Screening Of BHK-21 Suspension Cell Clone Strain And Preparationof Foot-and-Mouth Disease O-Type Plaque Virus

At present,China mainly adopts the policy of"prevention first"to control the occurrence of foot-and-mouth disease,and vaccination is one of the most important means of preventing and controlling foot-and-mouth disease.Therefore,improving the quality of foot-and-mouth disease vaccines is particularly important.BHK-21 cells,as the cell matrix for vaccine production,have been widely used in the production of foot-and-mouth disease vaccines through suspension culture technology.At the same time,the key to developing foot-and-mouth disease vaccines is the preparation of seed viruses,whose purity and antigen content directly affect the quality of foot-and-mouth disease vaccines.This experiment first applied soft agar cloning technology to single cell clone BHK-21 suspension cells.Through the determination of cell viability and observation of its growth status after cell resuscitation,BHK-21 suspension cells that meet the conditions were selected.They were made into single cell suspensions and inoculated into a 6-well flat bottom culture plate for single cell cloning.A total of 25 single cell clone strains were obtained from 145 to 170 hours,and the obtained clone cells were sequentially transferred to a 15m L centrifuge tube,a 50m L centrifuge tube,a 125m L shake flask,etc.for expanded culture,Finally,12 clone cells that could be expanded for culture were obtained for cryopreservation.Resuscitated clone cells were subjected to high-density clone screening,among which three clone strains had cell viability of over 90%,higher cell density compared to pre clone cells,and their biological characteristics met the requirements.Therefore,a high-density growth BHK-21 suspension cell library was established.Secondly,plaque cloning technology is used to screen for foot-and-mouth disease virus.Two cloned strains were obtained through the first plaque cloning,and five cloned strains were successfully obtained through secondary cloning.The plaque forming unit of the second clone of foot-and-mouth disease O/GX/09-7 strain was（12.9×10⁷and 10.8×10⁷）Compared to the first cloned plaque formation unit（2.5×10⁷）increased by four to five times.The TCID₅₀（6.60～7.00）and LD₅₀（8.61～9.15）of the foot-and-mouth disease O/GX/09-7 strain after cloning were significantly increased compared to the TCID₅₀（5.75～6.20）and LD₅₀（7.62～8.31）before cloning,resulting in highly virulent cloned strains.Finally,the cloned strain of foot and mouth disease virus GX-2-1 obtained from plaque screening was inoculated onto the BHK-21/XFKL/15 clone cells,and a high-quality strain of GX-2-1 XFKL was obtained.The 146S value detected was 12.24 ug/m L,which was 2.6 times higher than the pre cloned O/GX/09-7 strain 146S（4.55 ug/m L）and 1.9times higher than the foot and mouth disease GX-2-1 clone 146S（6.34 ug/m L）.Therefore,the effective antigen 146S content of the O/GX/09-7 strain was effectively increased,Providing high-quality seed virus for the production of foot-and-mouth disease vaccines is of great significance for improving the quality of foot-and-mouth disease vaccines.