摘要
利用PCR方法成功克隆了FPV 4b核心蛋白基因1 361 bp片段。序列分析表明:该序列与模板DNA(AF198100)的碱基序列同源性为99.5%,只有7个碱基差异(第215位C→A,第386位T→A,第388位G→A,第1 014位T→G,第1 034位T→G,第1 137位C→T,第1 143位A→G)。回收FPV 4b核心蛋白基因1 361 bp片段与pMD18-T载体相连构建重组质粒pMD18-T-4b,用EcoRⅠ酶切pMD18-T-4b后得到1个约360 bp大小的DNA片段,以其为模板制备了地高辛标记的DNA探针。对新标记的探针进行标记效率检测,结果显示其标记效率为100 mg/L。敏感性检测表明,该探针对同源DNA的检出限量为10 pg。特异性检测结果表明,用本试验所标记的探针对提取的FPV 282E4和FPV JL株DNA、重组质粒pMD 18-T-4b进行检测结果均呈阳性,而鸡马立克氏病病毒、鸡传染性喉气管炎病毒、CEF的核酸提取物均呈阴性,说明该探针具有较强的特异性。
A 1 361 bp fragment was obtained by PCR according to FPV 4b core protein gene.A recombinant plasmid was constructed by inserting the amplified 4b core protein gene 1 361 bp fragment into pMD18-T vector and identify by PCR and digestion with restriction enzyme. The positive plasmid （pMD18-T-4b） was selected and sequenced. Blast analysis showed the nucleotide homogeneity between the clones sequence and the published DNA sequence of 4b gene sequence of PFV （AF198100） was 99.5 %. A total of 7 nucleotide differences were found between them, the nucleic acid C, T, G, T, T, C, A at sites 215,386,388,1 014,1 034,1 137,1 143 were substituted by A,A,A,G,G,T,G. The 4b core protein gene was conserved according to the result of comparison. A 360 bp fragment was obtained by EcoR ~[ digestion of pMD18-T-4b and labeled with digoxigenin-dUTP, Determination of labeling eflqciency showed the yield of DIG-labeled DNA was 2 μg（ 100 mg/L） in this study. Dot-blot hybridization for FPV282E4 DNA, FPV JL DNA, pMD18-T-4b, MDV DNA, AILTV DNA and DNA from CEF was performed by using the probe. It showed that FPV282E4 DNA, FPV JL DNA and pMD18＇T-4b were positive, but MDV DNA, AILTV DNA and DNA from CEF were negative. The sensitivity detection indicated the probe could detect a minimal of 10 pg homology DNA.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2006年第4期440-443,共4页
Journal of Jilin Agricultural University
基金
国家"863"计划资助项目(101-05-03-01)
军队青年基金资助项目(01Q127)
