摘要
根据GenBank中A75/14株犬瘟热病毒(CDV)核衣壳蛋白(N)基因的核苷酸序列设计合成一对引物,经RT-PCR从病毒总RNA中扩增出1600bp的N基因,将其克隆于pMD18-T载体对其进行序列分析。结果表明A株CDV与其它毒株N基因序列的同源性较高。将N基因定向克隆于原核表达载体pPROEXTMHT,用重组质粒pPROEXTMHT-N转化感受态E.coliDH5a细胞,用IPTG于37℃诱导表达了分子量约63Ku的重组N蛋白,占菌体总蛋白18%。经Westernblot分析证实所表达的重组N蛋白具反应活性,将表达产物用ProBondTM纯化试剂盒进行了纯化。用所获得的重组蛋白免疫家兔制备的多克隆抗体可与CDV全病毒发生特异性反应,经琼扩试验测定其效价为1∶16。本实验为下一步用重组N蛋白作为诊断用抗原,建立特异的CDV抗体检测方法奠定了基础。
A pair of primers were designed according to the sequence of Nucleocapsid（N） gene of canine distemper virus A 75/14 strain, cDNA encloning for N gene of A isolate, was amplified and cloned into PMD18-T plasmid, Sequencing analysis indicated the homology between A strain and other strains is very high. Then the N gene was subcloned into Prokaryotic expression plasmid pPROEX^TM HT, After transformation of DH5a with recombinant plasmid carrying N gene （pPROEX^TM HT-N）, an 63 Ku expressed fusion protein was identified by SDS-PAGE after induced by IPTG, it accounted for 18 % of the total cellular protein. The immune reactivity of the recombinant protein was confirmed by Western blot, The expressed protein was purified by ProBond^TM purification system and used to prepare the multiclonal antibodies by subcutaneous injection of rabbit, the antibodies is specific to CDV, The results above laid a foundation for the development of ELISA test kit of CDV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第4期485-488,共4页
Chinese Journal of Preventive Veterinary Medicine
关键词
犬瘟热病毒
核衣壳蛋白
表达
反应活性
多克隆抗体
canine distemper virus
nucleocapsid
expression
immune reactivity
multiclonal antibodies
