摘要
目的 用基因疫苗pcDNA3_gtfB免疫大鼠 ,探讨基因疫苗整合入宿主细胞的可能性。方法 将 36只Wistar大鼠分为两组 ,一组为pcDNA3_gtfB颌下腺免疫组 ,另一组为PBS缓冲液颌下腺对照组。取Wistar大鼠颌下腺、肾脏、肝脏、心脏、肺脏、脑组织 ,以对照组 6种组织基因组DNA为模板 ,加入不同梯度拷贝数的pcDNA3_gtfB ,采用Pyrobest聚合酶PCR反应体系 ,确定PCR反应敏感性。以免疫组 6种组织基因组DNA为模板 ,检测pcDNA3_gtfB的整合率。结果 本实验PCR体系的检测水平为在 1 0 0 0 0个组织细胞核中能检测出一个pcDNA3_gtfB拷贝(1∶1 0 0 0 0 ) ,在此检测水平下 ,免疫组 6种组织基因组DNA中未发现整合。结论 pcDNA3_gtfB整合入宿主细胞基因组DNA的概率不超过 1∶1 0 0 0 0 。
Objective\ Gene vaccine security is of concern because of the possibility of insertion mutagenesis resulting in inactivation of tumor suppressor or activation of oncogene. The purpose of this study was to examine the potential of anti_caries DNA vaccine pcDNA3_gtfB integrating into the host cell genome.Methods\ Anti_caries DNA vaccine pcDNA3_gtfB was constructed by the previous study. The gtfB gene(904～4 578 bp, genebank M17361) was cloned from Streptococcus mutans GS_5. 36 Wistar rats were divided into 2 groups: submandibular gland_targeted injection(SGT) group and control group. Rats in SGT group were injected with 100 μg of plasmid pcDNA3_gtfB, rats in control group with PBS solution. Genomes from submandibular gland, kidney, heart, liver, lung, and brain tissues were isolated later in 12 weeks. Genomes from different tissues were purified by low_melting agarose electrophoresis. Using the purified genomes as template, plasmid integration were examined by PCR(upper primer: 5′_ATATGGTACCATGACCGAAGCGACATCTAAGCAAGA_3′, lower primer: 5′_ACTACTCGAGTTAGAACCATTGACCCTG AGCATTGC_3′). The sensitivity level of PCR was determined by adding gradient plasmid copies into genomes in control group.Results\ The examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level that could detect 1 copy integration in 10 000 nuclei.Conclusion\ The potential frequency of plasmid pcDNA3_gtfB integration into host cell genome would not exceed that of the spontaneous mutation. It was indicated that pcDNA3_gtfB was genetically safe as a promising anti_carious DNA vaccine.\;
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2003年第3期228-230,共3页
West China Journal of Stomatology
基金
国家自然科学基金资助项目 (编号 39770 797)
