CAJ | 학술논문

目的利用杆状病毒表达系统共表达狂犬病病毒CTN-1V株核蛋白(nucleoprotein,NP)及糖蛋白(glycoprotein,GP).方法 RT-PCR分别扩增CTN-1V株病毒GP、NP编码区基因,分别依次克隆入转移质粒pFastBacDual的PP10区及PPH区构建杆状病毒重组转移质粒P-NG,转化DH10Bac E.coli感受态细胞获得重组穿梭质粒A-NG,转染Sf9细胞获得重组杆状病毒Bac-NG,高效表达目的蛋白NP、GP,进行Western blot分析.结果重组杆状病毒穿梭质粒A-NG经PCR鉴定证明构建正确；狂犬病病毒NP、GP在重组杆状病毒感染的Sf9细胞中获得正确表达,表达的重组蛋白NP、GP相对分子质量(Mr)约为51×103、59×103；表达的重组蛋白NP、GP均可与抗RV小鼠血清特异性结合.结论成功共表达狂犬病病毒CTN-1V株NP、GP,表达产物具有良好的抗原性,为狂犬病病毒基因工程疫苗及诊断试剂的研究奠定了基础.
목적 이용간상병독표체계통공표체광견병병독CTN-1V주핵단백(nucleoprotein,NP)급당단백(glycoprotein,GP).방법 RT-PCR분별확증CTN-1V주병독GP、NP편마구기인,분별의차극륭입전이질립pFastBacDual적PP10구급PPH구구건간상병독중조전이질립P-NG,전화DH10Bac E.coli감수태세포획득중조천사질립A-NG,전염Sf9세포획득중조간상병독Bac-NG,고효표체목적단백NP、GP,진행Western blot분석.결과 중조간상병독천사질립A-NG경PCR감정증명구건정학；광견병병독NP、GP재중조간상병독감염적Sf9세포중획득정학표체,표체적중조단백NP、GP상대분자질량(Mr)약위51×103、59×103；표체적중조단백NP、GP균가여항RV소서혈청특이성결합.결론 성공공표체광견병병독CTN-1V주NP、GP,표체산물구유량호적항원성,위광견병병독기인공정역묘급진단시제적연구전정료기출.
Objective To co-express rabies virus CTN-1V strain nucleoprotein (NP) and glycoprotein(GP) using Bac-to-Bac baculovirus expression system.Methods The ORF of GP and NP were amplified from CTN-1V strain by RT-PCR and cloned into plasmid vector pFastBacDual PP10 and PPH,respectively.The constructed recombinant donor plasmid P-NG was transformed to DH10Bac E.coli and obtained the recombinant bacmid shuttle plasmid A-NG.Then the plasmid A-NG was transfected into Sf9 cells to construct recombinant baculovirus Bac-NG.The co-expressed protein was identified by Western blot.Resuits It was proved by PCR that recombinant bacmid shuttle plasmid A-NG was constructed correctly.The recombinant protein NP and GP,with a relative molecular mass of about 51 × 103 and 59 × 103,were co-expressed in Sf9 cells.Both expressed NP and GP showed the specific binding reactivity to murine sera against RV.Conclusion The NP and GP of rabies virus CTN-1V strain were successfully co-expressed and showed good immunogenicity,which could serve as a technical basis for new form of vaccine and diagnostic reagents for rabies virus.