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CD163 mutant and application thereof

A mutant and amino acid technology, applied to CD163 mutants and their application fields, can solve problems to be fully revealed and so on

Active Publication Date: 2019-08-09
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some studies have been done so far, the important amino acid sites involved in PRRSV infection in CD163 have yet to be fully revealed

Method used

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  • CD163 mutant and application thereof
  • CD163 mutant and application thereof
  • CD163 mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Alignment of CD163 Amino Acid Sequences and Site-directed Mutagenesis of Amino Acids in Different Species

[0028] Through the online software Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo) CD163 of different species, including porcine CD163 (UniProt entry Q2VL90), monkey CD163 (UniProt entry Q2VLG4) and human The amino acid sequences of CD163-like (UniProt entry Q9NR16) were compared.

[0029] The result is as figure 1 As shown, the Clustal Omega software shows that the partial amino acid sequences of different sources of CD163 are conservative, and some amino acids are non-conservative amino acids, and the 534th amino acid (numbering is based on UniProt entry Q2VL90) is a polar amino acid, but the charge is different ( Both pig and monkey sources are acidic glutamic acid E, and human source is basic lysine K).

[0030] Therefore, the amino acid at this site was selected for site-directed mutation, and E was mutated into K, ie E534K. Using the P...

Embodiment 2

[0039] Example 2. CD163 wild type and mutant recombinant expression

[0040] PK-15 cells were treated at a cell concentration of 2.0×10 524-well plate per cell / ml, 500 μl / well; remove the original cell culture medium, add DMEM medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin 500 μl (Gibco Company, USA), cultivated for 12 hours. The wild-type and mutant CD163 plasmids were transfected with Lipofectamine LTX reagent withPlus reagent transfection reagent (Invitrogen, USA) according to the operating instructions, 1 μg plasmid / well. After 24 hours of transfection, remove the cell culture medium, collect the cells with a cell scraper, and centrifuge at 3000 g for 5 minutes; resuspend with PBS, then centrifuge, and wash twice with PBS. PBS was discarded, and 100 μl of cell lysate (Shanghai Beyotime Biotechnology Co., Ltd.) was added to obtain whole cell lysate (WCL); centrifuged at 13000 g, 4°C for 15 min, to obtain WCL su...

Embodiment 3

[0042] Example 3. CD163 mutant inhibits PRRSV from infecting host cells

[0043] 3.1 Real-time quantitative PCR detection of PRRSV-infected host cells

[0044] Incubate the PK-15 cells expressing wild-type or mutant CD163 in Example 2 at 37°C for the PRRSV BJ-4 strain with a multiplicity of infection (MOI) of 1; discard the cell supernatant, wash the cells three times with PBS, Viruses from uninfected cells were washed away; 500 μl DMEM medium was added to the cell wells, and cultured at 37° C. for 12 hours.

[0045] Collect the cells, discard the cell supernatant, and wash the cells three times with PBS. Use RNA extraction reagent TRIzol (Invitrogen, USA) to extract the total RNA of PRRSV-infected cells, use PrimeScript TM The reverse transcription kit (TaKaRa Company) generates complementary DNA (cDNA) as a template for fluorescent quantitative PCR. The pMD-18T-ORF7 plasmid stored in the laboratory was used as a standard, and the 10-fold dilution was used as a template to...

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Abstract

The invention discloses a CD163 mutant and application thereof. According to the CD163 mutant, a 534th site of an amino acid sequence is changed from glutamic acid E to lysine K. By comparing amino acid sequences of CD163 of different species, amino acid at the 534th site is selected for site-directed mutation (the glutamic acid E is mutated into the lysine K, namely E534K), and an eukaryotic expression vector system is constructed; a wild type (WT) and the mutant CD163 are subjected to recombinant expression in a porcine kidney PK-15 cell; the cell is infected with a PRRSV classical strain BJ-4 (GenBank No.AF331831) and an HP-PRRSV strain HN07-1 (GenBank No.KX766378.1); through the identification of a fluorescent quantitative PCR and a virus titer experiment (50% tissue culture infectivedose, TCID50), the mutation of the amino acid at the 534th site has a significant inhibitory effect on PRRSV-infected host cells, and the CD163 mutant can be applied to breeding of anti-PRRSV pigs andresearch and development of anti-virus vaccines and drugs.

Description

technical field [0001] The invention relates to a CD163 mutant and application thereof, belonging to the fields of molecular biology, virology and cell biology. Background technique [0002] Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS), commonly known as porcine blue ear disease, is a reproductive disorder such as abortion, stillbirth, weak fetus and mummified fetus in pregnant sows, as well as respiratory symptoms in pigs of all ages. Characteristic highly contagious disease. PRRS is very harmful to my country's pig industry, and it is one of the infectious diseases that restrict the healthy development of domestic pig industry. Especially in 2006, the highly pathogenic PRRS (highly pathogenic PRRS, HP-PRRS), which was characterized by high fever, high morbidity and high mortality, broke out and prevailed in a large area in my country, which caused an unprecedented blow to the domestic pig industry. . The pathogen of ...

Claims

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Application Information

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IPC IPC(8): C07K14/705A01K67/027A61K38/17A61P31/14
CPCC07K14/70596A01K67/0275A61P31/14A01K2207/15A01K2227/108A61K38/00
Inventor 张改平李睿马红芳乔松林陈鑫鑫郭振华赵东李学伍
Owner HENAN ACAD OF AGRI SCI
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