猫疱疹病毒Ⅰ型US3基因剪接异构体的鉴定

发布时间：2018-11-24 20:52

【摘要】：为鉴定猫疱疹病毒Ⅰ型(FHV-1)US3基因的编码产物,本研究以提取FHV-1感染细胞的总RNA为模板,通过RT-PCR方法扩增US3基因转录产物,测序结果显示除了全长US3基因(1 056 bp)外,还出现了一个825 bp的片段(US3-X1)。该片段是由全长US3基因在226 bp~458 bp位置发生了自我剪接,导致234 bp DNA缺失而产生,但ORF并没有改变。此外,通过重叠延伸PCR将US3基因226 bp处碱基"A"突变为"G"(m US3),改变自我剪接位点。将全长US3基因、m US3和US3-X1基因分别克隆至p3×Flag-CMV-10中,转染F81细胞,经western blot检测表明FHV-1 US3可以表达两种不同分子量的异构蛋白,大小约为45 ku和50 ku。这种US3基因的剪接异构体现象在猪伪狂犬病毒和人单纯疱疹病毒中已有报道,而FHV-1 US3基因的剪接异构体的鉴定尚属首次。该研究结果为进一步研究FHV-1 US3异构体蛋白的功能奠定了基础。
[Abstract]:In order to identify the encoding product of US3 gene of cat herpesvirus type I (FHV-1), the total RNA of FHV-1 infected cells was used as template, and the transcription product of US3 gene was amplified by RT-PCR. In addition to the full-length US3 gene (1 056 bp), a 825 bp fragment (US3-X1) was found. The fragment was generated by self-splicing of the full-length US3 gene at 226 bp~458 bp, resulting in the deletion of 234 bp DNA, but the ORF did not change. In addition, the nucleotide "A" at 226 bp of US3 gene was mutated to "G" (m US3) by overlapping extension of PCR, and the self-splicing site was changed. The full-length US3 gene, m US3 gene and US3-X1 gene were cloned into p3 脳 Flag-CMV-10 and transfected into F81 cell line respectively. Western blot analysis showed that FHV-1 US3 could express two kinds of isomerism proteins with different molecular weight, about 45 ku and 50 ku.. The splicing isomer of US3 gene has been reported in porcine pseudorabies virus and human herpes simplex virus, but the identification of splicing isomer of FHV-1 US3 gene is the first time. The results laid a foundation for further study on the function of FHV-1 US3 isomer proteins.
【作者单位】：东北农业大学动物医学院;中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室;
【基金】：国家自然基金青年基金(31402201)
【分类号】：S852.65

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