A kind of cd169 gene related to pig immune traits and its preparation method and application

Abstract

The invention discloses a pig immune trait associated CD169 gene and a preparation method and use thereof. The preparation method comprises: extracting total DNA from the blood of a pig; designing premiers (a forward primer: 5'-GGGCATTCAAAGCCAAACTC-3', and a backward primer: 5'-CCTCGGGTGTAGCCCTCAAA-3') by using pig CD169 sequence (GeneBank registry number: N_010459.2) issued by GenBank as a template; performing polymerase chain reaction (PCR) amplification; purifying the product of PCR amplification, cloning the product of PCR amplification and sequencing the product of PCR amplification; andthus, obtaining the nucleotide sequence represented by SEQ ID No.1, wherein the sequence comprises a mutation locus C305-A305 that can be identified by HinfI endonucleases and therefore the polymorphism of the pig CD169 gene can be detected by a created restriction site-polymerase chain reaction- restriction fragment length polymorphism (CRS-PCR-RFLP) method. The gene is mainly used to mediate the adhesion and endocytosis of porcine reproductive and respiratory syndrome virus (PRRSV). After invading into a body, the PRRSV first bonds with receptor proteins (for example CD169) on the membrane of the cell and then moves into the cell under action of endocytosis with assistance from the CD169. The invention provides a new molecular marker for the marker-assisted selection of pigs.

Description

technical field [0001] The invention belongs to the technical field of livestock genetic engineering, and in particular relates to a CD169 gene related to pig immune traits, a preparation method of the CD169 gene related to pig immune traits, and an application of the CD169 gene related to pig immune traits. Background technique [0002] Since the discovery of porcine reproductive and respiratory syndrome virus (PRRSV), it has become prevalent in most parts of the world and has brought huge economic losses to the global swine industry. In order to gain a deeper understanding of the pathogenesis of porcine reproductive and respiratory syndrome and control the disease, researchers need to study the biological characteristics of the PRRSV receptor, including the involvement of virus adsorption, internalization, nucleocapsid disassembly, and genome release. receptor. However, traditional treatment methods have many limitations and cannot effectively solve the problem, and the r...

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Problems solved by technology

The sialic acid agglutinating activity plays an important role in the binding of PRRSV to the receptor CD169. After converting the N-terminal R116 amino acid into E116 amino acid by point mutation , although the structure of the whole protein did not change significantly, pSn lost the ability of sialic acid aggregation, resulting in the inability of the virus to adsorb and endocytize (Delputte PL et al. Porcine arterivirus attachment to the macrophage-specific receptor sialoadhesin is dependent on the sialic acid- binding activity of the N-terminal immunoglobulin domain of sialoadhesin. J Virol, 2007, 81(17): 9546-9550.), indicating that R116 is the binding of CD169 to the M / GP5 dimer of the virus important site of

In addition, R97 of the sialic acid binding region of CD169 can not only form a salt bridge with the carbohydrate of sialic acid, but also form a hydrophobic contact with the glycerol side chain of sialic acid. When R After 97 was replaced by lysine, it affected the hydrophobic interaction between CD169 and sialic acid, hindered the formation of salt bridge, and resulted in a significant decrease in the binding ability to sialyllactose

Similarly, conversion of R97 to alanine also causes CD169 to lose its ability to bind sialic acid

[0006] After treating PAM with the specific antibody MAb 41D3 of heparanase and pSn at the same time, the adsorption of PRRSV to PAM was completely blocked, indicating that both heparan sulfate and pSn Mediates viral adsorption to receptors, and other receptors involved in PRRSV adsorption may be absent on the PAM surface, or present but in such small numbers that they cannot be detected

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