Investigation Of The Role Of Vimentin In PRRSV Infection Of Cells

Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused byporcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly causedreproductive disorder, including of premature, abortion, fetal death, mummy and porcinerespiratory syndrome at variety of ages and high mortality. The disease was first discovered inamerican in1987and then prevailed in many countries and regions. In1996, Guo Baoqingfirst isolated a PRRSV strain from aborted fetuses and verified the evidence of PRRSV in ourcountry. The disease caused high morbidity and mortality and the clinical symptom becamemore complicated and the prevalence gradually expanded. At the present, PRRS has prevailedin the main world-while pig-raising country and districts and caused pig-raising largeeconomic losses in the world, and no effective standard vaccine candidates can control thehappening of PRRS. As one receptor of PRRSV, Vimentin plays an important role with theother cytoskeletal filaments in PRRSV infection, but the mechanism of action is stillunknown.To investigate the function of swine vimentin in PRRSV infection, the swine vimentingene from a PRRSV nonsusceptible cell lines PK-15cells was amplified by RT-PCR andcloned into pET-28a vector. The recombinant swine vimentin protein was expressed inEscherichia coli BL21(DE3) and was identified by SDS-PAGE and Western blot. Afterpurification, the target protein was used to immunize New Zealand rabbit to producepolyclonal antibodies. The recombinant swine vimentin protein and polyclonal antibodieswere used to test blocking function of PRRSV infection in Marc-145cells by virusneutralizations. The results showed that the swine vimentin gene was amplified successfullyfrom PK-15cells and cloned into pET-28a vector. Rabbit anti-vimentin antibodies wereproduced with the titer of1×105. PRRSV infection of Marc-145cells was blocked partially bythe recombinant vimentin protein while blocked completely by the antibodies.The vimentin gene was amplified by RT-PCR from Mare-145cells and inserted intopcDNA3.1/V5-His A plasmid together with EmGFP gene and IRES to construct therecombinant plasmid pcDNA3.1-GIV. The pcDNA3.1-GIV plasmid was transfected intoBHK-21cell line to establishment stably expressing vimentin in the presence of G418.Vimentin was detected by western blot and vimentin mRNA was detected by RT-PCR. Theresult indicated that the BHK-21-GIV cells could express exogenous vimentin. TheBHK-21-GIV cells were infected with500TCID50PRRSV, IFA result indicated that PRRSV infects BHK-21-GIV. This work establishment of a BHK-21-GIV cell line stably expressingvimentin from Marc-145cell line, and PRRSV can infect BHK-21-GIV cells.And4pairs of primers were designed based on the gene sequence of MA-VIM toamplify the gene sequence of vimentin by RT-PCR. The prospective fragments were obtainedand the size were as follows:807bp,876bp,1002bp and594bp. The4fragments were clonedinto pET-28a vector and expressed in Escherichia coli BL21(DE3). SDS-PAGE suggestedthat recombinant proteins were expression and the sizes as follows:34.3KD,38.2KD,42.6KDand26.5KD. The obtained recombinant proteins reacted with anti-His MAb specifically byWestern blot. I-ELISA indicated that the4fragments can react with PRRSV-N proteinspecifically.The study on PRRSV receptor benefits for the research of relationship between PRRSVand host cells, elucidates the procedure of PRRSV infect susceptible cell lines and themechanism of PRRSV pathopoiesis, which can provide new idea on PRRSV precaution andcontrol. These results provide additional information on the function of vimentin in PRRSVinfection of Marc-145cells.