甘薯卷叶病毒江苏分离物基因组全长序列测定及其外壳蛋白基因在大肠杆菌中的表达

摘要

为了制备甘薯卷叶病毒(SPLCV)的特异性抗体,克隆了甘薯卷叶病毒江苏分离物SPLCV(JS:XZ:3-2)的全长基因,并对其基因结构及分子变异情况进行分析,同时对外壳蛋白(CP)基因进行了克隆和表达.利用PCR方法扩增并克隆了SPLCV(JS:XZ:3-2)的全基因组,测序分析表明,SPLCV(JS:XZ:3-2)基因组全长为2 834 bp,含有6个开放阅读框架,与已发表的SPLCV其他分离物相比,全基因组核苷酸序列相似性为82.7％～94.4％.其中,CP基因由765个核苷酸组成,共编码254个氨基酸残基.SPLCV CP基因核苷酸序列与其他分离物的相似性在89.2％～90.6％,其中与SPLCV美国分离物(HQ333135)的相似性最高,为90.6％；与日本分离物(AB433788)的相似性最低,为89.2％；与中国大陆分离物(FJ515896)CP基因的核苷酸序列相似性为90.2％.将SPLCV CP基因克隆到原核表达载体pGEX - 4T-3上,获得重组质粒,经IPTG诱导表达、SDS - PAGE分析得到了大小约为54.3 kD的融合蛋白条带,说明CP基因在大肠杆菌中得到了高效表达.%The complete genome of sweet potato leaf curl virus (SPLCV) isolate JS : XZ : 3-2 from Jiangsu province was amplified by PCR. Analysis of the genomic sequence showed that the genome of SPLCVCJS : XZ : 3-2)was composed of 2 834 nt and included six open reading frames (ORFs). Compared with other isolates previously reported,the nucleotide sequence identity of the complete genome was 82. 7%-94. A%. The CP gene consisted of 765 nt and encoded 254 amino acid residues. The nucleotide sequence of CP gene was 89. 2%-90. 6% identical to other isolates previously reported. The highest identity was 90. 6% with the USA isolate(HQ333135) and the lowest identity was 89. 2% with the Japan isolate (AB433788). The nucleotide sequence of CP gene was 90. 2% identical to the China isolate(FJ515896). The CP gene was cloned into the expression vector pGEX-4T-3 far over-expression in prokaryotic cells. The SDS-PAGE result showed that a specific fusion protein about 54. 3 kD was produced after induction by IPTG.