犬瘟热病毒A株核衣壳蛋白基因的克隆、表达及特性分析

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-PCRgA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栽体pPROEXX^TM HT，用重组质粒pPROEX^TM HT-N转化感受态E．coli DH5a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Western blot分析证实所表达的重组N蛋白具反应活性，将表达产物用ProBond^TM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。

Cloning, expression and analysis of nucleocapsid protein gene of canine distemper virus A strain

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 pPROEXTM HT.After transformation of DH5a with recombinant plasmid carrying N gene(pPROEXTM 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 ProBondTM 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.