家蚕质型多角体病毒基因组片段1中的假定重叠基因检测

Verification of Hypothesized Overlapping Gene in Genomic Segment Ⅰ of Bombyx mori Cypovirus

生物信息学分析结果显示家蚕质型多角体病毒（BmCPV）基因组s1片段中含有-个假定的重叠基因ORFX。为了检测该重叠基因ORFX是否在RNA和蛋白质水平有相应的表达产物，用大肠杆菌表达的重组ORFX蛋白制备多克隆抗体，对感染BmCPV的家蚕中肠后部组织进行Westernblotting和免疫组化检测，结果在感染BmCPV第1～7天的家蚕中肠组织中没有检测到假定的ORFX蛋白。进一步基于假定重叠基因ORFX区域及其下游序列设计定量检测ORFX基因和s1片段转录本RNA总水平的引物RECPV-1／RECPV-2，以及定量检测S1片段转录本RNA水平的引物RECPV．3／RECPV-4，用引物Oligo（dT）／RECPV-2和Oligo（dT）／RECPV-4的反转录产物作为模板，对感染BmCPV的家蚕中肠组织中的S1片段转录本和假定的ORFX转录本进行定量PCR检测，结果在感染BmCPV第5～7天的中肠组织中均没有检测到假定的ORFX转录本。由此表明，BmCPV基因组S1片段中可能不存在假定的ORFX重叠基因。

Bioinformatic analysis revealed that there was a hypothesized overlapping gene （ORFX） in genomic Segment I of Bombyx mori cypovirus （ BmCPV）. In order to detect whether ORFX is expressed at the RNA and protein levels, we expressed the recombinant ORFX protein in E. coil and used it to prepare polyclonal antibody, which was then used to conduct Western blotting and immunohistochemical analyses for detection of the putative ORFX protein in posterior midgut of silkworm larvae infected by BmCPV. However, the putative protein was not detected in the midgut at day 1 to day 7 after infection. Furthermore, based on the ORFX sequence and its downstream sequence, a pair of primers （RE- CPV-1/RECPV-2） was designed to quantitate total transcriptional level of ORFX and Segment I, and another pair of primers （RECPV-3/RECPV-4） was designed to quantitate the transcriptional level of Segment I. The relative transcriptional level of ORFX and Segment I were determined by quantitative PCR in the midgut of silkworm larvae infected by BmCPV using the reverse transcription product from Oligo（dT）/RECPV-2 and Oligo（dT）/RECPV-4 primers as template. The results showed that ORFX transcript was not detected in the midgut of silkworm larvae at day 5 to day 7 after infection, suggesting that there might not be an overlapping gene in segment I of BmCPV genome.