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Chemiluminiscence detection kit for detecting swine foot-and-mouth disease O-type antibody by utilizing multi-epitope tandem protein

A technology for chemiluminescence detection and protein detection, which is applied in chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, etc. It can solve the problems of low sensitivity and long time consumption, and achieve good broad spectrum , simple operation and short reaction time

Active Publication Date: 2020-05-29
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional ELISA diagnostic method is time-consuming and has low sensitivity

Method used

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  • Chemiluminiscence detection kit for detecting swine foot-and-mouth disease O-type antibody by utilizing multi-epitope tandem protein
  • Chemiluminiscence detection kit for detecting swine foot-and-mouth disease O-type antibody by utilizing multi-epitope tandem protein
  • Chemiluminiscence detection kit for detecting swine foot-and-mouth disease O-type antibody by utilizing multi-epitope tandem protein

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0032] Further, the preparation method of the tandem epitopes of the different O-type FMDV strains comprises the following steps:

[0033] (1), screening of differential epitopes: by comparing the amino acid sequences of FMDV isolates on GenBank, select the sites with differences between different serotypes, then design primers, and form the respective epitope proteins by annealing Double-stranded DNA molecules were ligated with pGEX-4T-1 linearized plasmids that had been cut with the same enzymes, and then the ligated products were transformed into Escherichia coli BL21 (DE3) competent cells. After the expression was induced by IPTG, WB was used for verification. ELISA verification will be carried out after further purification of the available protein for verification.

[0034] (2), concatenation of differential epitopes: it is finally determined that the G-H loop (140-154 amino acids) is the dominant antigenic epitope of O-type FMD, which can distinguish O-type from A-type...

Embodiment 1

[0057] Example 1 Foot-and-mouth disease O-type differential epitope screening and construction of epitope tandem protein

[0058] 1. Screening of differential epitopes: The current research shows that there are 5 neutralizing epitopes of FMD type O, of which only the G-H loop and the C-terminus of VP1 are linear epitopes. In order to screen out epitopes that can distinguish type O from type A and Asia 1, this study compared the amino acid sequences of FMDV isolates on GenBank, and selected different serotypes with differences in the G-H loop and the C-terminus of VP1 sites, and sites with different O-type topologies (representative strains under different topologies), then design primers (see Table 1), and form double-stranded DNA molecules encoding each epitope protein by annealing . The specific operation is: add 6 μl of 100 μM upstream primer and 100 μM downstream primer into PCR tubes, heat in a 95° C. water bath for 5 minutes, and then slowly return to room temperature. ...

Embodiment 2

[0064] Example 2 Optimization of the chemiluminescent method for detecting porcine foot-and-mouth disease O-type antibody using multi-epitope tandem protein

[0065] The serum collected three times from pigs immunized with the monovalent inactivated vaccine of foot-and-mouth disease O / Mya98 / BY / 2010 was used as the standard positive control serum. (SPCE) detection kit (O-SPCE) detection blocking rate of 93%, using the French ID The competition percentage (S / N%) detected by FMD Type O Competition is 4%; the healthy pig serum that has not been immunized with any vaccine is used as the standard negative control serum, and the antibody titer of the serum detected by O-LPBE is The competition percentage (S / N%) detected by FMD Type O Competition is 102%, with The blocking rate of FMDV NS ELISA detection was -7%. Antigen coating concentration (4, 2, 1, 0.5, 0.25, 0.125 μg / ml) and serum dilution (1:10, 1:20, 1:40, 1:80) were optimized by checkerboard titration, as Figure 4 As sho...

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Abstract

The invention relates to a chemiluminiscence detection kit for detecting a swine foot-and-mouth disease O-type antibody by using multi-epitope tandem protein and belongs to the technical field of biology. The kit comprises a chemiluminiscence plate coated with M2 antigen, an enzyme-labeled secondary antibody, serum diluent, 20*PBST washing solution, positive control serum, negative control serum,chemiluminiscence substrate solution A and chemiluminiscence substrate solution B, wherein the M2 antigen is distinguishable multi-epitope tandem protein, and the multi-epitope tandem protein is obtained by connecting identification epitopes of different isolates in three topological types of the O-type foot-and-mouth disease in series, and the differential epitope is amino acid at the 140-154 position of a G-H ring. The kit has advantages of high sensitivity, good specificity, good broad spectrum, no cross reaction with immune A-type and Asia 1-type monovalent vaccine serum, simple operation,short reaction time, and good application prospect in foot-and-mouth disease O-type antibody detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chemiluminescent detection kit for detecting porcine foot-and-mouth disease type O antibody by using multi-epitope tandem proteins. Background technique [0002] Foot-and-mouth disease is an acute, febrile and highly contagious disease caused by foot-and-mouth disease virus (FMDV), mainly infecting artiodactyls. At present, the prevention and control of foot-and-mouth disease in my country is mainly based on the comprehensive prevention and control measures of immunization prevention and culling as a supplement, and at the same time combine the titer detection of immune antibodies to evaluate the immune effect. FMDV has seven serotypes: A, O, C, SAT1, SAT2, SAT3 and Asia 1. There are 5 epidemic strains of two serotypes of foot-and-mouth disease strains prevalent in my country, including 4 strains of O-type and 1 strain of A-type, which are respectively O-type Mya-98 st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N21/76
CPCG01N21/76G01N33/56983G01N33/6854G01N2333/09G01N2469/20
Inventor 常惠芸刘伟邵军军常艳燕
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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