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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 recombinant human adenovirus type 5 expressing the rabies virus (RABV) BD06 strain phosphoprotein (P). METHODS: The full-length P protein gene was obtained by double digestion of plasmid pMD18-T-BD06P and cloned into the shuttle plasmid pacAd5CMVK-Np A of adenovirus expression system to construct recombinant shuttle plasmid pac Ad5CMV-BD06P, Recombinant human adenovirus type 5 adenovirus Ad5-BP expressing RABV P protein was obtained after homologous recombination by HEK293AD cells. The Ad5-BP titer was calculated by K-ber method, and the transcription and expression of P protein gene in HEK-293AD cells were detected by RT-PCR, Western blot and direct immunofluorescence assay (dFA). Kunming mice were injected intramuscularly with 107 TCID50r Ad5-BP. After 14 days, blood was collected via tail vein and serum was separated. Anti-RABVP antibody and its activity were detected by IFA. Results The recombinant shuttle plasmid pac Ad5CMV-P was confirmed by restriction enzyme digestion and sequencing. The titer of recombinant adenovirus Ad-BP5 was 108 TCID50 / ml, which could mediate the transcription and expression of P protein in HEK293AD cells. Rats can stimulate the body to produce antibodies against RABVP protein, and with RABV has good reactivity. Conclusion Recombinant human recombinant adenovirus type 5 of P protein of RABVBD06 strain was constructed successfully. The recombinant protein can efficiently express P protein in mice and stimulate the production of antibody against RABVP protein. The recombinant protein has good reactivity with RABV. P protein in the process of viral infection laid the foundation for the role.