新城疫病毒F_(48)E_(9)株HN基因的克隆与酶切分析

Cloning and Identification of HN Gene of NDV F 48 E 9 Strain

为深入探讨新城疫病毒（ＮＤＶ）的结构与功能的关系，扩增和克隆了Ｆ４８Ｅ９株ＮＤＶ的ＨＮ基因。首先以提取的Ｆ４８Ｅ９株ＮＤＶ基因组ＲＮＡ为模板逆转录合成第一链ｃＤＮＡ，然后用ＨＮ基因特异性引物作ＰＣＲ扩增ＨＮｃＤＮＡ，结果得到１条分子量约１．８ｋｂ的ＤＮＡ带，与ＮＤＶＨＮ基因的大小一致。Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交进一步证实其为ＨＮ特异性ｃＤＮＡ。随后采用平端连接法将其克隆到ｐＳＶ·Ｓｐｏｒｔｌ质粒中，应用限制性内切酶ＥｃｏＲＩ、ＳｃａⅠ、ＭｌｕⅠ、ＡｐａⅠ、ＰｓｔⅠ、和ＳｍａⅠ对ＮＤＶＦ４８Ｅ９株ＨＮｃＤＮＡ重组质粒作单酶或双酶切，结果表明，ＮＤＶＦ４８Ｅ９株ＨＮ基因较ＨｉｔｃｈｎｅｒＢ１株的ＨＮ基因缺少１个ＭｌｕⅠ位点（７０５ｂｐ左右），多１个ＰｓｔⅠ位点（８０２ｂｐ左右）。

To further understand the structure and function of NDV HN gene, cDNA encoding the HN gene of NDV F 48 E 9 strain was cloned and identified. Virion RNA was extracted from purified NDV F 48 E 9 strain and used as a template for cDNA synthesis. Two primers were prepared referring to the sequence of NDV HitchnerB1 HN gene which located at 80 96 bp and 1 853 1 871 bp respectively. The total RNA of NDV was served as template for PCR. The PCR products were checked by agarose gel electrophoresis and its specificity was further confirmed by Southern blot hybridization with a HN specific probe from HN recombinant M6B12. The HN cDNA of NDV F 48 E 9 was then cloned into pSV·Sportl plasmid successfully and the HN Specific cDNA recombinant plasmids were analysed with Restriction Endonucleases (RE) (Eco RI, ScaⅠ, MluⅠ, ApaⅠ, PstⅠ and SmaⅠ) digestion. The results indicated that in the NDV F 48 E 9 strain HN cDNA exists an additional PstⅠ RE site at position 802 bp and absents one MluⅠ RE site at position 1 193 bp as compared with the previously published NDV HN sequences (such as HitchnerB1 strain).