犬瘟热病毒h基因克隆测序与原核表达

Cloning and Prokaryocyte Expression of Hemagglutinin Protein Gene of Canine Distemper Virus

根据GenBank中登录的犬瘟热病毒h基因序列设计一对引物,利用RT-PCR方法扩增出犬瘟热病毒弱毒疫苗株的h基因,将其插入到克隆载体pMD18-T中,经蓝白斑筛选及SalⅠ和XhoⅠ双酶切鉴定挑出阳性克隆进行序列测定,序列分析结果表明,该基因与GenBank上标准代表毒株序列完全同源;与扬州分离株、长春分离株、新疆分离株、日本分离株D85755同源性分别为90.6%、91.3%、90.5%、91%;与Onderstepoort、Convac株、美国分离株98-2666-2同源性较高,分别为97.7%、97.2%、98.1%。再将其定向克隆于原核表达载体pET-32a(+)中,转化感受态E.coliBL21细胞,经IPTG诱导表达分子质量约64 ku的重组H蛋白。

A pair of primers was designed based on the h gene sequence of canine distemper virus in GeneBank, the h gene of （CDV） strain isolated from an attenuated vaccine strain of canine distemper virus was amplified by reverse transcription polymerase ehain reaction （RT-PCR）. The amplified fragment was cloned into pMD18-T simple vector and the positive reeombinants were identified by blue-white screening and restriction endonuclease digestion, and the h fragments were sequenced and analyzed. Sequencing analysis indicates that the gene was the same to the representive canine distemper virus h gene. h gene was 90. 6%,91. 3%, 90. 5%, 91% identical to Yangzhou isolates, Changchun isolate, XinJiang isolate and Japanese isolat- eD85755 respectively. It was 97.7%, 97.2%, 98.1% indentical to Onderstepoort strain, Convac strain and American isolates 98-2666-2 respectively. Then the h gene was subctoned into Prokaryotic expression plasmid pET-32a（＋）. Reeombinantplasmid carrying h gene （pET-32a-h） was transformed into E. coli BL21（DE3） and induced with IPTG. A fusion protein about 64 ku was expressed.