| Ttype Ⅰ Interferon(IFN-Ⅰ)is an important antiviral cytokine in animals,which protects against viral infection by activating the JAK-STAT signaling pathway.During the course of evolution,the virus appears many ways to escape the innate immunity of the host.Therefore,to study the mechanism of innate immunity of virus escaping from host is of great significance for disease prevention and control and vaccine development.Porcine reproductive and respiratory syndrome virus(PRRSV)has clinical synergistic effect with diseases caused by Porcine circovirus,swine influenza virus,multiple system failure syndrome pathogen of weaned piglets,mycoplasma pneumoniae of pigs and haemophilus parahaemophilus of pigs.Since it was reported in China in 1995,the situation has spread to most parts of the country,causing serious economic losses to China’s pig industry and attracting widespread attention.This study investigated the prevalence of PRRSV in south China and the effect of FNAR 1 on PRRSV proliferation in MARC-145 cells,and provided a theoretical basis for the pathogenesis of PRRSV.In this study,534 samples of clinical materials suspected to have PRRS in south China were collected in 2018-2019 and detected by PCR.The positive detection rate was 30.7%,indicating that PRRSV had a high infection rate in south China.Some positive samples were amplified and sequenced by PCR,and 17 strains of ORF5 sequence and 12 strains of NSP2 sequence were obtained.The ORF5 nucleotide sequence homology was 78.1%-99.8%,and the NSP2 nucleotide sequence homology was 69.8%-99.6%.Genetic and evolutionary analysis showed that 4 ORF5 strains belonged to Lineage8.7 branch,6 were in Lineage1(NADC30-like)branch,and 6 were in Lineage3 branch.Genetic and evolutionary analysis of NSP2 gene showed that 4 strains belonged to Lineage8.7 branch and 8 strains were in Lineage1(NADC30-like)branch.In 2018-2019,PRRSV strains in South China were located in NADC30,QYYZ and JXA1 branches.In this experiment,IFNAR 1 gene of African green monkey was inserted into p ET28 a vector,transformed into E.coli BL21 and induced to express by PTG.SDS-PAGE showed that 65 k Da protein was correctly expressed,and the protein mainly existed in inclusion body.After the fusion protein was purified,New Zealand white rabbits were immunized to prepare polyclonal antibodies.ELISA was used to detect the antibody titer in the serum to determine the optimal package concentration of 0.2g /ml and the optimal serum dilution of > 1:1600,providing a material basis for the follow-up study of the JAK-STAT signaling pathway.The CRISPR/Cas9 technique was used to knock out the MARC-145 cell line of IFNAR 1,and it was found to inhibit PRRSV drug taking and replication after inoculation on the cells lacking IFNAR 1.IFNAR1 si RNA was transduced to MARC-145 cells and pcmv-flag-ifnar1 was transfected into cell lines deficient in IFNAR1.The effects of IFNAR1 overexpression and interference on PRRSV proliferation were analyzed.The results showed that the overexpression of IFNAR 1 cells promoted PRRSV adsorption and replication,and formed a metrological dependence.Interference with IFNAR 1 inhibited PRRSV adsorption and replication,and the titer of the virus was significantly lower than that of the control group.It was found that the adsorption and replication of PRRSV were enhanced after the inoculation of cell lines with stable expression of IFNAR 1.Thus,the host cell IFNAR protein has a promoting effect on PRRSV proliferation.In summary,this study investigated the prevalence of PRRSV in south China and provided a theoretical basis for the prevention and control of PRRSV.In this study,it was found that IFNAR 1 in MARC-145 cell line promoted PRRSV adsorption and replication,which laid a foundation for the pathogenesis of PRRSV. |
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