摘要
目的 :研究GM CSF和preS2对乙肝DNA疫苗的免疫增强作用。方法 :采用PCR方法 ,扩增HBVpreS2 +S基因约 84 6bp的片段和rhGM CSF(包括甘氨酸接头 )基因 4 5 0bp的片段。通过T A克隆技术和基因定向克隆 ,构建融合基因的真核表达质粒pcDNA3.1 S2S rhGM CSF ,并在HepG2细胞中表达。结果 :经酶切、PCR及DNA测序鉴定 ,融合基因表达质粒HB VpreS2S rhGM CSF成功地构建。将其转染HepG2细胞后 ,目的基因的转录通过RT PCR得到证实 ,而且表达的融合蛋白能与抗 HBs、抗 preS2和抗 GM CSF单克隆抗体 (mAb)均产生特异性反应。结论 :融合基因表达载体pcDNA3.1 S2S rhGM CSF的成功构建并表达 。
AIM: To construct GM-CSF and preS2 fusion gene expression plasmid to enhance the immunogenicity of hepatitis B DNA vaccine. METHODS: HBV preS2S gene(846 bp) and rhGM-CSF gene(384 bp) were amplified by PCR, respectively. The eukaryotic expression plasmid pcDNA3.1-S2S-rhGM-CSF was constructed by means of T-A clone and directional gene cloning techniques, and then the recombinant plasmid was expressed in HepG2 cells. RESULTS: Restriction enzyme digestion analysis, PCR amplification and/or DNA sequencing proved that the recombinant plasmid was constructed successfully. The transcription of target gene was confirmed by RT-PCR. The fusion protein expressed in HepG2 could reacted to the monoclonal antibodies (mAbs) against HBsAg, preS2 and GM-CSF, respectively. CONCLUSION: The successful construction and expression of pcDNA3.1-S2S-rhGM-CSF lay the foundation for further study of HBV DNA vaccine.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第5期548-551,共4页
Chinese Journal of Cellular and Molecular Immunology
