猪细小病毒VP2基因的克隆、测序与原核表达

利用PCR技术从猪细小病毒的RF DNA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。将所得的核心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pET28(b)构建成表达质粒pET15bVP2、pET17bVP2和pET28bVP2；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pET17bVP2f。利用上述四种质粒转化大肠杆菌BL21，IPTG诱导后SDS-PAGE检测表达情况，结果发现pET17bVP2f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。

Study on Clone,Sequence Analysis and Expression of Porcine Parvovirus VP2 Gene

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.