新城疫病毒特异性糖蛋白基因探针的制备及应用  

PREPARATION AND APPLICATION OF THE SPECIFIC PROBE FOR THE GLYCOPROTEIN GENES OF NEWCASTLE DISEASE VIRUS

作　　者：刘伟忠[1] 姜焱[1] 吴艳涛[1] 张如宽[1] 刘秀梵[1] 

机构地区：[1]扬州大学农学院动物医学系

出　　处：《畜牧兽医学报》1999年第1期44-49,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省自然科学基金;教委自然科学基金

摘　　要：应用ＲＴ－ＰＣＲ获取新城疫病毒（ＮＤＶ）Ｆ４８Ｅ８株的部分囊膜糖蛋白基因片段。扩增产物经琼脂糖凝胶电泳分析，长为９２６ｂｐ，与预期结果相符；将该基因片段定向克隆入质粒载体ｐＵＣ１９中，得到重组质粒ｐ９２６，酶切分析结果与已发表的ＮＤＶ毒株酶切位点一致。随后将该基因片段标记成为地高辛探针，该探针能检测出三株ＮＤＶ（Ｆ４８Ｅ８株、ＬａＳｏｔａ株、Ｕｌｓｔｅｒ株）基因组ＲＮＡ，不能检测出ＩＢＤＶ和ＩＢＶ基因组ＲＮＡ，表明其特异性良好。应用该探针进行Ｓｏｕｔｈｅｒｎ杂交试验鉴定ＮＤＶＦ４８Ｅ８株的Ｆ和ＨＮ基因克隆，结果质粒ｐＦ内１．７ｋｂ片段和质粒ｐＨＮ内１．９３ｋｂ片段为阳性，从而进一步证实ＮＤＶＦ４８Ｅ８株的囊膜糖蛋白基因均已成功克隆。A cDNA fragment of newcastle disease virus(NDV)strain F48E8 containing partial fusion(F)gene and partial haemagglutinin neuraminidase(HN)gene was obtained by RT PCR.The amplified fragment was 926 bps long as expected analyzed by agarose gel electrophoresis.It was directly cloned into plasmid vector pUC19 and the positive clone named as p926 was confirmed by restriction endonuclease analysis.Then the cDNA fragment was labeled as a DIG probe for detection of the envelope glycoprotein genes of NDV.The specificity of the DIG probe was tested by RNA dot blot hybridization.The probe detected the genomic RNA of three NDV strains(F48E8,La Sota,Ulster),and no hybridization signal was observed with RNA extracted from other avian viruses including IBDV and IBV.The clones(pF and pHN)containing envelope glycoprotein genes of NDV strain F48E8 were tested in a Southern hybridization assay with the DIG probe.The result showed that the 1.7kb long cDNA fragment of pF and the 1.93kb long cDNA fragment of pHN were positive,and further confirmed that the F and HN glycoprotein genes of NDV strain F48E8 were successively cloned.

关 键 词：新城疫病毒 糖蛋白 基因探针 

分 类 号：S852.65[农业科学—基础兽医学]