The Confirmatory Study Of Interaction Of LGALS1 With PRRSV Protein 2b

Porcine reproductive and respiratory syndrome virus(PRRSV)which contains single-strand positive-sense RNA is a member of the genus Arterivirus,family Arteriviridae,order Nidovirales.PRRSV can cause sow reproductive dysfunction and neonatal pig respiratory symptoms.The protein 2b,a small structural protein in PRRSV,was assumed to play a role as an ion channel in the case of virus hatching,and can cause the PRRSV particles to shell,and release RNA into the host cell,but does not work on stage of assembly of the virus.Studying the interaction between 2b protein and host cell protein contributes to understand the mechanism of PRRSV infection and the function of 2b protein comprehensively.In the previous study,using bait strains containing the PRRSV 2b gene and porcine alveolar macrophages(PAM)cDNA library conducted the yeast two-hybrid test,and the LGALS1 interacting with the 2b protein was successfully screened.This study has successfully constructed pGADT7-LGALS1 recombinant plasmid,and transferred them to the competent Y187 yeast,then conducted yeast return test with Y2 H yeast strain containing 2b gene.The result shows that colonies grown on the plate of SD /-Trp-Leu-His-Ade / AbA / X-?-gal were blue,indicating that interaction between the two proteins take places.In the GST-pull down test,the recombinant plasmid pEGX-KG-2b with GST tag and the recombinant plasmid pET-28a-LGALS1 with His tag were constructed,and then expressed by prokaryotic expression in Escherichia coli competent BL21,after the purification,the obtained protein was subjected to pull down test.The result shows that interaction takes place between PRRSV 2b and LGALS1.In the co-immunoprecipitation assay,recombinant plasmids pCMV-HA-2b and pCMV-Myc-LGALS1 were constructed and co-transfected into HEK293 cells,and cleaved those cells under non-denaturing conditions.This method confirms the interaction between PRRSV 2b and LGALS1.In this research,the interaction between LGALS1 and PRRSV 2b protein was successfully verified by yeast regression test,GST-pull down test and immunoprecipitation test,and provides a theoretical basis for the study of PRRSV infection pathways and proliferation processes.