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目的 分析Ag85B-ESAT6重组腺病毒(rADV-Ag85B-ESAT6)肺结核疫苗的遗传稳定性。方法将纯化的重组腺病毒rADV-Ag85B-ESAT6在HEK293细胞上连续传代20次,观察细胞病变情况;PCR扩增及序列比对分析重组腺病毒基因的稳定性;Western blot分析重组腺病毒Ag85B-ESAT6融合蛋白的表达稳定性。结果重组腺病毒rADV-Ag85B-ESAT6引起HEK293细胞病变的速度和形态均未发生明显变化;第10代和第20代重组腺病毒均可扩增出1 322 bp的外源基因片段和860 bp的重组腺病毒特征基因E2b片段,野生型腺病毒引物未扩增出特征条带;Western blot分析表明,第10代和第20代重组腺病毒rADV-Ag85B-ESAT6在相对分子质量约47 000处可见Ag85B-ESAT6融合蛋白表达条带。结论重组腺病毒rADV-Ag85B-ESAT6在体外传代20次,表现出良好的基因结构稳定性。
Objective To analyze the genetic stability of tuberculosis vaccine of Ag85B-ESAT6 recombinant adenovirus (rADV-Ag85B-ESAT6). Methods The recombinant adenovirus rADV-Ag85B-ESAT6 was serially passaged on HEK293 cells 20 times to observe the cytopathic effect. The stability of the recombinant adenovirus was analyzed by PCR and sequence comparison. The recombinant adenovirus Ag85B- ESAT6 fusion protein expression stability. Results The recombinant adenovirus rADV-Ag85B-ESAT6 caused no significant changes in the rate and morphology of HEK293 cells. The 10 322 and 1260-bp recombinant adenoviruses could amplify the exogenous gene fragment of 832 bp The recombinant adenovirus characteristic gene E2b fragment, the wild-type adenovirus primer did not amplify the characteristic bands; Western blot analysis showed that the 10th and 20th generation of recombinant adenovirus rADV-Ag85B-ESAT6 in the relative molecular mass of about 47 000 visible Ag85B-ESAT6 fusion protein expression band. Conclusion The recombinant adenovirus rADV-Ag85B-ESAT6 was passaged 20 times in vitro and showed good structural stability.