ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep

A technology of Echinococcus granulosus and detection kit, which is applied in the biological field, can solve the problems of low specificity, inappropriate acquisition of materials, and unsuitable diagnosis of antigens, etc., and achieve the effect of high sensitivity and good specificity

Pending Publication Date: 2021-09-03
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunological detection plays an irreplaceable role in early diagnosis. Hydatid cyst fluid (HCF) is the main source of Echinococcus granulosus antigen. Due to its low specificity and unsuitable materials, it is not suitable as a test for sheep CE. diagnostic antigen

Method used

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  • ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep
  • ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep
  • ELISA kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: Amplification of Eg14-3-3ζ coding gene and construction of recombinant plasmid

[0049] According to the Echinococcus granulosus 14-3-3ζ gene sequence in GenBank (protein accession number: EUB61917.1 SEQ ID NO.1; gene accession number: XM_024492439.1SEQ ID NO.2;), design primers and introduce restriction sites , Eg14-3-3ζF2 is: 5'-GC GTC GAC AGATGGCTGAGCTTCTGTCC-3' (the underline is the Sal I restriction site) SEQ ID NO.3, R2 is: 5'-ATT GCGGCCGC TTATTCAGCACCCTCGGT-3' (the underline is the Not I restriction site) SEQ ID NO.4.

[0050] The total RNA of Echinococcus granulosus Protoscolmus was extracted and reverse-transcribed into cDNA, which was used as a template to amplify the eg14-3-3ζ gene ( figure 1 ), with a size of about 771bp, in line with the expected size. The target fragment was recovered using a DNA recovery kit, and the eg14-3-3ζ gene fragment recovered from the gel and the pET-28a(+) vector were digested with SalI and NotI for 2 hours in ...

Embodiment 2

[0051] Embodiment 2: Expression and purification of recombinant protein

[0052] The successfully constructed recombinant plasmid pET-28a-eg14-3-3zeta was transformed into E.coli BL21(DE3) to express the recombinant protein. Under the induction of IPTG, the recombinant plasmid transformed bacteria successfully expressed recombinant Eg14-3-3ζ (recombinant Eg14-3-3ζ, rEg14-3-3ζ), and the recombinant protein was soluble ( image 3 ). After affinity purification by Ni-NTA-His column, high-purity protein was obtained with a relative molecular weight of about 35kDa ( Figure 4 ), which is the expected size. The concentrations of rEg14-3-3ζ were determined by BCA method to be 0.917mg / mL.

Embodiment 3

[0053] Example 3: Antigenicity Analysis of Recombinant Proteins

[0054] The antigenicity of rEg14-3-3ζ was analyzed by Western blot. The results of Western blot showed that rEg14-3-3ζ recognized Echinococcus granulosus-infected sheep serum, Echinococcus granulosus-infected dog serum and mouse anti-rEg14-3-3ζ IgG serum, and a single band appeared at about 35kDa ( Figure 5 ), without reacting with healthy sheep serum, healthy dog ​​and healthy mouse serum.

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Abstract

The invention discloses an ELISA (enzyme-linked immuno sorbent assay) kit for detecting echinococcus granulosus infection of livestock such as dogs and sheep. A coating antigen in the kit is a recombinant echinococcus granulosus 14-3-3 zeta protein. According to the invention, the echinococcus granulosus recombinant protein rEg14-3-3 zeta is cloned and expressed for the first time; western blot shows that the recombinant protein has good reactogenicity; it is found that after rEg14-3-3 zeta immunizes mice, good body fluid and cellular immune response is induced and generated, and Th1 type immune response is mainly induced and generated. Furthermore, an indirect ELISA method for detecting the echinococcus granulosus antibody is established on the basis of the rEg14-3-3 zeta protein, an ELISA kit is prepared and has good sensitivity, specificity and repeatability, and an effective method is provided for epidemiological investigation and diagnosis of echinococcus granulosus infection and cystic echinococcosis of livestock.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an ELISA kit for detecting Echinococcus granulosus infection in domestic animals such as dogs and sheep; in particular, it relates to an ELISA kit for detecting Echinococcus granulosus infection in dogs and cystic echinococcosis in sheep and other domestic animals Reagent test kit. Background technique [0002] Cystic echinococcus (CE) is a complex disease that is parasitized in humans by the middle tapeworm larvae of Echinococcus granulosus (Eg) in the family Taeniidae. and a chronic zoonotic parasitic disease caused in animals (Hu Huanhuan and Wu Weiping, 2010). The ultimate hosts of Echinococcus granulosus are canids such as dogs and wolves; the intermediate hosts are artiodactyls such as camels, sheep, cattle, and pigs, and occasionally can infect rodents, primates, and humans. [0003] The diagnosis of the final host is an important step in the prevention and control of cystic e...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/68G01N33/543G01N2333/43543
Inventor 陈兆国吕亚洁龚海燕米荣升黄燕
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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