为研究大熊猫轮状病毒(Giant Panda Rotavirus,GPRV)CH-1株外衣壳蛋白VP7基因的结构及功能,用MA-104细胞(恒河猴胎猴肾细胞)增殖GPRV,提取总RNA,运用RT-PCR扩增VP7基因,并测序,用生物信息学软件预测其功能,并构建系统进化树。结果显示...为研究大熊猫轮状病毒(Giant Panda Rotavirus,GPRV)CH-1株外衣壳蛋白VP7基因的结构及功能,用MA-104细胞(恒河猴胎猴肾细胞)增殖GPRV,提取总RNA,运用RT-PCR扩增VP7基因,并测序,用生物信息学软件预测其功能,并构建系统进化树。结果显示成功获得了长1042bp的GPRV VP7蛋白基因(GenBank登录号GU188284)。经生物信息学分析,此序列包含1个981bp的完整开放阅读框,编码326个氨基酸;预测GPRV VP7蛋白理论相对分子质量为37354.8u,等电点为4.76,半衰期为30h,不稳定系数为26.71,总平均亲水性为-0.015,疏水性介于-2.344～3.567;1—24位氨基酸可能是信号肽序列;跨膜结构分析表明VP7蛋白有2个跨膜区,其N端和C端都位于病毒膜外区;抗原表位预测显示VP7蛋白有12个抗原决定簇;结构预测显示其可能包含2个N-糖基化作用位点,5个蛋白激酶C磷酸化作用位点、4个酪蛋白激酶Ⅱ磷酸化作用位点、1个酪氨酸激酶磷酸化作用位点、4个N-豆蔻酰化位点、1个原核膜脂蛋白脂附着点和1个革兰氏阳性球菌表面蛋白‘锚’六肽。系统进化树分析显示GPRV VP7基因与人A组轮状病毒VP7基因的进化距离最近。本研究成功获得了GPRV VP7基因,为今后研究此基因的生物学功能以及建立该病毒的诊断方法奠定基础。展开更多
The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletro...The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletronmicroscope .It was concluded that the isolate was PCV-2 and was named SCH-A strain.The capsid protein gene(cp) in ORF2 was amplified from PK-15 cell line infected with SCH-A strain by PCR, and was cloned and sequenced.The nucleotide sequences of ORF2 gene of the SCH-A strain was compared with other porcine circovirus isolates.The results showed that the homologies of nucleotides were between 90.5% and 96.7% in comparision with the PCV-2 reference strains,but the homologies of nucleotides were only about 67% with the PCV-1 reference strains. SCH-A strain displayed the highest nucleotide homology((96.7)%) with the reference strain Hunan .Phylogenetic tree analysis showed ORF2 gene of the SCH-A isolate belongs to PCV-2 .展开更多
文摘The PCV-2 positive tissues by PCR-restriction fragment length polymorphism (PCR-RFLP) were inoculated to the PK-15 cells not contaminated by PCV. The viral particles were observed in the infected PK-15 cells by eletronmicroscope .It was concluded that the isolate was PCV-2 and was named SCH-A strain.The capsid protein gene(cp) in ORF2 was amplified from PK-15 cell line infected with SCH-A strain by PCR, and was cloned and sequenced.The nucleotide sequences of ORF2 gene of the SCH-A strain was compared with other porcine circovirus isolates.The results showed that the homologies of nucleotides were between 90.5% and 96.7% in comparision with the PCV-2 reference strains,but the homologies of nucleotides were only about 67% with the PCV-1 reference strains. SCH-A strain displayed the highest nucleotide homology((96.7)%) with the reference strain Hunan .Phylogenetic tree analysis showed ORF2 gene of the SCH-A isolate belongs to PCV-2 .
