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Polypeptide, application thereof and kit for detecting lawsonia intracellularis antibody

A technology of Lawsonia intracellulare and a kit, which is applied in the field of agricultural biology, can solve the problems of slow growth of pigs, increase feeding costs, and affect the feed-to-meat ratio, etc., and achieve easy expression and purification, high accuracy, and short time-consuming Effect

Pending Publication Date: 2021-10-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the current investigation and research in my country, the infection of porcine proliferative enteritis is on the rise in various regions of our country. Most of the pigs have no obvious clinical manifestations, and their feed intake and feces are normal. However, the pigs grow slowly, which seriously affects the feed-to-meat ratio. , increased feeding costs, and caused serious losses to the pig industry
[0004] At present, the detection method of LI antibody is in the development stage, the operation is complex, time-consuming, low sensitivity, low specificity, low accuracy, and there is no domestic commercial detection kit

Method used

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  • Polypeptide, application thereof and kit for detecting lawsonia intracellularis antibody
  • Polypeptide, application thereof and kit for detecting lawsonia intracellularis antibody
  • Polypeptide, application thereof and kit for detecting lawsonia intracellularis antibody

Examples

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Embodiment 1

[0032] Embodiment 1: preparation polypeptide

[0033] Taking the polypeptide having the amino acid sequence shown in SEQ ID No.1 as an example, the preparation method of the polypeptide of the present invention is described.

[0034] 1. Obtain the polypeptide coding gene

[0035] In this study, the method of PAS (PCR-based Accurate Synthesis) was used to remove the DNA sequence corresponding to the signal peptide structure on the basis of the nucleic acid sequence shown in SEQ ID No.3 and optimize the rare codons, and then add BamH at the front and rear ends of the sequence. I and HindIII enzyme cutting sites to obtain the polypeptide coding gene having the DNA sequence shown in SEQ ID No.2.

[0036] The polypeptide encoding gene amplified above was ligated with the pMD19-T vector, and the ligation reaction was performed overnight at 4°C. The ligated products were transformed into Trans5α competent cells, spread on ampicillin-resistant LB plates, and cultured overnight at ...

Embodiment 2

[0047] Example 2: Preparation of a kit for detecting Lawsonia intracellulare antibodies

[0048] This example uses the ELISA kit as an example to describe the application of the recombinant polypeptide prepared in Example 1 in the preparation of reagents or devices for detecting antibodies to Lawsonia intracellulare.

[0049] The ELISA kit of this embodiment mainly includes: washing solution, sample diluent, stop solution, substrate chromogenic solution, enzyme-labeled secondary antibody, LI positive serum and LI negative serum, and enzyme plate.

[0050] The washing solution is a phosphate buffer (0.01M, pH7.4) with a final concentration of Tween-20 of 0.05% (v / v), and the preparation method is as follows: Weigh 0.2g of KH 2 PO 4 , 0.2g of KCl, 2.9g of Na 2 HPO 4 12H 2 O and 8g of NaCl were dissolved in water and the volume was adjusted to 1000mL, and then 0.5mL of Tween-20 was added to obtain a washing solution.

[0051] Sample diluent: 1-2% skim milk powder aqueous s...

Embodiment 3

[0087] Embodiment 3: the usage method of kit

[0088] 1. Determine the best antigen-antibody reaction time

[0089] Add 100 μL of LI positive serum or negative serum diluted according to the optimal dilution determined in Example 2 to the corresponding reaction wells, incubate at 37°C for 30, 60, and 90 min, discard the liquid in the wells, and use 200 μL Add washing solution at an amount per well, repeat washing three times, each time for 3-5 minutes, then add HRP-labeled goat anti-pig IgG diluted at 1:10000 in an amount of 100 μL / well, and incubate at 37°C for 1 hour; discard the liquid in the well , add 300 μL of washing solution to each well for washing, repeat the washing 3 times, each time for 5 minutes, shake off the liquid in the well after washing; add the substrate color development solution according to the amount of 100 μL / well, develop color at 25°C for 15 minutes in the dark, and stop the addition solution (50 μL / well) to terminate the reaction.

[0090] Read...

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Abstract

The invention provides a polypeptide, an application thereof and a kit for detecting a Lawsonia intracellularis antibody. The polypeptide has an amino acid sequence as shown in SEQ ID No. 1 or an amino acid sequence which has more than 85% of homology with an amino acid sequence as shown in SEQ ID No. 3. The polypeptide is preferably a recombinant polypeptide obtained through prokaryotic cell exogenous expression and purification. According to the application of the polypeptide, the polypeptide is used for preparing a preparation or a product for detecting the Lawsonia intracellularis antibody. The polypeptide disclosed by the invention is simple in preparation method and good in immunoreactivity, and the kit for detecting the Lawsonia intracellularis antibody, which is prepared on the basis of the polypeptide, has the advantages of simplicity in operation, convenience in use, high sensitivity, strong specificity, high accuracy, good repeatability and the like.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a polypeptide and its application and a kit for detecting Lawsonia intracellulare antibodies. Background technique [0002] Lawsonia intracellularis (LI) parasitizes in intestinal cells and can cause porcine proliferative enteropathy (PPE). Clinically, it often presents acute type, subclinical type and chronic type; among them, the chronic type is the most common; the disease has a long incubation period, high infection rate, low mortality rate, and is sporadic globally. [0003] The disease was first reported in 1931, and then there were reports of the disease in major pig producing countries such as Europe, America, Australia, and Asia. According to the current investigation and research in my country, the infection of porcine proliferative enteritis is on the rise in various regions of our country. Most of the pigs have no obvious clinical manifestations,...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/70G01N33/569G01N33/543
CPCC07K14/00C12N15/70G01N33/56911G01N33/543G01N2469/20
Inventor 李彬王丹丹周俊明汪伟倪艳秀祝昊丹朱雪蛟周金柱范宝超
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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