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Marker gp50 of cerebral polycephaly and a kit for diagnosing cerebral polycephalic

A diagnostic kit and polycephalic disease technology, which is applied in the field of kits for diagnosing cerebral polycephalic disease, can solve problems such as no research reports on cerebral polycephalic larvae, and achieve the effect of specific diagnosis

Active Publication Date: 2018-03-23
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, FAST-ELISA (Falcon assay 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, and their sensitivity and specificity Both were high, 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 of cerebral polycephaly and a kit for diagnosing cerebral polycephalic
  • Marker gp50 of cerebral polycephaly and a kit for diagnosing cerebral polycephalic
  • Marker gp50 of cerebral polycephaly and a kit for diagnosing cerebral polycephalic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0059] Take the brain polycephaly worm body preserved in liquid nitrogen, and extract the total brain polycephaly RNA according to the procedure provided by the total RNA extraction kit of Shanghai Huashun Biological Co., Ltd., and store it at -70°C for later use.

[0060] According to the instructions of the Fermentas kit, the extracted total RNA from Taenia polycephalum brain polycephaly was reverse transcribed to synthesize cDNA. The reaction system is as follows: first add total 10μL RNA to the PCR tube; 1μL DEPC-Water; 1μLOligo dT(18 ); water bath at 65°C for 5min, ice bath for 3min, add 4μL 5×Reaction buffer; 2μL, dNTP(10mM); 1μL AMV; 1μL RNase inhibitor; the reaction program is 42℃60min, 70℃5min, and then store at 8℃.

[0061] According to the Unigene17013 sequence in the Taenia polycephalus transcriptome data and the Taenia solium GP50 diagnostic antigen gene (Accession No: AY214...

Embodiment 2

[0068] Example 2. Construction of Prokaryotic Expression Vector

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

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

[0071] Upstream primer: 5’-CCG GAATTC GAAAATGCCCCAA-3' (including 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 a 1% agarose gel, Gold-view stained, the target band was cut, purified and connected to the pMD19-T vector. According to the instructions of the plasmid extraction kit, the plasmids of the cloned strains identified as positive by PCR were extracted. Double digestion with B...

Embodiment 3

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

[0080] A single white colony BL21(DE3) of pET32a(+)-GP50 plasmid was inserted into 2mL LB culture medium (containing Amp 100μg / mL), and cultured in a shaker at 37°C overnight; each 1mL culture medium was inoculated into two other 100mL bottles In LB broth containing Amp (100μg / mL), culture for 4h at 37℃ in a shaker; add IPTG to one bottle to a final concentration of 1mmol / L, and the other bottle for control culture (no IPTG); take 1mL of bacterial solution respectively Transfer to two new centrifuge tubes, centrifuge at 12,000r / min for 2min, remove the supernatant to collect the bacteria. Polyacrylamide gel electrophoresis (SDS-PAGE) detection, see the results Figure 4 .

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

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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 specifically relates to a marker GP50 of cerebral polycephaly and a kit for diagnosing cerebral polycephaly. Background technique [0002] Cerebral polycephaly (Coenuriasis), also known as cerebral hydatid disease, is caused by the midtaen stage larva of Taenia multiceps (Taenia multiceps), which parasitizes the brain and spinal cord of cloven hoofed animals such as cattle and sheep. A kind of parasitic disease. The disease has a worldwide distribution. It often causes the death of diseased animals and causes huge economic losses to the animal husbandry. At the same time, human infections have also been reported. [0003] The clinical manifestations of polycephaly are diverse, and typical clinical symptoms do not appear for a period of time after the animal is infected, so it is difficult to diagnose in the early stage of infection. The diverse clinical manifestations have led to the comple...

Claims

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Application Information

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