科研动态

目前在昆虫杆状病毒表达系统（baculovirus expression vector system，BEVS）中表达A型塞内卡病毒（Senecavirus A，SVA）结构蛋白VP1尚未见报道。本研究将VP1基因通过限制性酶切位点插入杆状病毒载体pFastBac I，然后将鉴定正确的重组载体转化至DH10Bac感受态细胞，经蓝白斑筛选，对鉴定正确的菌落提取质粒。将质粒转染Sf9昆虫细胞，待有明显细胞病变后收获重组杆状病毒rBac-I-VP1，并进行病毒滴度测定。随后通过蛋白免疫印记（Western Blot）和间接免疫荧光（IFA）鉴定VP1蛋白表达情况。结果显示，重组杆状病毒rBac-I-VP1滴度为6.8×105 pfu/mL；Western Blot可检测到相对分子质量约27 kDa的表达产物，且其能够被SVA兔阳性多克隆血清识别；IFA试验显示，VP1蛋白能够在Sf9细胞内表达。结果表明，本研究利用BEVS表达的SVA VP1蛋白具有良好的免疫反应性，为其后期功能研究及SVA诊断试剂研制提供了技术基础。

Expression and Identification of Senecavirus A Structural Protein VP1 in Insect Baculovirus 

The expression of senecavirus A（SVA）structural protein VP1 in baculovirus expression vector system（BEVS）has been not reported till now. In the study，VP1 gene was inserted into baculovirus vector of pFastBac I through the restriction enzyme site，the identified recombinant vector was transformed into DH10Bac，and the correct colonies were screened by blue and white spots to extract the plasmids that were transfected into Sf9 insect cells，and the recombinant baculovirus rBac-I-VP1 was obtained after obvious cytopathic changes. The expression of VP1 protein was identified by Western Blot and indirect immunofluorescence（IFA）. The results showed that the titer of recombinant baculovirus rBac-I-VP1 was 6.8×105 pfu/mL；the expression products with relative molecular weight of about 27 kDa could be detected by Western Blot，which could be recognized by SVA rabbit positive serum；it was shown by IFA that，VP1 protein could be expressed in Sf9 cells. It was concluded that the SVA VP1 protein expressed by the BEVS was with good immunoreactivity，which provided a basis for future study on related functions and the development of diagnostic reagents.

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