狂犬病病毒CVS-11株培养工艺的优化

Optimization of culture technology for rabies virus CVS-11 strain

目的通过优化培养工艺提高狂犬病病毒CVS-11株的滴度。方法 BSR细胞(1.5×106个细胞/孔)以不同感染复数(MOI 0.100、0.050、0.010、0.005和0.001)接种CVS-11,培养72 h收获,测定病毒滴度;以0.005和0.001 MOI,分别接种不同密度的BSR细胞(1×105、2×105、3×105、4×105、5×105、6×105、8×105和10×105个细胞/孔)培养CVS-11,于72 h收获,测定病毒滴度;以5×105个细胞/9.6 cm2和0.001 MOI,分别在48、72、96、120和144 h收获病毒,测定病毒滴度;并通过正交实验获得最佳培养条件;由6孔板(9.6 cm2/孔)放大到T75 cm2瓶培养,验证正交实验结果。结果细胞密度对CVS-11病毒滴度有一定的影响,以4×105细胞/9.6 cm2获得的CVS-11病毒滴度最高。以相同的细胞数量和收获时间,CVS-11病毒的滴度随着MOI的减小而增大。固定细胞数和MOI,病毒滴度在培养早期随时间的延长而增加,直至96 h后,随着时间的延长滴度下降。正交实验的结果表明3个因素对滴度的影响顺序是MOI>细胞数量>收获时间,放大培养验证了正交试验的结果。结论以4×105个细胞/9.6 cm2,0.001 MOI,培养96 h获得的CVS-11滴度最高。

Objective To optimize the culture process in order to improve the titer of rabies virus CVS-11 strain. Metho-d s BSR cells（ 1. 5 × 106 cell / well） were infected with CVS-11 strain at different MOI as 0. 100,0. 050,0. 010,0. 005 and 0. 001. The cultured cells were harvested at 72 h after the infection and virus titer wasdetected. Different amount of BSR cells（ 1 × 105,2 × 105,3 × 105,4 × 105,5 × 105,6 × 105,8 × 105 and 10 × 105 cells /9. 6 cm2） were inoculated with CVS-11 strain at MOI of 0. 005 and 0. 001. The cellswere harvested at 72 h after infection and virus titer wasdetected.BSR cells（ 5 × 105 cell / well） were infected with CVS-11 at a MOI of 0. 001,the cells were then harvested at different time as 48,72,96,120 and 144 h,and virus titer was detected. The optimal culture condition was obtained by an orthogonal experiement and its result was further verified through amplifying the culture system from a 9. 6 cm2 plate to a T75cm2 flask.Results The cell density had a significanteffect on the titer for CVS-11 strain and the highest titer was obtained at the density of 4 × 105 cell /9. 6 cm2. When fixing cell density and the harvesting time,the CVS-11 titer showed an increasing tendency with a decreasing of MOI. When fixing cell density and MOI,the virus titer significantly increased as the time extended at early stage of infection until 96 h,and then decreased as the time extended. The orthogonal experiement result showed that the virus titer reached maximum as the cell number is 4 × 105 cells /9. 6 cm2,MOI 0. 001,and harvested at 9 6 h. The three factors to affect the virus titer was in an order MOI cell amount harvest time. Enlarged culture experiment confirmed the result of the orthogonal experiement. Conclusion The virus titer will reach maximum when the cell amount is 4× 105 cells /9. 6 cm2,with MOI 0. 001,and harvestes at 96 h.