Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis

A diagnostic kit and a technology for polycephaly, which are applied in the field of kits for diagnosing polycephaly in the brain, can solve problems such as no research reports on polycephaly in the brain, and achieve the effect of specific diagnosis.

Active Publication Date: 2016-10-12
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, FAST-ELISA (Falconassay screening test–enzyme-linked immunosorbent assay, FAST-ELISA) and QuickELISA diagnostic methods based on GP50 recombinant protein have been established for porcine cysticercosis in pigs and humans. Higher, confirming the diagnostic value of GP50 recombinant protein in porcine cysticercosis, but there is no relevant research report on brain polycephalic GP50 (TmGP50) protein

Method used

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  • Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis
  • Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis
  • Marker GP50 for coenuriasis, as well as coenuriasis diagnosing kit for diagnosing coenuriasis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Extraction of brain polycephalic total RNA and amplification of GP50 gene

[0059] The brain polycephalic worms stored in liquid nitrogen were taken, and the total RNA of brain polycephalic larvae was extracted according to the procedure provided by the total RNA extraction kit of Shanghai Huashun Biological Co., Ltd., and stored at -70°C for later use.

[0060] According to the instructions of the Fermentas kit, the total RNA of the extracted Taenia polycephala brain polycephalus was reverse-transcribed to synthesize cDNA. The reaction system was as follows: first, add 10 μL total RNA to the PCR tube; 1 μL DEPC-Water; 1 μL Oligo dT (18 ); 65°C water bath for 5 min, ice bath for 3 min, then add 4 μL 5×Reaction buffer; 2 μL, dNTP (10 mM); 1 μL AMV;

[0061] According to the Unigene17013 sequence in the T. polycephala transcriptome data and the open reading frame of the T. suis GP50 diagnostic antigen gene (Accession No: AY214922.1) in Genbnk, a pair of primers...

Embodiment 2

[0068] Embodiment 2, the construction of prokaryotic expression vector

[0069] 1. Design, amplification and cloning of GP50 gene expression primers

[0070] According to the GP50 gene sequence, Primer5.0 was used to design primers, and restriction endonuclease sites BamH I, Xho I and protective bases were added to the 5' ends of the upstream and downstream primers respectively. The primer sequences are as follows:

[0071] Upstream primer: 5'-CCG GAATTC GAAAATGCCCCAA-3' (with BamH Ⅰ restriction site);

[0072] Downstream primer: 5'-CGG CTCGAG TCACAAAACCATTGGTATCA-3' (containing Xho Ⅰ restriction site).

[0073] See Example 1 for the PCR reaction system and amplification procedure. The PCR amplified product was electrophoresed in 1% agarose gel, stained with Gold-view, the band of interest was excised, purified and connected to pMD19-T vector. According to the instructions of the plasmid extraction kit, the plasmids of cloned strains identified as positive by PCR were e...

Embodiment 3

[0079] Example 3. Induced expression and SDS-PAGE analysis of recombinant plasmid pET32a(+)-GP50 in Escherichia coli

[0080] Insert a single white colony BL21(DE3) of the pET32a(+)-GP50 plasmid into 2mL LB culture solution (containing Amp 100μg / mL), culture overnight on a shaker at 37°C; In the LB culture solution containing Amp (100 μg / mL), culture on a shaker at 37°C for 4 hours; add IPTG to one of the bottles to a final concentration of 1 mmol / L, and the other bottle as a control culture (without IPTG); take 1 mL of the bacterial solution respectively Transfer to two new centrifuge tubes, centrifuge at 12,000r / min for 2min, remove the supernatant and collect the bacteria. Polyacrylamide gel electrophoresis (SDS-PAGE) detection, see the results Figure 4 .

[0081] Figure 4 The results showed that the recombinant expressed protein was located in the bacterial inclusion body, and the induced expression product was a fusion protein coupled with a His-tag protein of about ...

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Abstract

The invention belongs to the field of animal disease diagnosis, and particularly relates to a marker GP50 for coenuriasis, as well as a coenuriasis diagnosing kit for diagnosing coenuriasis. According to a coenuriasis indirect ELISA diagnosing method, the sensibility reaches 95%, and the specificity reaches 92.6%. Monitoring of a serum antibody of a goat artificially infected with taenia multiceps discovers that the GP50 antibody values in the whole detection period after infection are all positive. Therefore, the GP50 recombinant protein can be used as the coenuriasis marker, and is used for diagnosing coenuriasis. According to the coenuriasis indirect ELISA diagnosing kit, the coenuriasis can be quickly, sensitively and specifically used for diagnosing coenuriasis, and a foundation is laid for early diagnosis and the treatment effect evaluation of coenuriasis infection.

Description

technical field [0001] The invention belongs to the field of animal disease diagnosis, and in particular relates to a marker GP50 for cerebral polycephalus and a kit for diagnosing cerebral polycephalus. Background technique [0002] Coenuriasis, also known as cerebral hydatid disease, is caused by the mid-stage larvae of Taenia multiceps, Coenurus cerebralis, parasitizing the brain and spinal cord of cattle, sheep and other artiodactyls. of a parasitic disease. The disease is distributed worldwide, and often causes the death of sick animals, causing huge economic losses to animal husbandry, and at the same time, people are also reported to be infected. [0003] The clinical manifestations of polycephalus are diverse, and typical clinical symptoms do not appear within a period of time after animal infection, so it is difficult to diagnose in the early stage of infection. Various clinical manifestations lead to the complexity of clinical diagnosis and the urgent need for mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 杨光友黄兴
Owner SICHUAN AGRI UNIV
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