Enzyme-linked immunosorbent assay (ELISA) test kit for testing lawsonia intracellularis (LI) of pigs

A detection kit, Lawsonia technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low LsaA protein expression, low utilization rate, etc., and achieve short detection time, high utilization rate, and good stability. Effect

Inactive Publication Date: 2018-01-12
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Codon utilization analysis found that the codon utilization of LsaA protein was low in the prokaryotic expression system (E. low expression

Method used

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  • Enzyme-linked immunosorbent assay (ELISA) test kit for testing lawsonia intracellularis (LI) of pigs
  • Enzyme-linked immunosorbent assay (ELISA) test kit for testing lawsonia intracellularis (LI) of pigs
  • Enzyme-linked immunosorbent assay (ELISA) test kit for testing lawsonia intracellularis (LI) of pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Synthesis of opt-LsaA gene and construction of recombinant expression vector pET-32a-opt-LsaA

[0045] According to the codon preference of Escherichia coli, the present invention conducts DNA analysis and RNA structure prediction on the LsaA gene sequence of Lawsonia intracellularis (accession number AF498259) registered in GenBank, and synthesizes the code without changing the amino acid sequence of the LsaA protein. The LsaA gene of the LsaA protein is artificially optimized to obtain the opt-LsaA gene, and the nucleotide sequence of the artificially optimized and synthesized opt-LsaA gene is shown in SEQ ID NO.2.

[0046] Add the NdeI restriction endonuclease recognition site at the 5' end of the opt-LsaA gene sequence artificially optimized and synthesized above, and add the XhoI restriction endonuclease recognition site at its 3' end; opt-LsaA The gene and the pET-32a vector were double-digested with NdeI and XhoI restriction endonucleases, and the dige...

Embodiment 2

[0047] Example 2: Induced expression and purification of LsaA recombinant protein

[0048] The correct recombinant expression vector pET-32a-opt-LsaA sequenced in Example 1 was transformed into E.coli BL21 (DE3) competent cells, and then the transformed E.coli BL21 (DE3) was coated to contain 100 μg. mL -1 Amp LB solid culture plate, culture overnight at 37°C. Pick a single colony on the next day containing 100 μg·mL -1 In LB liquid medium of Amp, 37°C 250r·min -1 , cultured to OD 600nm About 0.8, add IPTG to the final concentration of 0.1mmol·L -1 , 37℃, 250r·min -1 Shaking induced culture for 6h. Centrifuge the induced bacterial solution at 4000rpm for 10min, discard the supernatant, and collect the bacterial pellet; resuspend the pellet with 0.1M PBS, sonicate for 5min (sonication for 9s, stop for 6s) to break the bacterial cell, and then centrifuge at 10000rpm at 4°C for 10min , collect the supernatant and the precipitate respectively, resuspend the precipitate with...

Embodiment 3

[0049] Embodiment 3: Identification of LsaA recombinant protein

[0050] The antigenicity of the purified LsaA recombinant protein was analyzed by Western blot. The specific operation was as follows: the LsaA recombinant protein separated by SDS-PAGE electrophoresis was transferred to a nitrocellulose membrane, and the nitrocellulose membrane was placed in a 5 % (w / v) skimmed milk powder blocking solution overnight at 4°C. Porcine anti-Lawsonia intracellulare positive serum was diluted with PBS buffer containing 5% (w / v) skimmed milk and 0.5% (v / v) Tween-20 at a volume ratio of 1:200 as the primary antibody , incubate at 37°C for 2h, wash the membrane and dilute horseradish peroxidation with PBS buffer containing 5% (w / v) skimmed milk and 0.5% (v / v) Tween-20 according to the volume ratio of 1:5000 Phytosome-labeled rabbit anti-pig IgG was used as a secondary antibody, incubated at 37°C for 2 hours, and finally developed with a DAB color development kit (purchased from Wuhan B...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay (ELISA) test kit for testing lawsonia intracellularis (LI) of pigs. The ELISA test kit for testing the LI of the pigs comprises a solid phase carrier and an antigen enveloped in the solid phase carrier, wherein the antigen is LsaA recombinant protein, and the amino acid sequence of the LsaA recombinant protein is as shown in SEQ ID No. 1. The kit has the characteristics of being higher in sensitivity, high in specificity, good in stability, and the like, and has the advantages of convenient use, simple operation, short detection timeand low use cost; the kit can be used for carrying out serological investigation, antibody detection, vaccine immune effect evaluation and the like on the LI infection condition of the pigs, and is suitable for large-scale popularization and use.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to an ELISA detection kit for Lawsonia intracellulare. Background technique: [0002] Lawsonia intracellularis (LI) is an obligate intracellular parasitic bacterium that mainly parasitizes in the epithelial cells of the intestinal mucosa of pigs and causes proliferative enteritis in pigs. Clinically, it leads to loss of appetite, diarrhea and growth retardation in affected pigs, causing huge economic losses to the pig industry. Since porcine proliferative enteritis was reported in 1931, it has existed and been prevalent in major pig-raising countries in the world. There is not much attention paid to Lawsonia intracellulare infection in my country, and there is no comprehensive and clear understanding of the prevalence of this bacteria in pig herds in China. The main reason for this situation is not only the lack of attention, but also the lack of detection methods. S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/569
Inventor 赵军王川庆吴艳阳高冬生常洪涛陈陆李永涛杨霞王新卫刘红英
Owner HENAN AGRICULTURAL UNIVERSITY
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