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当前位置: 首页 > 文章 > FPV 4b核心蛋白基因的克隆及其探针制备 吉林农业大学学报 2006,28 (4) 440-443
Position: Home > Articles > Cloning and Probe Labeling of Fowlpox Virus 4b Core Protein Gene Journal of Jilin Agricultural University 2006,28 (4) 440-443

FPV 4b核心蛋白基因的克隆及其探针制备

作  者：
郭建顺;金宁一;张学东;冯立文;沈晓峰;李文红
单  位：
吉林大学畜牧兽医学院;军事医学科学院军事兽医研究所
关键词：
鸡痘病毒;4b核心蛋白基因;克隆;基因探针
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。
译  名：
Cloning and Probe Labeling of Fowlpox Virus 4b Core Protein Gene
作  者：
GUO Jian-shun~(1,2),JIN Ning-yi~2,ZHANG Xue-dong~1,FENG Liwen~1,SHEN Xiao-feng~1,et al.1.College of Veterinary Medicine and Animal Science,Jilin University,Changchun 130062,China;2.The Military Veterinary Institute,Academy of Military Medicine of PLA,Changchun 130062,China)
关键词：
FPV;4b core protein gene;cloning;gene probe
摘  要：
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 EcoRⅠdigestion of pMD18-T-4b and labeled with digoxigenin-dUTP.Determination of labeling efficiency showed the yield of DIG-labeled DNA was 2 μg(100 mg/L) in this study.Dotblot 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.

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