Lutze-Wallace C, Sapp T, Sidhu M, Wandeler A
Biologics Evaluation Laboratory, Animal Diseases Research Institute, Agriculture and Agri-Food Canada, Ontario.
Can J Vet Res. 1995 Apr;59(2):157-60.
The genetic stability of a live human adenovirus 5: rabies glycoprotein recombinant vaccine has been assessed upon 20 serial passages in a permissive cell line of human origin. Restriction endonuclease analysis and the polymerase chain reaction were used to examine the integrity of the expression cassette for the rabies glycoprotein and the viral vector at the site of insertion of the cassette. It was found that the restriction endonuclease profile was identical for each sample assayed. A more detailed analysis of the expression cassette following amplification by the polymerase chain reaction revealed no changes in the size and number of fragments originating from the coding sequence for the glycoprotein nor the signals controlling the expression of the protein product. The amplified product obtained from the 10th and 20th passages was subjected to nucleotide sequencing. Additionally, 20 plaques isolated from the 20th passage of the virus expressed the rabies glycoprotein as demonstrated by fluorescent antibody staining with glycoprotein specific monoclonal antibodies. These results suggest that the recombinant vaccine maintains the integrity of the heterologous sequences upon passage in tissue culture.
一种人源腺病毒5型：狂犬病糖蛋白重组活疫苗在人源允许细胞系中连续传代20次后，对其遗传稳定性进行了评估。采用限制性内切酶分析和聚合酶链反应来检测狂犬病糖蛋白表达盒以及病毒载体在表达盒插入位点处的完整性。结果发现，所检测的每个样品的限制性内切酶图谱均相同。对聚合酶链反应扩增后的表达盒进行更详细分析发现，源自糖蛋白编码序列的片段大小和数量以及控制蛋白质产物表达的信号均未发生变化。对第10代和第20代传代获得的扩增产物进行了核苷酸测序。此外，用糖蛋白特异性单克隆抗体进行荧光抗体染色表明，从病毒第20代分离出的20个噬斑均表达狂犬病糖蛋白。这些结果表明，重组疫苗在组织培养传代过程中维持了异源序列的完整性。
