【摘要】 目的构建和包装稳定表达泛素化HBcAg融合基因和Tapasin基因的重组慢病毒,为慢性乙型肝炎免疫治疗的实验研究奠定基础。方法设计引物通过聚合酶链式反应(PCR)扩增获得目的基因Ub-HBcAg,Ub-HBcAg基因与经酶切线性化的慢病毒表达载体质粒pLenti-CMV-HA-3Flag-P2A-EGFP定向连接,其产物pLenti-CMVUb-HBcAg-HA-3Flag-P2A-EGFP转化大肠杆菌菌株DH5α感受态细胞,挑选阳性克隆进行PCR鉴定和直接测序序列分析。将化学合成的T2A-Tapasin基因插入至pLenti-CMV-Ub-HBcAg-HA-3Flag-P2A-EGFP,其产物p Lenti-CMV-Ub-HBcAg-HA-T2A-Tapasin-3Flag-P2A-EGFP经直接基因测序分析后与包装质粒pLP1、pLP2以及包膜质粒p LP/VSVG共转染人胚肾293T细胞,得到携带有Ub-HBcAg基因和Tapasin基因的重组慢病毒颗粒LV-Ub-HBcAg/Tapasin,重组慢病毒颗粒转染293T细胞,实时荧光定量PCR检测慢病毒浓缩液的滴度,Western blot法检测目的基因的表达。结果成功构建和包装了表达泛素化HBcAg融合基因和Tapasin基因的重组慢病毒,实时荧光定量PCR证实重组慢病毒的滴度达3.25×10~8TU/mL。结论成功构建稳定表达泛素化HBcAg融合基因和Tapasin基因的重组慢病毒,为慢性乙肝的免疫治疗提供了实验基础。更多还原

【Abstract】 Objective To construct and package a recombinant lentivirus stably expressing the ubiquitinated HBcAg fusion gene and the Tapasin gene and to lay the foundation for the experimental study of immunotherapy for chronic hepatitis B. Methods The Ub-HBcAg gene was amplified with designing primers by polymerase chain reaction(PCR).The purified Ub-HBcAg fragment was cloned into the lentivirus vector p Lenti-CMV-HA-3 Flag-P2 A-EGFP which was ligated by restriction endonuclease. The product pLenti-CMV-Ub-HBcAg-HA-3 Flag-P2 A-EGFP was transformed into E. coli DH5α cells. The positive clones were chosen for PCR identification and direct sequencing analysis. Then, T2 ATapasin sequence was chemically synthesized and cloned into pLenti-CMV-Ub-HBcAg-HA-3 Flag-P2 A-EGFP. The product recombinant pLenti-CMV-Ub-HBcAg-HA-T2 A-Tapasin-3 Flag-P2 A-EGFP plasmid was confirmed by direct gene sequencing. Lentiviral particles of LV-Ub-HBcAg/Tapasin were produced by triple transfection of 293 T cells with pLenti-CMV-Ub-HBcAg-HA-T2 A-Tapasin-3 Flag-P2 A-EGFP using packaging plasmids pLP1, pLP2 and envelope plasmid pLP/VSVG. The recombinant lentiviral particles were transfected into human embryonic kidney 293 T cells. The titer of lentivirus was detected by real-time fluorescent quantitative PCR and the expression of the target gene was determined by Western blot. Results Recombinant lentivirus vectors expressing ubiquitinated HBcAg fusion gene and Tapasin gene were successfully constructed and packaged. The titer was 3.25 ×10~8 TU/mL detected by real-time fluo-rescent quantitative PCR. Conclusion The stable expressing of ubiquitinated HBcAg fusion gene and Tapasin gene recombinant lentivirus vectors are successfully established, providing experimental basis for immunotherapy of chronic hepatitis B.更多还原