To investigate the adaptation of poliovirus Sabin 2 in human embryonic lung diploid fibroblast（KMB17） and its relation to nucleotide mutations of 5′ noncoding region（5′NCR）,the Sabin 2 vaccine virus was passaged...To investigate the adaptation of poliovirus Sabin 2 in human embryonic lung diploid fibroblast（KMB17） and its relation to nucleotide mutations of 5′ noncoding region（5′NCR）,the Sabin 2 vaccine virus was passaged serially in KMB17 for seventeen passages.The infectious titers of Sabin 2 virus increased along with the passages,the highest titer reached 8.0 LgCCID50/ml at passage 13（P13）.During passage,there were three nucleotide mutations appeared in 5′NCR,G changed to C at position 20,C to U at position 189 and G to A at position 481.The results showed that Sabin 2 poliovirus adapted well in KMB17 after 17 passages.The nucleotide mutation at position 481 that appeared from passage 3 on indicated the possibility of virulent reversion.展开更多
目的检测疫苗生产用人二倍体细胞2BS和MRC-5是否存在外源因子猪圆环病毒1型(porcine circovirus type 1,PCVl)和猪圆环病毒2型(PCV2)污染。方法对已建立的PCR方法进行灵敏度验证后,检测人二倍体细胞2BS和MRC-5中PCV1和PCV2污染情况。结...目的检测疫苗生产用人二倍体细胞2BS和MRC-5是否存在外源因子猪圆环病毒1型(porcine circovirus type 1,PCVl)和猪圆环病毒2型(PCV2)污染。方法对已建立的PCR方法进行灵敏度验证后,检测人二倍体细胞2BS和MRC-5中PCV1和PCV2污染情况。结果两种细胞上清中最低均可检出1 pg的PCV1和PCV2 DNA,灵敏度较高;两种细胞中PCV检测结果均为阴性。结论在生产用人二倍体细胞2BS和MRC5中,均未检出PCV1和PCV2污染。展开更多
目的研究2种低血清培养基对人胚肺二倍体细胞(2BS株)的培养效果及甲型肝炎病毒(hepatitis A virus,HAV)在低血清培养基中的增殖情况。方法使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清培养细胞,显微镜...目的研究2种低血清培养基对人胚肺二倍体细胞(2BS株)的培养效果及甲型肝炎病毒(hepatitis A virus,HAV)在低血清培养基中的增殖情况。方法使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清培养细胞,显微镜下观察细胞形态、贴壁情况,细胞计数并绘制生长曲线;优化低血清培养基B的血清浓度;使用5层细胞工厂连续传代培养,放大低血清培养基细胞培养工艺;使用低血清培养基连续进行3次细胞传代后接种HAV,检测病毒滴度。结果使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清,细胞生长密度峰值分别为1.55×10^(5)、2.07×10^(5)和1.30×10^(5)个/cm^(2),统计学分析表明,低血清培养基B+5%新生牛血清达到细胞生长密度峰值高于低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清,差异具有统计学意义(P均<0.05);低血清培养基B+5%新生牛血清同B+3%新生牛血清连续培养3代细胞生长密度差异均无统计学意义(P=0.47、0.07、0.12);使用5层细胞工厂培养细胞,低血清培养基A+2%新生牛血清、B+5%新生牛血清连续培养3代,细胞生长密度均高于E-MEM+10%新生牛血清;低血清培养基B+3%新生牛血清与低血清培养基B+5%新生牛血清的病毒滴度分别为9.50和9.33 lgCCID_(50)/mL,低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清的病毒滴度分别为8.50和9.00 lgCCID_(50)/mL。结论低血清培养基适用于培养2BS细胞增殖HAV,为低血清培养基在甲肝疫苗生产中的应用奠定了基础。展开更多
文摘To investigate the adaptation of poliovirus Sabin 2 in human embryonic lung diploid fibroblast（KMB17） and its relation to nucleotide mutations of 5′ noncoding region（5′NCR）,the Sabin 2 vaccine virus was passaged serially in KMB17 for seventeen passages.The infectious titers of Sabin 2 virus increased along with the passages,the highest titer reached 8.0 LgCCID50/ml at passage 13（P13）.During passage,there were three nucleotide mutations appeared in 5′NCR,G changed to C at position 20,C to U at position 189 and G to A at position 481.The results showed that Sabin 2 poliovirus adapted well in KMB17 after 17 passages.The nucleotide mutation at position 481 that appeared from passage 3 on indicated the possibility of virulent reversion.
文摘目的研究2种低血清培养基对人胚肺二倍体细胞(2BS株)的培养效果及甲型肝炎病毒(hepatitis A virus,HAV)在低血清培养基中的增殖情况。方法使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清培养细胞,显微镜下观察细胞形态、贴壁情况,细胞计数并绘制生长曲线;优化低血清培养基B的血清浓度;使用5层细胞工厂连续传代培养,放大低血清培养基细胞培养工艺;使用低血清培养基连续进行3次细胞传代后接种HAV,检测病毒滴度。结果使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清,细胞生长密度峰值分别为1.55×10^(5)、2.07×10^(5)和1.30×10^(5)个/cm^(2),统计学分析表明,低血清培养基B+5%新生牛血清达到细胞生长密度峰值高于低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清,差异具有统计学意义(P均<0.05);低血清培养基B+5%新生牛血清同B+3%新生牛血清连续培养3代细胞生长密度差异均无统计学意义(P=0.47、0.07、0.12);使用5层细胞工厂培养细胞,低血清培养基A+2%新生牛血清、B+5%新生牛血清连续培养3代,细胞生长密度均高于E-MEM+10%新生牛血清;低血清培养基B+3%新生牛血清与低血清培养基B+5%新生牛血清的病毒滴度分别为9.50和9.33 lgCCID_(50)/mL,低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清的病毒滴度分别为8.50和9.00 lgCCID_(50)/mL。结论低血清培养基适用于培养2BS细胞增殖HAV,为低血清培养基在甲肝疫苗生产中的应用奠定了基础。
