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Protein antigen, coding gene thereof, and application of protein antigen and coding gene thereof to identification of mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody

A technology of Mycoplasma hyopneumoniae and protein antigen, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of infection and indistinguishability, and achieve the effect of high sensitivity and high sensitivity

Pending Publication Date: 2019-08-16
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Mhp is widely present in various pig farms, pigs may be infected with Mhp when they are just born, which causes all pigs in the herd to produce antibodies against Mhp regardless of whether the Mhp vaccine is used, but these antibodies are The existing Mhp serum antibody detection kits cannot distinguish whether it is produced by inactivated vaccine immunization or natural infection

Method used

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  • Protein antigen, coding gene thereof, and application of protein antigen and coding gene thereof to identification of mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody
  • Protein antigen, coding gene thereof, and application of protein antigen and coding gene thereof to identification of mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody
  • Protein antigen, coding gene thereof, and application of protein antigen and coding gene thereof to identification of mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] This example is used to illustrate the preparation method of the protein antigen provided by the present invention

[0122] 1. Construction of recombinant bacteria

[0123] Entrust Chengdu Qingke Zixi Biotechnology Co., Ltd. to synthesize the nucleotide sequence shown in SEQ ID NO: 1 and the amplification primers of SEQ ID NO: 1. The upstream and downstream 5' ends of the amplification primers were respectively introduced with restriction endonucleases The sequences of enzymes BamHI and XhoI restriction sites (underlined) and corresponding protective bases (underlined wavy lines) are as follows:

[0124] Upstream primer (SEQ ID NO:8):

[0125] Downstream primer (SEQ ID NO:9):

[0126] The PCR reaction system is 50 μL: Primestar Max Premix 25 μL, DNA template 0.5 μL, upstream primer (10 pmol / μL) 0.5 μL, downstream primer (10 pmol / μL) 0.5 μL, ddH 2 O 23.5 μL.

[0127] The PCR reaction conditions were: pre-denaturation at 98°C for 5 min; pre-denaturation at 98°C ...

Embodiment 2

[0139] This embodiment is used to illustrate the collection and detection of pig serum

[0140] In the Mycoplasma hyopneumoniae negative pig farms, piglets were immunized with Mycoplasma hyopneumoniae vaccine Xiyifeng on the 28th day after weaning on the 21st day, boosted once on the 42nd day, and blood was collected from the anterior vena cava on the 63rd day. A total of 28 serum samples were collected.

[0141] 30 samples of commercial pig blood at the age of 90-200 days were collected from a pig farm positive for Mycoplasma hyopneumoniae.

[0142] The collected serum was detected by the Mycoplasma hyopneumoniae antibody kit of IDEXX Company, and 20 copies of Mycoplasma hyopneumoniae antibody-positive serum (hyperimmune serum) after vaccine immunization and 20 copies of Mycoplasma hyopneumoniae-positive pig farm antibody-positive serum (convalescent serum) were preserved.

Embodiment 3

[0144] This example is used to illustrate the determination and operation steps of the optimal reaction conditions of the ELISA kit

[0145] 1. Determination of optimal antigen coating concentration, blocking solution and blocking time

[0146] Dilute the purified protein with carbonate buffer. Concentrations were 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 100 μL was added to each well of the ELISA plate, incubated at 37 °C for 1 hour and then overnight at 4 °C. The washing solution was washed 5 times for 3 min each time, and 200 μL of PBS containing 2.5% by weight of skim milk powder was added to block for 0.5 h. The washing solution was washed 5 times for 3 min each time, and 3 parts of hyperimmune serum and 3 parts of convalescent serum were diluted 1:1000 with PBS containing 2.5% by weight skimmed milk powder, 100 μL was added to each well, and incubated at 37°C for 0.5 h. The washing solution was washed 5 times for 3 min each time, and the HRP-labeled ...

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Abstract

The invention relates to the field of biotechnology, in particular to a protein antigen, a coding gene thereof and application of the protein antigen and the coding gene thereof to identification of amycoplasma hyopneumoniae inactivated vaccine antibody and a natural infection antibody. An amino acid sequence of the protein antigen is shown as SEQ ID NO:1. A detection system disclosed by the invention can rapidly and conveniently identify the mycoplasma hyopneumoniae inactivated vaccine immune antibody and the natural infection antibody at high specificity and high sensitivity, does not needany complex instrument, can meet the current demand of identification of a mycoplasma hyopneumoniae high-immunity serum antibody and a serum antibody at a recovery period well, can be popularized andapplied on a large scale, and has a wide market prospect and great economical and social benefits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein antigen and its coding gene and their application in distinguishing mycoplasma hyopneumoniae inactivated vaccine antibody and natural infection antibody. Background technique [0002] Mycoplasma pneumonia of swine (MPS), also known as swine asthma or swine asthma, is a chronic, contagious disease caused by Mycoplasma hyopneumoniae (Mhp), which spreads in pigs and pigs through the respiratory tract. Transmission between herds is one of the most common diseases in pig farms. The disease usually does not cause death in pigs, but it can cause symptoms such as fever, cough, and difficulty breathing, resulting in loss of appetite, growth retardation, reduced feed conversion, and delayed time to market. Once the disease is introduced into pigs, it is difficult to completely eliminate it, and Mhp usually causes porcine respiratory disease complex (PRDC) with other viruses or bacte...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K14/30C12N15/31C12R1/35
CPCC07K14/30G01N33/6854
Inventor 丁红雷徐作波文玉康
Owner SOUTHWEST UNIVERSITY
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