摘要
用纯化的产蛋下降综合征（ＥＤＳ－７６）病毒（Ｈ９１毒株）悬液提取的核酸，经琼脂糖凝胶电泳只出现１条分子量较大、背景清晰的核酸带。用ＢａｍＨＩ酶消化产生４个片段，大小分别为１７ｋｂ、１０ｋｂ、４．０ｋｂ和２．０ｋｂ。将其中的２．０ｋｂ片段克隆进ｐＵＣ１８ＤＮＡ载体中，重组质粒ＤＮＡ用ｄｉｇｏｘｉｇｅｎｉｎ标记作为ＤＮＡ探针，在ｄｏｔｂｌｏｔ中该探针不与鹅胚尿囊液核酸抽提物、马立克氏病病毒（ＭＤＶ）ＤＮＡ、鸡传染性贫血病毒（ＣＡＶ）ＤＮＡ、ＳＰＦ鸡基因组ＤＮＡ等核酸反应，只与３株ＥＤＳ病毒（ＥＤＳ标准毒株ＡＶ－１２７、ＥＤＳＨ９１毒株和从健康鹅体中分离的ＥＤＳＹ８１毒株）ＤＮＡ呈阳性反应。人工感染产蛋母鸡和雏鸡后２４ｈ即可用该探针从泄殖腔棉拭子样品中检测出ＥＤＳ病毒ＤＮＡ，在感染后３５ｈ仍能从部分感染鸡样品中检出。对部分探针检测阳性鸡的泄殖腔棉拭子样品用ＳＰＦ鸡胚分离病毒，并用ＨＡ和ＨＩ试验检测分离病毒的特异性；在感染过程中，同时检测了血清中ＨＩ抗体的消长情况。结果表明，人工感染鸡排毒至少可以维持２个月左右，而且血清中ＨＩ抗体即使很高，也不能阻止感染鸡的排毒。
By using cesium chloride gradient ultra-centrifugation, egg drop syn- drom virus(EDS-76)particles were purified form allantoic fluids of goose embryos inoc- ulated with the EDS- 76 strain H91 isolated from a layer flock. Under electron micro- scope,the round virus particles in diameter of 70-85 μm without envelopes were ob- served in the preparation. The phenol-extracted DNA from the purified virus suspension showed a clear band of more than 30 kb in the agarose gel electrophoresis. By Bam HI digestion,the virus genomic DNA was cut into 4 fragments of 17 kb,10kb,4 kb and 2 kb.The fragment of 2 kb was cloned into the plasmid pUC18,and the recombinant plasmid DNA was labeled with digoxigenin and used as the probe,In the hybridization,the probe reacted only with 3 strains of EDS-76 virus, but not with DNA samples from Marek’s disease virus, chicken anemia virus, normal goose embryo allantoic fluid and SPF chickens. By dot blot hybridization, the probe could detect EDS-76 specific DNA in cloacal swab samples from chicks and laying hens during a period of 24 hours to 35 days after infection,The virus was also reisolated from some dot blot positive swap samples when inoculated into SPF chicken embryos, and the virus isolates were confirmed by HA and HI tests. During the period,the HI antibody titers in sera from infected birds were determined.The results indicated that Chickens could shed viruses for at least 2 months after infection even though the HI titers to the virus maintianed at high levels in the infected birds.
出处
《中国兽医学报》
CAS
CSCD
1995年第3期219-223,共5页
Chinese Journal of Veterinary Science
基金
江苏省科委自然科学基金
