口蹄疫病毒非结构蛋白3ABC基因重组杆状病毒的构建与表达

通过设计特异引物,扩增获得口蹄疫病毒3ABC编码区的目的基因片段,将其用SalI和NcoI双酶切后,与经同样处理的带有一段信号肽序列的穿梭质粒pMelBac-B连接,经筛选、鉴定及DNA序列分析后,获得重组质粒pMel-3ABC.将重组质粒与线性化的杆状病毒骨架Bac-N-Blue共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒.用重组病毒感染Sf9昆虫细胞,通过间接ELISA方法,检测细胞培养基上清液中表达的口蹄疫病毒抗原.结果表明,口蹄疫病毒基因片段3ABC正确克隆到杆状病毒载体上,重组病毒可在昆虫细胞中表达目的蛋白.

Construction of recombinant baculovirus expressing of foot-and-mouth disease virus nonstructural protein 3ABC gene in Sf9 insect cell

A pair of specific primes 3ABC. The fragment was subcloned into were designed a pMelBac- B quence. After being identified by PCR, enzymatic dige for amplification of FMDV non-structural protein shuttle plasmid with a melittin secretion signal sestion and DNA sequence analysis, the recombinant plasmid was named as pMel-3ABC. Then, the pMel-3ABC plasmid and linearized Bac-N-BlueTM DNA were co-transfected into Sf9 insect cell mediated by cellfectin reagent. Recombinant baculoviruses were screened by plaque assay,and pure virus clone contained only the target 3ABC coding sequence was identified by PCR. The Sf9 cell was infected with the recombinant baculovirus for expression of 3ABC protein. The target protein was confirmed to be expressed in the supernatant of Sf9 cell culture by indirect ELISA. The result showed that the gene 3ABC was cloned into baculoviruses,and the recombinant baculo-virus could express proteins of FMDV in Sf9 cells. The expressed proteins had good bioactivity which could react with sera derived from FMDV infected animals. This study was a good groundwork for establishment of diagnosis method to discriminate the FMDV infected animals from vaccinated ones.