狂犬病病毒磷蛋白重组人5型腺病毒的构建及鉴定  

Construction and identification of recombinant human adenovirus 5 expressing rabies virus phosphoprotein

作　　者：靳红亮 王述超[2] 高菽蔓 张守峰[2] 严妍[1,2] 王东方[1,2] 刘晔[2] 扈荣良[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2015年第10期1023-1027,共5页Chinese Journal of Biologicals

基　　金：国家高技术研究发展(863)计划(2011AA10A212)

摘　　要：目的构建表达狂犬病病毒(rabies virus,RABV)BD06株磷蛋白(phosphoprotein,P)的重组人5型腺病毒,并进行鉴定。方法质粒p MD18-T-BD06P经双酶切后,获得完整的P蛋白基因,将其克隆至腺病毒表达系统穿梭质粒pac Ad5CMVK-Np A,构建重组穿梭质粒pac Ad5CMV-BD06P,与骨架质粒pac Ad5 9.2-100经PacⅠ酶切线性化后,共转染HEK293AD细胞,经同源重组,获得表达RABV P蛋白的重组人5型腺病毒r Ad5-BP。采用K覿ber法计算r Ad5-BP滴度,RT-PCR、Western blot、直接免疫荧光方法(direct immunofluorescence assay,d FA)检测P蛋白基因在HEK-293AD细胞的转录及表达水平。以107 TCID50r Ad5-BP肌肉注射免疫昆明小鼠,14 d后,经尾静脉采血,分离血清,IFA法检测抗RABVP蛋白抗体及其反应活性。结果经酶切及测序鉴定证明重组穿梭质粒pac Ad5CMV-P构建正确;重组腺病毒r Ad5-BP滴度可达108 TCID50/ml,能够介导P蛋白在HEK293AD细胞中的转录及表达,接种小鼠后可刺激机体产生针对RABVP蛋白抗体,且与RABV具有良好的反应活性。结论成功构建了RABVBD06株P蛋白重组人5型腺病毒,该病毒能够介导P蛋白在小鼠体内有效表达,刺激机体产生针对RABVP蛋白抗体,且与RABV具有良好的反应活性,为进一步研究RABV P蛋白在病毒感染过程中的作用奠定了基础。Objective To construct and identify a recombinant human adenovirus 5 expressing phosphoprotein（P） of rabies street virus BD06 strain（RABVP）. Methods Entire P protein gene of rabies virus BD06 strain was obtained from plasmid p MD18-T-BD06 P by digestion with restriction endonuclease and cloned into shuttle plasmid pac Ad5CMVK-Np A.The constructed recombinant shuttle plasmid pac Ad5CMV-BD06 P and backbone plasmid pac Ad5 9. 2-100 were linearized with Pac Ⅰ, then cotransfected into HEK293 AD cells to obtain recombinant human adenovirus 5 expressing RABVP,named as r Ad5-BP, by homologous recombination. The titer of r Ad5-BP was calculated by K覿ber method, while the transcription and expression levels of P by RT-PCR, Western blot and direct immunofluorescence assay（d FA）. Kunming mice were inoculated i.m. with 107TCID50 r Ad5-BP, of which serum samples were collected 14 d later, and determined for anti-RABVP antibody and its reactive activity by d FA. Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pac Ad5CMV-P was constructed correctly. The obtained r Ad5-BP virus reached a titer of108TCID50/ ml, which mediated the transcription and expression of P protein in HEK293 AD cells elicited anti-RABVP antibody in mice, and showed good reactive activity with RABV. Conclusion Recombinant human adenovirus expressing phosphoprotein of RABV BD06 strain was constructed successfully, which laid a foundation of further study on the role of RABVP in viral infection.

关 键 词：狂犬病病毒 磷蛋白 人5型腺病毒 

分 类 号：R373.9[医药卫生—病原生物学] Q782[医药卫生—基础医学]