Establishment Of BHK-21Cell Line Stably Expressing CDV Receptor Canine Signaling Lymphocyte Activation Molecule

Canine distemper (CD), caused by canine distemper virus, is an acute and highly contagiousdisease of the order Carnivora. The clinical symptoms of the disease are such as two-phase heat,conjunctivitis, acute coryza, severe gastroenteritis and neurological. The disease spreaded worldwidely. In recent years, reports about CD in rare animals such as the giant panda, the lberian lynxand dogs vaccinated confirmed the host range expansion and antigenic mutation of CDV, thisphenomenon had attaracted numerous scholars’s attention about the unknown host range andbiological characteristics of CDV. Biological characteristics study of CDV field strains were on thebasis of virus isolation and identification technologies, but the growth of CDV on usual cell linessuch as Vero and MDCK cells might not allowed, might occurred without the formation ofcytopathic effects (CPE), might attenuated the virulence during passages, led to the low rate ofvirus isolation, limited the separation and etiology biology, pathogenic mechanisms research ofCDV field strains. So the establishment of cell line proliferated CDV easily had importantapplication value for further study and vaccine virus production of CDV.Signalling Lymphocyte Activation Molecule (SLAM, also called CD150) serves as a maincell receptor for CDV. This study was aimed to establish a cell line, used BHK-21cells as theparental cell, expressing canine SLAM stably, which could be used to isolate and propagate CDV.This research amplified the gene encoding canine SLAM without the signal peptide, sub-cloned itinto eukaryotic expression vector pDisplay to get the recombinant expression plasmidpDisplay/SLAM. The pDisplay/SLAM plasmid was transfered mediated by Lipofectamine2000reagent (Invitrogen, USA) in BHK-21cells. The positive stably transfectant BHK-21/SLAM cellswere screened by G418, subcloned by gradient dilution, and identified by immunofluorescence (IF)and RT-PCR. The detection of BHK-21/SLAM cells mRNA of the5th,10th,15thand20thpassagesby RT-PCR, demonstrated the stably transcription of the canine SLAM gene; The IF analysis of the20thpassages of BHK-21/SLAM cells confirmed the stably expression of the canine SLAM protein;The Western Blot analysis also confirmed the expression of the canine SLAM protein inBHK-21/SLAM cell lines. There was no significant difference of growth characteristics betweenBHK-21/SLAM and BHK-21cells, only with the higher total cell number per unit area than BHK-21cells.Reference strain Snyder Hill were infected to the BHK-21/SLAM and BHK-21cells,experiment showed no CPE in BHK-21cells, while had CPE in BHK-21/SLAM cells at about12h after infection; The result of virus titration by BHK-21/SLAM cell line showed, CDV referencestrain Snyder Hill had a titre of103.9TCID50per0.1ml at about24h after infection; The nucleicacid content of CDV Snyder Hill strain appeared in BHK-21/SLAM cells at24h (107.9copies/μl)was significantly higher than that in BHK-21cells at48h (107.2copies/μl). The wild strain CDV-Z7was infected to both cells, experiment showed CPE in BHK-21/SLAM cells, while no CPE inBHK-21cells.This research established the BHK-21/SLAM cell line expressing canine SLAM receptorstably, virus proliferated in BHK-21/SLAM cells had higher titer and more obvious CPE comparedwith that in BHK-21cells. The cell line would play an active role in virus isolation, biologicalcharacteristics study and vaccine virus production of CDV.