摘要
为了构建出表达新布尼亚病毒(SFTSV)Gn基因的重组5型腺病毒,试验根据GenBank上发表的SFTSV Gn基因序列(登录号为ADZ04482.1)设计合成引物,采用特异性PCR方法在Gn基因前添加了KOZAK序列和tPA信号肽序列;然后通过酶切、连接等方法构建重组穿梭质粒PGA-KOZAK-tPA-Gn,与腺病毒骨架质粒pAd5-ΔE1ΔE3-5E4在BJ5183感受态细胞中进行同源重组,得到重组腺病毒质粒rAd5-KOZAK-tPA-Gn,用PacⅠ酶酶切重组腺病毒质粒,并将线性化的质粒转染至HEK 293细胞中进行病毒包装、扩繁和纯化;采用PCR扩增、Western-blot等技术检测病毒基因的表达情况,并测定重组腺病毒效价。结果表明:通过PCR扩增获得了1 546 bp的KOZAK-tPA-Gn基因,并成功构建了重组穿梭质粒;SFTSV Gn基因在重组腺病毒传代过程中稳定存在;重组腺病毒在HEK 293细胞中表达出分子量约为61 ku的SFTSV Gn蛋白;测得重组腺病毒效价为1×10^(-7.63)/0.1 mL TCID_(50)。说明试验成功构建出表达SFTSV Gn基因的重组5型腺病毒。
In order to construct a recombinant adenovirus type 5 expressing the novel Bunia virus(SFTSV) Gn gene,primers were designed and synthesized based on the SFTSV Gn gene sequence published in GenBank(ADZ04482.1),and the KOZAK sequence and tPA signal peptide sequence were added to the Gn gene by specific PCR method.Then,a recombinant shuttle plasmid PGA-KOZAK-tPA-Gn was constructed by enzyme digestion and ligation,and homologous recombination with the adenovirus skeleton plasmid pAd5-ΔE1ΔE3-5E4 was performed in BJ5183 receptor cells to obtain the recombinant adenovirus plasmid rAd5-KOZAK-tPA-Gn.The recombinant adenovirus plasmid was cleaved using Pac Ⅰ enzyme,and the linearized plasmid was transfected into HEK 293 cells for virus packaging,propagation,and purification.PCR,Western-blot and other techniques were used to detect the expression of viral genes,and the titer of the recombinant adenovirus was determined.The results showed that 1 546 bp KOZAK-tPA-Gn gene was obtained by PCR amplification,and the recombinant shuttle plasmid was successfully constructed.SFTSV Gn gene was stable during the passage of recombinant adenovirus.Recombinant adenovirus expressed SFTSV Gn protein with molecular weight of 61 ku in HEK 293 cells.The titer of the recombinant adenovirus was measured to be 1×10~(-7.63)/0.1 mL TCID_(50).These results indicated that recombinant adenovirus type 5 expressing SFTSV Gn gene was successfully constructed.
作者
张璇
王新宇
刘宇婷
涂影叶
梁耀文
易昌华
殷国平
ZHANG Xuan;WANG Xinyu;LIU Yuting;TU Yingye;LIANG Yaowen;YI Changhua;YIN Guoping(Nanjing Hospital Affiliated to Nanjing University of Traditional Chinese Medicine,Nanjing 210003,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2024年第9期8-12,17,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金委员会重大研究计划项目(92169106)。
