Abstract:

Abstract:Objective To establish a method to screen a mouse reactive suppressive oligodeoxynucleotide (ODN) in vitro for future research in mice. Methods     Mouse splenocytes were divided into  control and  CpG 2216 groups. The supernatants of splenocytes induced by CpG 2216 with different concentrations(0,0.25,0.50,1.00，2.00，4.00，8.00，16.00 and 32.00 mg·L^-1) for different incubation time (3,6,12,24,48,72 and 96 h)were collected and detected by VSV protection bioassay to confirm the optimal concentration and incubation time of CpG 2216 and the dilution of the supernatant. Then the mouse splenocytes were divided into control group, CpG 2216 group, CpG 2216 + different concentrations of suppressive ODN(A151) group to find an optimal concentration of A151. At last,the  mouse splenocytes were divided into  control group, CpG 2216 group, CpG 2216 + suppressive ODN(developed by our lab) group. The anti-viral activities of the supernatants were detected by VSV protection bioassay using above conditions to practice the screening method established. Results  The screening method established was as followed: mouse splenocytes(cell density 5×10⁶·mL^-1) were cultivated with 2 mg·L^-1 CpG 2216 and/or 16 mg·L^-1 suppressive ODN for 24 h. The supernatants were collected and added to L929 cells by 1∶4 dilution. After 18 h incubation, the cells were cultivated  with  VSV for 48 h and A578 was detected to screen suppressive ODN. Conclusion Using CpG 2216 as the stimulator and A151 as the positive suppressive ODN, the conditions of screening experiment are established successfully and could be used for further development of novel suppressive ODN in mice.

Key words: suppressive oligodeoxynucleotide, CpG oligodeoxynucleotide, mice, inbred BALB C, splenocyte, anti-viral activity