A Study of Recombinant Dunaliella Salina Vaccine against Dental Caries

Objectives:To transform the gene encoding chimera SBR-CT¦¤A1 into dunaliella salina by ultrasonic treatment to construct the recombinant dunaliella salina vaccine against caries. Methods: A strain of dunaliella salina cells from a single colony were isolated and developed. Their sensitivity to the kanamycin and herbicide phosphinothricin were observed. Recombinant plant expressing plasmid pROSB carrying the gene encoding chimera SBR-CT¦¤A1 and bar gene was transformed into the dunaliella salina cells by ultrasonic treatment at different power levels (22w,33w,44w,55w,66w,77w,88w) and time intnervals (10sec,30sec,1min,3min,5min, 10min) . The genomic DNA of dunaliella salina which could propagate in solid and liquid media containing herbicide phosphinothricin was extracted by CTAB method and were analyzed by PCR and Southern blotting .Dunaliella salina without either ultrasonic treatment or plasmid addition or both were used as the control. Results: Wild dunaliella salina had no sensitivity to kanamycin but was sensitive to herbicide phosphinothricin . Herbicide phosphinothricin completely suppressed the growth and proliferation of dunaliella salina cells in liquid medium at a dose of 6.0mg/L and 8.0mg/L in solid medium. Ultrasonic treatment with higher power and shorter time or lower power and longer time could provide higher transformating rate. There were no dunaliella salina growing in control groups. 1.9kb fragments which consisted with the gene encoding SBR-CT¦¤A1 in size were amplified from the transgenic dunaliella salina genome by PCR. Positive signal fragments were gained by Southern blotting.Conclusions: Herbicide phosphinothricin could be used for screening transgenic dunaliella salina which carrying foreign bar gene. Dunaliella salina could be transformed effectively by ultrasonic treatment. The gene encoding chimera SBR-CT¦¤A1 was integrated into the genome of dunaliella salina.