Infection Of PRV In Lung Tissue Of CGAS Gene Knockout Mice

Pseudorabies virus（PRV）,also known as Suid Alphaherpesvirus 1（Su HV-1）or Aujeszky disease virus（AD）,is a member of the herpesviridae,herpesviridae and varicella virus genus.Pseudorabies（PR）is an infectious disease caused by PRV in a variety of animals with fever,itching（except pigs）and encephalomyelitis.In addition to its natural host pigs,pseudorabies virus can infect a wide range of mammals.In view of the importance of cGAS in immune regulation of host resistance to virus infection,this study used CRISPR/Cas9 gene editing technology to construct cGAS gene knockout mice,and tested the effects of cGAS gene knockout mice infected with PRV on PRV proliferation compared with wild-type mice.To prove the role of cGAS in inhibiting virus proliferation.The main research results are as follows:1.cGAS knockout had no effect on lung and brain morphology of C57BL/6N miceIn this study,cGAS gene knockout mice were constructed using CRISPR/Cas9 gene editing technology.The gene sequencing and the lung and brain tissues of homozygous mice and wild-type mice were used to detect the cGAS gene knockout by Western Blot.The gene sequencing results showed that the total knockout length between exon 1 and exon 5 was 3663 bp.Western Blot results showed that cGAS gene had been completely knocked out.H&E staining was used to detect the lung and brain tissue morphology of cGAS knockout mice and control mice.The results showed that cGAS knockout had no effect on the lung and brain tissue morphology.2.cGAS knockout improves PRV virus titerIn order to study the effect of cGAS knockout on the virus titer of PRV,PFU method was used in this study to detect the changes in virulence of PRV infected C57BL/6N mice and C57BL/6N-cGAS^-/-mice.The results of PFU test showed that the virus titer of C57BL/6N-cGAS^-/-mice was significantly higher than that of C57BL/6N mice with the increase of PRV infection time.The results showed that knockout of cGAS could significantly improve the virus titer of PRV.3.cGAS knockout promoted the transcription of PRV-g B and PRV-TK m RNA and the expression of PRV-g B and PRV-g E proteinsTo investigate the effect of cGAS knockout on the transcriptional translation of PRV-g B and PRV-TK and the expression levels of PRV-g B and PRV-g E proteins.In this study,q RT-PCR and Western Blot were used to detect m RNA transcription levels of PRV-g B and PRV-TK and the protein expression levels of PRV-g B and PRV-g E in the lung tissues of mice infected with PRV.The results showed that the m RNA expression levels of PRV-g B and PRV-TK and the protein expression levels of PRV-g B and PRV-g E in C57BL/6N-cGAS^-/-mice were significantly higher than those in C57BL/6N mice with the increase of PRV infection time.The results indicated that knockout of cGAS promoted the transcription of PRV-g B and PRV-TK genes and the expression of PRV-g B and PRV-g E proteins.4.cGAS knockout inhibited PRVregulated mrna transcription of IFN-β,ISG15 and ISG20In this study,q RT-PCR was used to detect the m RNA expression levels of IFN-β,ISG15 and ISG20 in C57BL/6N and C57BL/6N-cGAS^-/-mice infected with PRV.The results showed that the m RNA expressions of IFN-β,ISG15 and ISG20 in C57BL/6N mice were significantly higher than those in C57BL/6N-cGAS^-/-mice with the increase of PRV infection time,indicating that cGAS^-/-inhibited the transcription of IFN-β,ISG15 and ISG20 m RNA.5.cGAS knockout reduces the survival rate of miceTo detect the difference in survival rate of C57BL/6N and C57BL/6N-cGAS^-/-mice infected with PRV,statistical analysis showed that C57BL/6N and C57BL/6N-cGAS^-/-mice died after 3 days of infection.However,the mortality rate of C57BL/6N-cGAS^-/-mice was significantly higher than that of the control group,and all the mice died after 5 days of virus infection.The results showed that knockout of cGAS could promote the death of mice.