, equipment and the like, overcomes the limit of the existing detection method, and can be used for rapid detection of the peste des petits ruminants virus antibody and investigation of serum epidemiology.

Description

technical field [0001] The invention relates to the field of animal virus antibody detection, in particular to a rapid detection test strip for the recombinant protein antigen of Peste des Petits Ruminants virus and the antibody of Peste des Petits Ruminants virus prepared by using the recombinant protein antigen. Background technique [0002] Peste des petits ruminants (PPR), also known as pseudorinderpest in small ruminants, pneumoenteritis, stomatitis pneumonia complex, is an acute, severe infectious disease. The World Organization for Animal Health (OIE) lists Peste des Petits Ruminants as an animal disease that must be reported, and the Ministry of Agriculture of my country lists Peste des Petits Ruminants as a first-class animal disease. Peste des petits ruminants has seriously affected the healthy development of ruminant breeding industry and the international trade of related animals and animal products, causing huge economic losses to the affected areas. Peste des...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/40C12N15/11G01N33/569
CPCC07K14/005C12N2760/18422G01N33/56983G01N33/6893G01N2333/115
Inventor 孙洁花群义杨俊兴艾军吕建强林庆燕廖立珊唐金明刘建利陶虹
Owner SHENZHEN AUDAQUE DATA TECH
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