摘要
本研究是在前期获得狂犬病弱毒株SRV9的基础上,经小鼠脑内传至5代,将处理过的鼠脑第五代毒种接种BHK-21细胞,经过建立初步生产工艺获得高滴度的狂犬病弱毒疫苗。
首先分别用10mL的细胞培养瓶、1000mL的方瓶、2000mL的小转瓶、10L的大转瓶培养BHK-21细胞的同时接种狂犬病病毒SRV9弱毒株,经反复确定条件后获得高滴度的狂犬病弱毒疫苗。
分别对小鼠及犬做了安全性试验,结果表明狂犬弱毒疫苗对犬无论是肌注还是口服均是安全的,对18日龄以上的小鼠不具有致病性。犬的免疫原性试验结果表明,该疫苗对犬具有良好的免疫原性,
本研究为我国生产具有良好免疫原性和安全性的口服狂犬疫苗奠定了坚实的基础,现在已经可以在实验室自主制备。
BHK-21 cells were cultured in 100ml, 1000ml cell culture bottles and 2000ml, 10L roll cell culture bottles, and simultaneous inoculation proliferative SRV9 low virulent strain from mice brain. Definite culture temperature and vaccination dosage repeatedly, at last gain high titer live attenuated rabies vaccine to establish craft of lab production to finish production of live attenuated rabies vaccine .
To assure the safety of live attenuated rabies vaccine for animals, eighteen dogs were parted averagely to six groups for immunity, dogs in the first group were immunized by intramuscular with 1milliliter live vaccine and 1 milliliter repeated after 7 days in the second group , and 3 months dogs in the third were immunized by intramuscular with 1 milliliter live vaccine. Fifth group were immunized by mouth with 10 milliliter live vaccine and sixth group is control group. The fifth and six group were bred together.We used the RT-PCR and direct immunofluorescence to examine rabies virus which we collect swab after immunize dogs, used RT-PCR examine dogs which immunized by mouth rabies virus after blind passage three generation in BHK-21 cell, rabies virus was found. In the safety experiment of mice, 1-21 day-aged icr mice were injected inner brain with 30 micromilliter SRV9 low virus. The ice of above 18 day-age were all alive and healthy without any symptoms. And it was confirmed by direct immunofluorescence. The results indicated that live attenuated rabies vaccine made by SRV9 stain for above 18 day-age were safety. We passaged SRV9 stain five generation in icr mice brain, and amplication G gene, amico acids sequence comparison. When compared to isolated five SRV9 low virus strain, primitive SRV9 low virus strain, amino acid was 99.2%, 99.0%, 90.2%, 99.4%, 99.0%.
For the SRV9 low virulent strain suiting to immune animals by mouth and intramuscular, safety and immunogenicity research were studied in this experiment according to WHO charrette about the field test requirementand standard for inoculating rabies vaccine by mouth to dogs , wildlife and cats which hadn’t be studied in America. To assure immunogenicity of live attenuated rabies vaccine for animals, fifteen dogs were parted averagely to five groups for immunity, dogs in the first group were immunized by intramuscular with 1milliliter live vaccine and 2 milliliter in the second group, and dogs in the third and fourth group were immunized by mouth with 10 and 20 milliliter live vaccine respectively. The first and second group were bred together, meanwhile the third, the fourth and the control group were together. After 7 days, all dogs were alive and healthy. Neutralizing antibody all above 0.5IU/ml were detected in the serum samples by fluorescent antibody virus neutralization test, which arrived to the protective level.
The results indicated that live attenuated rabies vaccine made by SRV9 stain for dogs and cats was safety, and immunogenicity was wonderful.
引文
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