| 摘要: |
| 【目的】分离并鉴定陕西省牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)。【方法】采集腹泻犊牛的肠系膜淋巴结,处理后接种于MDBK细胞,盲传,逐日观察是否有细胞病变产生。采用病毒蚀斑技术对病毒克隆纯化后,提取其RNA,进行RT-PCR鉴定。将扩增的NS5b基因克隆入pMD18-T载体中,采用碱裂解法小量提取质粒,并对质粒进行EcoRⅠ和SalⅠ双酶切、SalⅠ单酶切及PCR鉴定,对阳性克隆进行核苷酸序列测定及分析。采用病毒蚀斑试验测定分离毒株的半数组织培养感染量(TCID50)。【结果】盲传4代后细胞出现典型、规律的细胞病变。RT-PCR扩增出预期长度(360 bp)的基因片段,核苷酸序列分析结果表明,所扩增的基因为牛病毒性腹泻黏膜病病毒NS5b基因,其与BVDV VEDEVAC株NS5b基因核苷酸序列有99%同源性。BVDV陕西株的半数组织培养感染量(TCID50)为3.6。【结论】成功分离了牛病毒性腹泻病毒陕西株,为陕西省BVDV的防治提供了理论依据。 |
| 关键词: 牛病毒性腹泻病毒 NS5b基因 RT-PCR 细胞病变 陕西省 |
| DOI： |
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| 基金项目:陕西省科技攻关项目（2005K01-G20-2）；国家“十一五”科技支撑计划奶业专项“西北农区生态型奶业生产技术和奶牛性控技术研究与开发”（2006BAD4A11） |
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| Isolation and NS5b gene sequencing of BVDV in Shaanxi Province |
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| Abstract: |
| 【Objective】 The study was to isolate and identify BVDV in Shaanxi Province. 【Method】 The supernatant of the intestine absorbent gland was innoculated into MDBK cells and reproduced blindly.The virus was purified by the virus plaque formation,and the RNA was isolated and detected by RT-PCR.The fragments of NS5b gene was successfully cloned into the pMD18-T vector,the recommbanant plasmid was identified by incision enzyme digestion and PCR，and the positive clone was sequenced.The TCID50 of the BVDV in Shaanxi Province was tested by the virus plaque formation.【Result】 The unknown virus isolated from mesenteric lymphadenitis showed obvious cytopathic effects (CPE) after blindly subculturing 4 times.The cloning virus was isolated by virus plaque technique.The nucleotides sequence of NS5b gene of the cloning virus shared 99％ homology with strain VEDEVAC,and its TCID50 was 3.6.【Conclusion】 The cloning virus was Bovine viral diarrhea virus (BVDV)，and its nucleotides sequence of NS5b gene shared 99％homology with strain VEDEVAC. |
| Key words: Bovine viral diarrhea mucosal disease NS5b gene RT-PCR cytopathic effect Shaanxi Province |
