家蚕二分浓核病毒ns1基因序列的改造、表达和鉴定

探讨翻译起始区(TIR)部分密码子发生同义突变后,对家蚕二分浓核病毒(BmBDV)ns1基因表达的影响,以及对BmBDV NS1蛋白毒性进行鉴定,设计特异性上游引物,对BmBDV ns1基因中第3、4、9和10个密码子进行同义突变,利用原核表达系统对野生型和改造后的ns1序列进行表达,通过SDS-PAGE电泳对这两种序列的表达产量进行分析。利用Protein Iso~(TM)GST Resin从超声破碎的菌液上清中纯化融合有GST的NS1蛋白,进而对纯化的靶蛋白在细胞水平和家蚕体内进行毒性分析。结果表明:TIR突变后的BmBDV ns1序列,其与野生型序列的表达产量之间没有明显差异;BmBDV NS1蛋白具有抑制细胞增殖和诱导家蚕致死的生化活性。

Modification, Expression of Bombyx mori Bidensovirus ns1 Gene and Characterization

In order to investigate the effects of part of codons after happening synonymous mutation in the translational initiation region (TIR) of BmBDV (Bombyx mori bidensovirus) ns1 gene on its expression, as well as to characterize the virulence of BmBDV NS1 protein, specific upstream primer was specially designed, and carry out four synonymous mutations at 3rd, 4th, 9th and 10th codons of the BmBDV ns1 gene; and express the ns1 sequence using prokaryotic expression method on its wild type and the one after modification, then analyze the expression quantity of these two sequences with SDS-PAGE electrophoresis. NS1 protein merged with GST was purified from ultrasound-breaking cell broth using ProteinIso^TM GST Resin, and carried out virulence analysis on target protein at cell level and in B. mori in vivo. The results showed that there is no significant difference between expression quantity of BmBDV ns1 sequence after TIR mutation and sequence of wild type; BmBDV NS1 protein had biochemical activities of the inhibition against cell proliferation and silkworm death-inducing.