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Neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit

A neosporosis and antibody detection technology, which is applied in the direction of measuring devices, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of poor pertinence and high price, and achieve the effect of highlighting technological progress

Inactive Publication Date: 2014-06-18
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

①There are "Neospora caninum ELISAkit" produced by Italy's Euroclone company and the "Neospora caninum Antibody Test kit" produced by American IDEXX company, which are expensive, not very specific, and have certain limitations

Method used

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  • Neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit
  • Neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit
  • Neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0051] A kit construction

[0052] 1 method

[0053] 1.1 DNA template extraction

[0054] Take 200 μL of anticoagulated whole blood and place it in a 1.5 mL centrifuge tube. Add 450 μL of erythrocyte lysate (10mmoL / L Tris-Cl, 1mmoL / L EDTA, 1mg / L SDS, pH8.0) and mix by inversion, place at 4°C for 30min, centrifuge at 12000r / min for 10min, discard the supernatant, repeat this step , until the precipitate was white. Add 300 μL extract solution (20mmoL / L Tris-Cl, pH7.6), 1.5 μL proteinase K (0.1mg / ml), suspend the precipitate, invert and mix well, and bathe in 55°C water bath for 3h. Add 100 μL of isopropanol, mix well, centrifuge at 12000 r / min for 5 min, and discard the supernatant. Add 500 μL of 75% ethanol, centrifuge at 12000 r / min for 5 min, and discard the supernatant. After drying, add 30 μL of sterilized TE water, detect the extraction effect by 10 mg / L agarose gel electrophoresis, and store at -20°C.

[0055] 1.2 Amplification of Neospora NcSRS2 gene

[0056] PCR ...

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Abstract

The invention provides a neosporosis rELISA antibody assay kit. The neosporosis recombinant enzyme-linked immunosorbent assay (rELISA) antibody assay kit is characterized in that designing a primer set according to a neospora caninum NcSRS2 gene of the GenBank, introducing the primer set to an enzyme digestion site, removing a signal peptide zone of an NcSRS2 protein, extracting a DNA from Xinjiang cow positive serum, carrying out polymerase chain reaction (PCR) amplification of the DNA to obtain a tNcSRS2 genetic fragment having length of 975bp, cloning the tNcSRS2 genetic fragment into a pMD-18-T carrier, carrying out enzyme digestion and sequencing, further directionally connecting the desired fragment obtained by the previous step to a pGEX-4T-2 expression carrier, transferring the pGEX-4T-2 expression carrier with the desired fragment into escherichia coli, carrying out inducible expression and purification, wherein expressed fusion protein molecular weight is about 62kDa, and carrying out ELISA antigen coating by the expressed fusion protein to obtain the neosporosis rELISA antibody assay kit. A use method of the neosporosis rELISA antibody assay kit comprises the following steps that a coated enzyme label reaction plate is taken out, is subjected to first washing, is enclosed, is subjected to second washing, is added with a sample needing to be detected, is subjected to third washing, is added with a secondary antibody, is subjected to fourth washing, and undergoes a color reaction; and when the color reaction is finished, an OD450 / 630 value of the coated enzyme label reaction plate is read and a result is determined. Compared with a commercial ELISA assay kit, the neosporosis rELISA antibody assay kit has a higher coincidence rate above 95% The neosporosis rELISA antibody assay kit also is suitable for the ELISA of a neospora caninum antibody in ruminant serum and blood plasma.

Description

technical field [0001] The invention relates to gene recombination technology, in particular to the cloning and expression of Neospora tNcSRS2 gene and the construction of rELISA antibody detection kit. Background technique [0002] Neosporosis is a protozoan disease newly discovered in 1998. Dogs are the ultimate hosts of Neospora, and many mammals such as cattle and sheep, as well as wild animals, can be used as intermediate hosts. The disease is distributed worldwide and has been reported in more than 50 countries. The infection rate of dairy cows in foreign countries is 10-40%, up to 82%. It has been confirmed that Neosporosis is the main source of abortion in affected animals. Xinjiang is an important animal husbandry production base in my country, and cattle breeding is the main pillar industry in our region. The positive rate of serum Neospora antibody is 40%; nearly 5-20% of pregnant cows have unexplained cases every year. Abortion, brucellosis and other infectious...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N15/70C12N15/62C07K19/00
Inventor 巴音查汗·盖力克杨帆岳城王振宝陈亮郭庆勇王连芳季新成
Owner XINJIANG AGRI UNIV
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