摘要
根据对虾白斑综合症病毒(whitespotsyndromevirus,WSSV)1个可能编码蛋白的开放读码框(openreadingframe,ORF)的序列(WSSVA基因),结合pQE30载体的多克隆位点,设计1对引物进行PCR,扩增出大小为0.28kb的A基因片段。把目的片段克隆到pGEM-TEasy载体上,构建出重组质粒pGT-A,再用引物两端酶酶切出目的片段,并按正确的读码框顺序插入到pQE30表达载体上的乳糖操纵子中,构建出带有目的片段的重组质粒pQE30-A,然后将重组质粒转化大肠杆菌M15细胞,经IPTG诱导,SDS-PAGE和Westernblot显示有与A基因预期大小11kD相吻合的融合蛋白带。结果表明,来源于WSSV的这一ORF是1个可表达的基因。
An ORF for coding protein was found in sequence analysis of WSSV DNA(named WSSV A gene). Its DNA sequence is the same to the ORF110 which was reported by Van Hulten et al(2001), and its function is unknown. A pair of primers was designed according to the known sequences of the ORF and the MCS of the pQE30 vector. The fragments of 0.28 kb (A gene) were amplified by polymerase chain reaction (PCR). The PCR products were cloned into the pGEMT Easy vector to get the recombinant plasmid pGT-A and then the recombinant expression plasmid pQE30A was obtained after pGTA was digested and the target gene was cloned into the expression vector pQE30. The recombinant expression plasmids were transferred, respectively, into the E. coli strain M15. After the recombinant strain was induced by IPTG, the target expression products of A gene showed a fusion protein band by SDSPAGE, corresponded to molecular mass positions of 11 kD. A further confirmation was obtained by Western blot. The result indicates the ORF is an expressible gene.
出处
《中国水产科学》
CAS
CSCD
北大核心
2003年第1期10-13,共4页
Journal of Fishery Sciences of China
基金
资助项目:国家'八六三'高技术研究发展计划资助项目(2001AA621030)
中国水产科学研究院重点项目(99-01-02).
