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Application of Taenia polycephala antigen B

A technology for taenia polycephalus and polycephaly, which is applied in the biological field, can solve the problem of antigen thermal stability to be improved, etc., and achieve good immunogenicity and reactogenicity, high sensitivity and specificity, and good thermal stability. Effect

Active Publication Date: 2020-11-20
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, ELISA diagnostic methods based on recombinant Taenia polycephala antigens (Tm-P2, Tm-HSP70, Tm-GP50, Tm-GST and Tm-HSP60) have been reported and have achieved good detection results, but the antigens used for production The thermal stability has been to be improved

Method used

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  • Application of Taenia polycephala antigen B
  • Application of Taenia polycephala antigen B
  • Application of Taenia polycephala antigen B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Gene Amplification

[0019] 1. Extraction of total RNA and cDNA synthesis of Taenia polycephala

[0020] The Taenia polycephala adult worms, protoscole and hexacarcinus preserved in liquid nitrogen were taken out and ground with a mortar, and then the total RNA was extracted according to the instructions of the Tiangen Animal Tissue RNA Extraction Kit. Total protein was extracted from adult worms according to the instructions of Suleibao Mammalian Protein Extraction Kit.

[0021] 2. Amplification of the Tm-AgB gene of Taenia polycephala

[0022] The primers of Tm-AgB refer to the transcriptome data Unigene17133, and the primers are set with Primer Premier 5.0 software, and all primers are synthesized by Shanghai Sangong:

[0023] Upstream of Tm-AgB: 5'-CGGGATCCATGAAAGCTACATTGTT-3'BamHI

[0024] Downstream of Tm-AgB: 5'-CCAAGCTTCTAGTTCTCCTCATCCAT-3'HindIII

[0025] The amplification program is shown in Table 1:

[0026] Table 1

[0027]

[0028] 3. Cl...

Embodiment 2

[0048] Example 2: Western Blot

[0049] Detect the immunoreactivity of rTm-AgB with the sera of diseased animals by immunoblotting, the specific steps are as follows:

[0050] (1) After the protein electrophoresis, take the corresponding gel part where the protein is located, and put it into the transfer buffer for equilibrium, a total of 3 times, 4 minutes each time.

[0051] (2) Soak nitrocellulose filter membrane (NC membrane) and 24 layers of filter paper in transfer buffer for 5 min.

[0052] (3) Place the cathode electrode plate, 24-layer filter paper, gel, NC membrane, and 24-layer filter paper in the Bio-Rad semi-dry transfer tank in order, and cover the anode electrode plate.

[0053] (4) Connect the electrotransfer device to the electroporator, and add transfer buffer at 35mA for transfer for 30min.

[0054] (5) After the transfer, the NC membrane was taken out, soaked in 5% skimmed milk powder TBST, and blocked overnight at 4°C.

[0055] (6) After blocking, cut t...

Embodiment 3

[0060] Embodiment 3: the establishment of indirect ELISA method

[0061] 1. Indirect ELISA operation steps

[0062] (1) Dilute with antigen coating solution in proportion, add 100 μL per well to 96 microplate plate for coating;

[0063] (2) Pour off the coating solution, pat dry the liquid in the well, wash with PBST, 5min each time, repeat four times;

[0064] (3) Add blocking solution to seal.

[0065] (4) After washing, the serum was diluted in proportion with PBS, and 100 μL of each well was added to the enzyme-labeled wells for incubation, and the liquid was discarded.

[0066] (5) After washing, add 100 μL diluted HRP-labeled goat or sheep anti-rabbit secondary antibody to each well and incubate.

[0067] (6) Add the soluble one-component substrate TMB to the well under the condition of avoiding light to carry out the color reaction;

[0068] (7) Add 100 μL of 2M H2SO4 to the well to terminate the reaction, and measure the OD value when the UV absorbance is 450 nm. ...

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Abstract

The invention, which relates to the field of biotechnology, discloses a series of related applications of taenia multiceps antigen B as a diagnostic antigen for cerebral coenurosis. The related experimental results show that the taenia multiceps antigen B can be identified by the goat serum infected with cerebral coenurosis but does not make reaction with the negative serum, thereby realizing thegood immunogenicity and reactogenicity. The high sensitivity and specificity are represented in a indirect ELISA method. The taenia multiceps antigen B has the high thermal stability by being comparedwith other proteins. The results prove that the taenia multiceps antigen B can be used a diagnostic antigen for cerebral coenurosis and can be applied to a detection kit.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of Taenia polycephala Antigen B. Background technique [0002] Cerebral coenurosis (Cerebral coenurosis) is a parasitic disease that is seriously harmful to cattle and sheep caused by the middle-stage larvae of Taenia multiceps. Brain polycephalus mainly parasitizes the central nervous system of cattle and sheep, and can also parasitize subcutaneous or muscle tissue. Humans can also act as intermediate hosts in the case of eating Taenia polycephala eggs by mistake. This disease often leads to the direct death of cattle and sheep, causing huge economic losses to the livestock industry in Europe, the United States, Africa and Asia. The accurate diagnosis of infected animals is one of the key directions of current research. [0003] Diagnostic methods for cerebral polycephalus include anatomy, imaging, and laboratory diagnosis. With the development of science and techn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/92G01N33/53G01N33/535C12N15/70
CPCC12N15/70G01N33/53G01N33/535G01N33/92
Inventor 杨光友刘俞辰古小彬谢跃
Owner SICHUAN AGRI UNIV