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ns2是黑胸大蠊浓核病毒的一个非结构基因,所编码的蛋白质大小为30kD,是一个功能未知的基因。为了对该基因进行深入的功能研究,从感染了黑胸大蠊浓核病毒的蟑螂的后肠组织中通过RT-PCR得到ns2基因编码序列,将其构建于原核表达载体pET-28a,转化大肠杆菌BL21(DE3)获得融合表达产物。此融合蛋白经分离纯化后,免疫新西兰大白兔,制备其多克隆抗体。采用Western印迹技术,用该抗体检测ns2基因的真核表达产物,证明该抗体有较好的针对NS2蛋白的专一性,可用于对NS2结构和功能的研究。同时,将此编码序列克隆至果蝇细胞表达载体pAC,得到重组质粒后转染果蝇S2细胞表达重组蛋白,通过共聚焦显微镜用该抗体检测该蛋白在S2细胞中的亚细胞定位,发现NS2蛋白主要定位于细胞质,核内仅有少量分布。
ns2 is a nonstructural gene of N. capitatum densovirus and encodes a protein of 30 kD, a gene of unknown function. In order to further study the function of this gene, the ns2 gene coding sequence was obtained from the gut of the cockroach infected with the black-throat cockroach densovirus by RT-PCR. The coding sequence of the ns2 gene was constructed into the prokaryotic expression vector pET-28a and transformed into the large intestine Bacillus BL21 (DE3) to obtain a fusion expression product. The fusion protein was isolated and purified, immunized New Zealand white rabbits to prepare polyclonal antibodies. Western blotting was used to detect the eukaryotic expression product of ns2 gene. The results showed that the antibody was specific to NS2 protein and could be used to study the structure and function of NS2. At the same time, the coding sequence was cloned into the expression vector pAC of Drosophila. The recombinant plasmid was obtained and transfected into Drosophila S2 cells to express the recombinant protein. The subcellular localization of this protein in S2 cells was detected by confocal microscopy. It was found that NS2 Protein mainly located in the cytoplasm, nuclear only a small amount of distribution.