猪伪狂犬病病毒FZ株的分离鉴定及gD基因序列分析

Isolation and identification of porcine pseudorabies virus FZ strain and sequence analysis of the gD gene of the strain

采集福州郊区病仔猪的鼻腔黏液、脑、脾脏、肾脏、淋巴结等组织病料,研磨上清接种到非洲绿猴肾(Vero)细胞和狗肾(MDCK)细胞进行病毒分离,通过对分离病毒株进行形态学、血清学、荧光抗体试验、动物接种试验、PCR试验等鉴定,确定所分离的病毒为猪伪狂犬病病毒(PRV),命名为PRV-FZ株.根据GenBank收录的PRV gD基因的序列设计一对引物,通过PCR方法扩增出约1579 bp的目的片段,并将其克隆到PCAGGS载体进行酶切鉴定、核苷酸序列测定和分析.结果表明,目的片段包含一个1203 bp的开放阅读框,编码400个氨基酸组成的多肽,与GenBank中有代表性的5株参考毒株相应基因序列比较显示,核苷酸和氨基酸序列同源性分别为99.1%-99.8%和98.5%-99.5%.用Bioedit和TMPRED软件预测PRV-FZ gD基因的跨膜区,表明存在2处显著意义的跨膜区;用Predictprotein软件结构域预测,表明该蛋白存在2个cAMP-/cGMP-依赖蛋白激酶磷酸化位点、5个蛋白激酶C磷酸化位点、1个酪蛋白激酶Ⅱ磷酸化位点及3对二硫键.

A wild-type pseudorabies virus（PRV） was isolated from an affected porcine in Fuzhou.Supernatants from cell extracts of nose cavity mucus,brain,spleen,kindy,lymph were inoculated to the Vero cells and MDCK cells and significant cytopathogenic effect was produced.The isolated virus was confirmed by serological tests,PRV-Fluorescence antibody tests,experimental infection of animals and PCR tests.The virus isolate was designated as PRV-FZ.A pair of oligonucleotide primers were designed according to the sequences published by the GenBank in order to amplifiy gD gene of the PRV-FZ.A PCR product of approximately 1579 bp was amplified from PRV-FZ strain.The PCR product was inserted into PCAGGS vector and sequenced.Nucleotide sequencing revealed that this fragment contained an open reading frame of 1203 bp encoding a 400 aa protein.The gene was sequenced and compared with those of five other PRV strain in GenBank.The results showed that the gD gene of PRV-FZ shared 99.1%-99.8% nucleotide homology and 98.5%-99.5% amino acid homology with strains.Meanwhile there are two notable transmembrane region in PRV-FZ gD by Bioedit and TMPRED;Structure prediction shows that there are two CAMP_PHOSPHO_SITE,five PKC_PHOSPHO_SITE,one CK2_PHOSPHO_SITE and three pires of disulfind.