The Role of Porcine Lck Protein in PCV2 Replication in vitro
In order to identify important host factors in cell entry and establishment of infection of porcine circovirus type 2(PCV2),the effects of porcine lymphocyte specific tyrosine kinase Lck expression and activity on PCV2 infection replication were analyzed,and Lck was identified as a new target for the regulation of PCV2 replication.In this study,based on analysis of the struc-tural characteristics and sequence conservation of porcine Lck,the Lck SH3 domain was selected as the immunogen gene sequence,and the polyclonal antibody against porcine Lck was prepared in mice using the protein expressed the E.coli expression system.Then,the effect of PCV2 in-fection on the expression and activity of Lck in PK-15 and 3D4/21 cells was detected using West-ern blot.Finally,the effects of porcine Lck expression and phosphorylation on PCV2 replication in PK-15 cells transiently transfected with overexpressed vector or treated with Lck specific inhib-itor(A770041)were detected by Western blot and RT-qPCR,respectively.The results showed that mouse derived polyclonal antibodies against pig Lck were successfully prepared.There was no significant change in the expression level of Lck,but the phosphorylation level of Lck was sig-nificantly up-regulated in the early stage of PCV2 infection in PK-15 and 3D4/21 cells.Overex-pression of Lck significantly increased PCV2 Cap protein expression and viral copy number,while inhibition of Lck phosphorylation level with A770041 could significantly downregulate the expres-sion of PCV2 Cap protein,viral copy number and virus titer.These results indicated that Lck positively regulated PCV2 replication,which lays an important foundation for exploring the new molecular mechanism of PCV2 infection.
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维普
