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Polypeptide capable of specifically bonding porcine reproductive and respiratory syndrome viruses, screening method thereof and use thereof

A technology for respiratory syndrome and pig reproduction, applied in the field of animal virology, can solve the problems of complicated operation steps, low sensitivity and low specificity, and achieve the effects of simple operation, high sensitivity and high specificity

Active Publication Date: 2013-01-09
国家兽用生物制品工程技术研究中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the use of polyclonal antibodies for indirect immunofluorescence detection of PRRSV is cumbersome, with low specificity and low sensitivity.

Method used

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  • Polypeptide capable of specifically bonding porcine reproductive and respiratory syndrome viruses, screening method thereof and use thereof
  • Polypeptide capable of specifically bonding porcine reproductive and respiratory syndrome viruses, screening method thereof and use thereof
  • Polypeptide capable of specifically bonding porcine reproductive and respiratory syndrome viruses, screening method thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0040] Example 2 The identification of the bacteriophage screened and the binding characteristics of PRRSV

[0041] 1. Pick a single clone for ELISA identification

[0042] After the third round of screening product (elution product) among the embodiment 1 is diluted, the plates are spread, and each phage monoclonal is picked and inoculated respectively in the early logarithmic growth phase (OD 600 about 0.3) of ER2738 (Ph.D.-12 TM PhageDisplay Peptide Library Kit (purchased from New England Biolabs) was amplified for 4.5 hours, centrifuged to get the supernatant, measured phage titer, and adjusted the concentration to 10 11 pfu / 10 μL.

[0043] Coat the purified PRRSV on the ELISA plate (positive value P), and at the same time coat the lysed supernatant of Marc-145 cells as a negative control (negative value N), coat overnight at 4°C, and block with blocking solution at 4°C for 1 hour the next day The same phage clone was added to the two coated wells, and then the rabbit ...

Embodiment 3

[0061] Example 3 Detection of the inhibitory ability of positive phages to PRRSV

[0062] 1. Amplification of Positive Phage Clones

[0063] Get 5 μ L of the positive phage clone supernatant that embodiment 2 step 1 obtains, add to 20 mL logarithmic growth early stage (OD 600 ~0.3) host strain ER2738 (Ph.D.-12 TM Phage Display Peptide Library Kit (purchased from New England Biolabs), amplified for 4 hours, centrifuged at 10,000 rpm for 10 minutes, supernatant was taken, precipitated twice with PEG-NaCl solution, and finally dissolved with 200 μL of TBS to obtain amplified Positive phage is cloned, and its titer is detected again, and the method is the same as the operation of step 2.2 in the embodiment 1.

[0064] 2. Determination of half tissue infection dose (TCID50)

[0065] Seed Marc-145 cells in 96-well plate in advance, and when the monolayer of cells adheres to 80% of the well, add 10 μL of 1.0×10 11 PFU of phage was mixed with 90 μL of 10-fold serially diluted PRR...

Embodiment 4

[0068] Example 4 FITC-labeled short peptide detection of PRRSV

[0069] 1. Artificial synthesis of FITC-labeled segmented peptides

[0070] Comprehensive comparison of ELISA and TCID 50 As a result of the detection, the phage clone Ph.D-PRRSV-4 was selected, and the short peptide sequence displayed by it was His Trp Trp Ser Trp Pro Ser Tyr Thr Gln Ser Ser, so the short peptide was artificially synthesized: His Trp Trp Ser Trp Pro Ser Tyr Thr Gln Ser Ser-FITC.

[0071] 2. Direct immunofluorescence detection of PRRSV

[0072] Marc-145 cells were grown on 24-well cell culture plates at 5.0 × 10 6 Cells / well were plated, and 200 TCIDs were added to each well after 24 hours to grow into a monolayer of cells 50 The PRRSV virus, and another two wells were kept as controls, and continued to culture for 48h until obvious lesions. After discarding the culture medium, wash with PBS 3 times, fix with ethanol for 10 min, and wash with PBS 3 times. Lyse with 0.3% (v / v) Triton for 5 mi...

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Abstract

The invention relates to a polypeptide capable of specifically bonding porcine reproductive and respiratory syndrome viruses (PRRSV), a screening method thereof and use thereof, and belongs to the field of animal virology. The polypeptide is His Trp Trp Ser Trp Pro Ser Tyr Thr Gln Ser Ser. The invention also provides a screening method of the polypeptide, which comprises: acquiring viruses; screening out phage monoclones with high affinity with the PRRSVs; and sequencing the phage monoclines. The PRRSV inhibition capabilities of the phage monoclines are tested, phage monoclines which can inhibit the duplication of the PRRSVs obviously are selected, and the polypeptide of the selected phage monoclines is the target polypeptide. The polypeptide can be used in PRRSV detection. When marked by an FITC marker, the polypeptide can be used for detecting PRRSVs. The screening method of the polypeptide of the invention is short in period and simple in operation and compared with a monoclonal antibody making method, is quicker and more convenient. The screened polypeptide can be used for detecting the PRRSVs with simple operation and has high specificity and sensitivity.

Description

technical field [0001] The invention relates to a polypeptide specifically binding to porcine reproductive and respiratory syndrome virus and a method for obtaining the polypeptide through screening, belonging to the field of animal virology. Background technique [0002] Porcine reproductive and respiratory syndrome is an infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). , Piglets and fattening pigs have respiratory symptoms. The disease broke out in my country at the end of 1995, and it has become the most important disease that endangers the pig industry in my country. People have conducted in-depth research on the functions of PRRSV structural and non-structural proteins, the pathogenic mechanism of the virus, the mechanism of persistent infection of the virus, and the immune mechanism of the body against the virus. PRRSV infects the macrophage system of the pig body, causing the body's cellular immune function to be low,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/04G01N33/569G01N21/64
Inventor 徐海于辙吕芳侯继波
Owner 国家兽用生物制品工程技术研究中心
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