山羊痘病毒基因组提取、酶切及TK基因克隆测序分析-预防兽医学专业论文.docxVIP

PAGE PAGE 2 Extraction and restriction endonuclease analysis of genomic DNA and sequence analysis of TK gene from goatpox virus Abstract Capripox caused by capripoxvirus, is an acute feverish and highly contagious disease in sheep or goat. Goat pox is an ancient disease that is currently endimic in the Middle East, the Indian subcontinent, Bangladesh, and Central and Northern Africa, The desease is defined as a heavy disease of animal by OIE, based on further economic loss in the endemic countries. The Study on Real-time PCR Technique for Goat poxvirus Detection multiplication properties of goatpox virus on Vero cell Cultures, restriction endonuclease analysis of genomic DNA and sequence analysis of TK gene from goatpox virus were carried out in the experiment. Real-time PCR Technique for Goat poxvirus Detection Using SYBR Green I A pair of primers were designed for detection of Goat pox virus (GPV) ITR (Inverted Terminal Repeat) gene by using SYBR Green I real-time PCR technique. Established and optimized detection of the ITR gene by SYBR Green I real-time PCR technique and compared sensitive with conventional PCR. The results showed that: SYBR Green I real-time PCR technique can detect 7×102 copies of the DNA template, this assay was more sensitive than the conventional PCR assay reported previously. Study on multiplication properties of goatpox virus on Vero cell Cultures Two goat poxvirus strains (B and LD) were inoculated in Vero cells, cell cultures post inoculation every 12 h were detected by real-time quantitative PCR and draw growth curve in the Vero cells.The results showed that:two strains have the similar characteristics of the multiplication; the contents of goat poxvirus in the Vero cells increased at 12 h post inoculation, the obviously cytopathic effect(CPE) was observed between 72 h ～ 96 h and the highest contents of goat poxvirus were detected. Through this result, we have a better understanding of the multiplication properties of goat