番鸭源禽1型副粘病毒PX2/03株P基因的克隆及原核表达

根据GenBank中水禽源1型副粘病毒(ZJ1)P基因序列设计2对引物,运用RT-PCR技术从1株雏番鸭源禽1型副粘病毒(PX2/03)的基因组中扩增出P全基因的cDNA.测序结果表明,P基因全长1441 nt,包含1个完整的开放阅读框架,编码395个氨基酸.与GenBank中已发表的P基因比对发现,该基因与近年来分离的鹅源禽1型副粘病毒分离株的亲缘关系很近.以分别含BamHⅠ、XhoⅠ的1对引物对该目的基因进行亚克隆后插入到pET32 a原核表达载体中,构建了pETP原核重组载体,将该重组载体转化入BL21(DE3)后,成功诱导表达了分子质量大小约为62 ku的P融合蛋白,经W estern b lotting检测证实表达产物具有免疫活性.

Cloning and prokaryotic expression of phosphoprotein gene of avian paramyxovirus type 1 of PX2/03 strain isolated from Muscovy duck

The nucleotide acid of the phosphoprotein(P) gene of avian paramyxovirus type 1(APMV-1),PX2/03 strain,isolated from Muscovy duckling has been determined by sequencing cDNA clone derived from viral genomic RNA.The result showed that the length of the whole P gene is 1441 nt,containing an open reading frame(ORF) which encoded a polypeptide of 395 amino acids.Comparing the P gene of PX2/03 with those of some other APMV-1 strains submitted to GenBank database,it shared close relationship with those of the stains isolated from geese.Subsequently,the P gene was subcloned directly into a prokaryotic expression vector pET-32a,yielding the recombinant pETP.After transformation of pETP into E.coli BL21 (DE3) and induction with the IPTG,the P fusion protein with molecular weight of 62 ku was properly expressed.Western blotting analysis showed that the recombinant protein shared good immunogenicity.