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白斑综合症病毒WSSV(White Spot Syndrome Virus),是严重危害虾类养殖业的主要病原之一。本实验根据已知南美白对虾WSSV ORF147序列设计1对特异性引物,从患疑似白斑病毒病的罗氏沼虾中提取总DNA,用PCR法扩增得到1特异性片段。将该片段克隆进pET-28a(+)载体,测序表明该片段全长1 475 bp,最大开放式阅读框为1 380 bp,编码459个氨基酸,预计其相对分子质量为51.9 kDa;与GenBank登录的WSSV ORF147序列(登录号AF369029)进行比对,核苷酸同源性为99%,证实为WSSV ORF147片段。将该片段在大肠杆菌E coli中进行表达,能获得相应的特异多肽条带。根据测序结果推导WSSVORF147多肽在N端有信号肽序列,并且在氨基酸序列的122~144区间形成跨膜螺旋区。
WSSV (White Spot Syndrome Virus) is one of the major pathogens that seriously endanger the shrimp farming industry. In this experiment, we designed a pair of specific primers based on the known sequence of WSSV ORF147 from Penaeus vannamei. Total DNA was extracted from M. rosenbergii with suspected white spot virus and 1 fragment was amplified by PCR. The fragment was cloned into pET-28a (+) vector and sequenced. The full length of the fragment was 1 475 bp, the largest open reading frame was 1 380 bp encoding 459 amino acids, and its molecular weight was estimated to be 51.9 kDa. Of the WSSV ORF147 sequence (accession number AF369029), nucleotide homology was 99%, confirmed as WSSV ORF147 fragment. The fragment was expressed in E. coli E. coli to obtain the corresponding specific polypeptide bands. Based on the sequencing results, the WSSVORF147 polypeptide was found to have a signal peptide sequence at the N-terminus and a transmembrane helix region was formed between 122 and 144 of the amino acid sequence.