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目的 探讨逆转录病毒载体介导的HBsAg抗原的表达及其稳定性。方法 将HBVS基因插入逆转录病毒载体pLXSN中 ,构建成重组逆转录病毒载体。用电穿孔法转染PA317细胞 ,包装成假病毒颗粒 ,在不同温度下冻存。于不同时间用假病毒颗粒感染HepG2 、NIH3T3及 2 93细胞 ,用RT PCR及ELISA法检测HBsAg表达 ;以感染后G4 18抗性克隆形成数确定假病毒颗粒的活力。结果 在各个时间段HBsAg表达量均差异无显著性。在 - 2 0℃冻存时 ,6个月后克隆数即下降过半 ,12个月后仅形成少数克隆 ,2 4个月后无克隆形成 ;在 - 4 0℃冻存时 12个月后HepG2 、NIH3T3、2 93形成的克隆数分别为 12 1、332和 89,2 4个月后分别为 4 2、137和 4 3,与冻存前比较差异有显著性 ;- 70℃冻存时 ,2 4个月后克隆数分别为 15 9、4 6 3和 112 ,与冻存前比较差异无显著性。结论 HBsAg重组逆转录病毒颗粒在 - 70℃保存 2年后 ,病毒活力未受明显影响 ,HBsAg表达量亦无明显改变。
Objective To investigate retroviral vector-mediated expression of HBsAg antigen and its stability. Methods The HBVS gene was inserted into the retroviral vector pLXSN to construct a recombinant retroviral vector. PA317 cells were transfected by electroporation, packaged into pseudovirions and stored at different temperatures. HepG2, NIH3T3 and 293 cells were infected with pseudovirions at different times. HBsAg expression was detected by RT-PCR and ELISA. The number of G418-resistant clones formed after infection was used to determine the viability of pseudovirions. Results There was no significant difference in the expression of HBsAg between different time points. At -20 ℃, the number of clones decreased more than half after 6 months, only a few clones formed after 12 months and no clones formed after 24 months. After 12 months of freezing at -40 ℃, HepG2 , NIH3T3,293 clones were 12 1,332 and 89, respectively 4,2,137 and 4 3 4 months after cryopreservation, compared with the pre-cryopreservation difference was significant; - 70 ℃ cryopreservation, The number of clones after 24 months was 15 9, 463, and 112, respectively, which was not significantly different from those before cryopreservation. Conclusion HBsAg recombinant retroviral particles stored at -70 ℃ for 2 years, the virus activity was not significantly affected, HBsAg expression did not change significantly.