| Porcine Reproductive and Respiratory Syndrome(PRRS,also known as blue ear disease),Porcine Epidemic Diarrhea(PED)and other diseases have severely plagued our country’s pig industry.They caused huge losses to our country’s economy.Due to the complex pathogenic mechanism and rapid virus mutation,vaccine immunization cannot be used to completely prevent or control them.Researchers successfully bred PRRS-resistant pigs by editing the CD163 gene(PRRSV receptor),which can effectively resist PRRSV infection.It can effectively resist PRRSV infection,which provides strategies and confidence for pig breeding against PED or other diseases.However,other important genes(except CD163)which can be used for PED resistance breeding and studying the mechanism of PRRS-resistance or PRRSV-host interaction,still need to be researched and excavated.In the early stage of our laboratory,55 candidate genes related to PRRSV infection were high throughput screened out by using the whole-genome gene knockout technology.In this study,three genes(IL20RB,ATP6V0A1 and STX10)were selected from 55 candidate genes based on gene function and signaling pathway.We preliminarily verified the role of these genes in the infection of PRRSV and PEDV.In order to explore the effect of candidate genes on multiple virus infections quickly and efficiently,another eight genes were selected.To knock out Marc145 cell line(African green monkey kidney cells,which can be infected by a variety of porcine viruses),we constructed g RNA vectors for eight candidate genes knocked out separately(our laboratory confirmed which can significantly regulate PRRSV proliferation)to explore the role of them on multiple virus infections.The main results are as follows:1.We studied the inhibiting effect of IL20 RB,ATP6V0A1 and STX10 gene-knockout cells on the proliferation of PRRSV.The pig cell line(PK15-CD163-Cas9,our laboratory prepared before)was used for gene knockout and infection with PRRSV.We got cell with three genes knockout separately by using CRISPR/Cas9 technology(PK15-CD163-Cas9-ATP6V0A1,PK15-CD163-Cas9-IL20 RB and PK15-CD163-Cas9-STX10),they were used to infect with PRRSV(experimental group)and normal cell(control group)was infected too.The ORF7 expression level of PRRSV was detected by fluorescence quantitative PCR.The expression level of ORF7 in the experimental group was significantly lower than the control group,which indicates that knockout IL20 RB,ATP6V0A1 and STX10 can Inhibit PRRSV infection significantly;2.We analyzed the function of IL20 RB,ATP6V0A1 and STX10 gene separately knocked-out to PEDV infection: In order to explore the effect of three genes on the proliferation of PEDV,we used CRISPR/Cas9 technology and IPEC-J2-Cas9 cell line(our laboratory prepared before)to prepare for gene knockout cell(IPEC-J2-Cas9-ATP6V0A1,IPEC-J2-Cas9-IL20 RB and IPEC-J2-Cas9-STX10).Three gene knocked-out cell(experimental group)and normal cells(control group)were used to be infected with PEDV.Detect the N protein transcription level of PEDV by fluorescence quantitative PCR.Compared with the control group,the N protein transcription level of PEDV was significantly reduced in the experimental group,which proved that knocking out IL20 RB,ATP6V0A1 and STX10 respectively can significantly inhibit the proliferation of PEDV;3.By using CRISPR/Cas9 technology,we constructed gene knock-out g RNA expression vector for 8 genes.Marc145 cell can be infected by a variety of porcine viruses and is one of the most commonly used cell models for porcine virus research.In order to explore the effect of the selected pig candidate genes on more virus infections Efficiently,we selected another eight genes(VTN,F11 R,UBB,LGALS2,MAP4K3,NDUFB3,PPP2 CA and KXD1,which have been confirmed in regulating PRRSV proliferation significantly)from the 55 candidate genes.According to the NCBI database,they are homologous to the above eight pig genes.We designed 2 g RNA for each gene.Fifteen g RNA expression vectors(one of the carriers is not well connected)were lentivirus-packaged and prepared to construct gene knockout cells and identify important genes effect on multiple porcine virus infections.The results of this study will lay the foundation for animal disease-resistant breeding,prevention and treatment drugs,vaccine design and development,as well as the in-depth study of the functions of these genes and the mechanism of pig resistance to PRRSV or PEDV infection.In addition,it is necessary to study of the effects and mechanisms of these three genes on multiple and different virus infections. |
