新城疫病毒HN基因的克隆与原核表达

根据GenBank公布的标准Lasota株的HN基因序列,设计了一对引物,经RT-PCR从标准Lasota疫苗毒中特异性扩增出约1590bp的HN基因。将扩增得到的基因定向克隆到原核表达载体pET-30a(+)中,获得重组质粒pET-NDV/HN,将重组质粒转化大肠杆菌BL21(DE3)中,得到重组菌BL21(pET-NDV/HN)。将重组菌诱导表达后,通过SDS-PAGE分析可见约64.3kDa的特异条带,说明融合蛋白大小与预期理论值相符;Western blotting结果进一步表明表达的蛋白可与NDV阳性血清发生特异性结合,说明其具有良好的免疫原性。

Cloning and Prokaryotic Expression of hemagglutinin-neuraminidase Gene of Newcastle Disease Virus

The HN gene of NDV-Lasota strain was amplified by RT-PCR,which the primers were designed on the basis of HN gene sequence of NDV Lasota strain,and then inserted into the prokaryotic vector pET30a and transformed into Escherichia coli BL21,then identified by SDS-PAGE and Western blot.The prokaryotic expression vector pET-HN was constructed,and SDS-PAGE showed that the fusion protein had a molecular weight 64.3 kDa,which could be specifically recognized by serum against NDV.It is apparent that the HN gene was highly conservative and was expressed in E.coli in high level,also the prokaryotic expression products of this gene had a fine reactiongenicity of immunity.