作　　者： ; ; ; ; ;

机构地区： 华中农业大学畜牧兽医学院

出　　处： 《畜牧兽医学报》 2003年第2期195-198,共4页

摘　　要： 利用PCR技术从猪细小病毒的RFDNA模板中扩增了含有VP2 全基因的 2 0kb的基因片段 ,将PCR产物克隆至 pMD18 T载体 ,利用双脱氧末端终止法测定了VP2 全基因的核苷酸序列。将所得的核苷酸序列和推导的氨基酸序列分别与GenBank中的相应序列进行同源性比较 ,同源性均在 99%以上 ,从而说明该蛋白的碱基序列和氨基酸序列均具有高度的保守性。将VP2 全基因分别克隆入原核表达载体pET15 (b)、pET17(b)和 pET2 8(b)构建成表达质粒 pET15bVP2 、pET17bVP2 和 pET2 8bVP2 ;将含有VP2 基因起始位点至EcoRⅠ切点间的 0 8kb片段克隆入 pET17(b)中构建成表达质粒 pET17bVP2 f。利用上述四种质粒转化大肠杆菌BL2 1,IPTG诱导后SDS PAGE检测表达情况 ,结果发现 pET17bVP2 f质粒在 45kD处有一特异性表达带 ,而其它几种质粒均未看到特异性表达带 ,推测VP2 基因 3′端的某些结构可能对VP2 全基因的表达有一定影响。这一结果对研究VP2 基因的结构与功能具有重要意义。 The completed VP 2 gene was amplified from PPV RFDNA by PCR method.Then the products were cloned into pMD18-T vector and the sequence was determined.The homology analysis of this gene with others reported in GenBank showed that this gene was high conserved.The completed VP 2 gene were cloned into pET15(b),pET17(b) and pET28(b) vectors respectively and 0.8 kb fragment of VP 2 gene were cloned into pET17(b) vector,the recombinant plasmid pET15bVP 2,pET17bVP 2,pET28bVP 2 and pET17bVP 2f were constructed.Then these plasmids were introduced into E.coli BL 21.After induction by IPTG,a high expression was found in products of pET17bVP 2f,while no expression protein was detected for other plasmids.These results will be useful for further research of VP 2 gene.

关 键 词： 猪细小病毒 基因 基因克隆 序列分析 原核表达

领　　域： [农业科学] [农业科学] [农业科学]