Identification Of Host Cell Replication-Related Protein ANXA8 Of Porcine Astrovirus Type Ⅰ

Astrovirus is an extremely common causative agent of intestinal disease.It has complex hosts,wide spread and is distributed all over the world.Its genome is unstable,high incidence of variation,and its pathogenicity and cell tropism are constantly changing.It has the potential to greatly endanger production and break through the interspecific barrier and become a zoonotic pathogen.However,little is known about the proteins that affect the replication of astrovirus in vivo,which adds obstacles to the in-depth study of the infection and replication process of astrovirus.Porcine Astrovirus 1(PAst V1)is a classical strain of porcine stellate virus,which is representative,and is highly similar to the classical strain of human stellate virus in pathogenicity.It is an ideal model for the study of stellate virus.ANXA8 protein is one of Annexin family.In previous experiments,it has been found that it may affect the replication of PAst V1.Therefore,this experiment selects PAst V1 and ANXA8 protein as materials to explore the relationshipbetween them through siRNA inhibition,overexpression and immunofluorescence confocal imaging,that is,whether the expression of ANXA8 protein affects the replication of PAst V1,And participate in the replication process of PAst V1 in host cells,so as to lay a foundation for exploring the mechanism of infection and replication of astrovirus and analyzing its transmission and proliferation mechanism.In the first stage of this experiment,pCAGGS-ANXA8 eukaryotic expression plasmid was constructed and transfected into PK-15 cells,which can overexpress ANXA8 protein in cells;The recombinant protein pET32a-ANXA8 with functional activity was purified from the supernatant of bacterial lysate and used to incubate the virus in subsequent experiments to produce competitive inhibition with ANXA8 protein in PK-15 cells;pGEX6p1-ANXA8 protein was purified to produce mouse anti-ANXA8 antibody with high specificity and sensitivity,which was used to block ANXA8 protein in some PK-15 cells and reduce its interaction with virus.In the second stage,the effect of ANXA8 protein on the replication efficiency of PAst V1-GX1 strain in PK-15 cells was studied.The results showed that inhibition of ANXA8 protein transcription by siRNA could significantly reduce the titer and replication level of PAst V1-GX1.Overexpression of ANXA8 can increase the amount of virus in cell supernatant without affecting the amount of virus in cells.The results of immunofluorescence confocal imaging showed that the capsid protein of the virus was Co-located with some ANXA8 protein in cells.When the exogenous protein or protein antibody was added to a certain dose,it could significantly inhibit virus replication.The expression of porcine ANXA8 protein in non sensitive cell line Vero cells did not increase the replication level of the virus in Vero cells.In conclusion,this experiment found that ANXA8 protein is closely related to the replication of PAst V1-GX1 strain in PK-15 cells,and can promote the replication of PAst V1-GX1 strain in host cells.It is speculated that ANXA8 protein may participate in the packaging,transport or release of pastv-gx1 in cells,but this promotion is race specific and cannot increase the sensitivity of Vero to viruses.