Research On Function Of Prokineticin 2 In The Inlfammation Of The ORFV

Orf disease is a zoonosis caused by the orf virus(ORFV),which can cause infections in goats,sheep,many wild animals and humans.It shows redness pimples,blisters,pustules and scabs in the lips,nose,tongue,and breasts of affected animals.Orf disease is widely distributed around the world,and the occurrence and prevalence of this disease are common in places where sheep are raised.The occurrence and prevalence of this disease are common in northwest,northeast,and southern of China.Because the disease has a high infection rate on the sheep and is susceptible to recurrence after infection,it is difficult to remove the sheep once it is infected.Therefore,the disease has caused serious damage to China’s sheep industry.At present,the comprehensive prevention and control of Orf disease is almost in a blank state because of lack of basic data on the disease and its pathogenic.In-depth understanding of ORFV-mediated inflammatory mechanisms and body reactions is the scientific basis for prevention and control of the disease.Prokineticin 2(PK2)is an endogenous small molecule secreted protein in mammals.It interacts with the receptor G protein-coupled receptor family and participates in important physiological functions such as angiogenesis,inflammation,and immune regulation.Prokineticin 2 and its mediated downstream effects are closely related to the occurrence of inflammation.At present,there is no study on the role of PK2 in the inflammatory process of the infection of ORFV.The basic data of the protein in the host inflammation induced by ORFV is the basis for the development of prevention and treatment methods based on the protein.Therefore,the PK2 gene of Hainan black goat was cloned,to construct PK2 eukaryotic overexpression plasmid and RNAi interference plasmid.The role of PK2 protein in ORFV inflammation was studied.The study is as follows:1.Amplification of Black goat PK2 gene and sequence analysisThe PK2 gene of Hainan black goat was amplified by RT-PCR using Hainan black goat tissue total RNA as a template and submitted to GenBank accession no: KY655329.The related bioinformatics analysis of PK2 showed that the goat PK2 gene is 327 bp in length and encodes 109 amino acids with a protein molecular mass of 11.74 Ku;the lowest nucleotide and amino acid sequences were 41.6% and 24.8% homologous to deer,and 96.3% amino acid homologous to cattle;based on the phylogenetic tree of PK2,Hainan black goats and cattle have the highest affinity;PK2 protein exists in the Prokineticin domain and is a structurally conserved protein;The protein has a transmembrane region and containing a signal peptide.2.Construction of overexpression plasmid and RNA interference plasmid of PK2The pMD18-T-PK2 plasmid was used as a template to design specific primers containing two restriction sites of Bgl II and EcoR I,pEGFP-N1-PK2 eukaryotic overexpression plasmid was constructed.pEGFP-N1-PK2 was transfected into OFTu cells.The green fluorescence was detected by fluorescence microscopy and IFA detection,indicating that the overexpression vector was successfully constructed.Oligonucleotide primers of the PK2 gene were designed,and pSUPER-PK2 interference vector was constructed.The pSUPER-PK2 transfected OFTu cells were observed by fluorescence microscopy and ELISA detection.The results showed that the inhibitory efficiency of PK2 mRNA expression in pSUPER-PK2 transfected 24 h was 51%.3.Research on the role of PK2 during the ORFV infection in host cellsOFTu cells infected with ORFV,PK2 gene has a tendency of up-regulation,indicating that PK2 is one of the virus-infected effector proteins.Infection of OFTu cells transfected with PK2 overexpression and interfering expression plasmids by ORFV.The results showed that,after overexpression of PK2,interleukin-1β(IL-1β)was significantly inhibited;after PK2 was interfered,the expression of IL-1β and interleukin-18(IL-18)was significantly upregulated,pattern recognition receptor proteins(NLRP3)also showed a trend of up-regulation.This indicates that PK2 protein may play a role in the inflammatory process of ORFV through NLRP3 signaling pathway.