摘要
以苜蓿丫纹夜蛾核型多角体病毒 (AcNPV)的核衣壳蛋白基因vp39设计引物 ,用PCR技术扩增了家蚕核型多角体病毒中国株 (BmNPV Ch)～ 1.3kb片段 ,并测定了其全序列长 12 30bp。推导的氨基酸 351个 ,其与BmNPV日本株 (BmNPV Ja)vp39基因核苷酸序列同源性达 97.5% ,氨基酸同源性达 97.1%。该片段在E .coliBL2 1中诱导表达能产生分子量约为 38kD的特征性蛋白带 ,证明所扩增片段为BmNPV Ch的vp39基因。经与BmNPV Ja比较 ,其 6 2 5位有 3个核苷酸(CGA) ,985～ 910位有 6个核苷酸 (GTCGGC)的插入 ,未造成码组移动。有 17处点突变 ,但取代突变 (replacementmutation)仅 7处 ,对两株病毒VP39蛋白的亲疏水性和酸碱性质未产生显著影响。由于点突变带来氨基酸改变 ,使BmNPV Ch的vp39蛋白的α 螺旋由 13.2 %增加到 15.4 % ,无规卷曲由 7.2 %减少到 5.8% ,β 折叠和β 转角维持不变 ,约为 79.6 % ,仍保持以β 折叠为主要二级结构单元的片层状折叠结构。
The nuclear capsid protein gene (vp39) of Bombyx mori nuclear polyhedrosis virus (Chinese isolate, BmNPV Ch) was amplified by PCR and inserted into pGEM 3zf(+). The amplified vp39 gene (1 230 bp) was sequenced with silver staining dideoxy chain termination. The coding region is 1 053 bp, and code for 351 amino acids. Comparing with the vp39 gene of BmNPV Ja (Japanese isolate), the homology of the nucleotide and the amino acid sequences is 97.5% and 97.1% respectively. The BmNPV vp39 gene was inserted into the expression vector pRSET A, and transformed into E.coli BL21. This gene from BmNPV Ch is 9 bp longer than that of BmNPV Ja. The insertion of nine nucleotides is found in vp39 gene sequence of BmNPV Ch (CGA at 625 site and GTCGGC at 985-910). Although there are 17 site mutations, only 7 site mutations are replacement mutations. There was no effect to the hydrophilicity and charge of two isolates of VP39. The replacement of anino acids caused by the site mutations made the changes of the secondary structure of Bmvp39 Ch. e.g.α helix increased from 13.2% to 15.4%; rundom coil decreased from 7.2% to 5.8%. β sheet and β turn of two isolates were similiar (about 79.6% ). It is indicated that the main secondary structure of the VP39 protein is fold sturcture containing high proportion of β sheet and β turn. This structure may be concordant with its function.
出处
《中国病毒学》
CSCD
2000年第1期52-58,共7页
Virologica Sinica
基金
国家自然科学基金
国家教委博士点基金
关键词
核衣壳蛋白基因
家蚕
核型多角体病毒
点突变
Nuclear capsid gene (vp39)
Gene cloning, BmNPV
Site mutation
Secondary structure
