神经生长因子高亲和力受体(TrkA)cDNA片段的克隆  

HNGFR (TrkA) gene cDNA cloning by pGEM -Easy T-Vector and Sequencing

作　　者：姜声扬[1] 丁斐[1] 沈爱国[2] 庄勋[3] 

机构地区：[1]南通医学院江苏省神经再生重点实验室,南通226001 [2]南通医学院流行病学教研室 [3]南通医学院卫生毒理学教研室,南通226001

出　　处：《南通医学院学报》2003年第2期133-134,137,共3页ACTA Academiae Medicinae Nantong

基　　金：江苏省高校自然科学研究指导性计划项目 (编号 :0 1KJD330 0 0 1)

摘　　要：目的 :克隆大鼠背根神经节中 NGF高亲和力受体基因。方法 :从出生 1周大鼠背根神经节中抽提总RNA,设计 Trk A引物 ,进行 RT- PCR扩增。扩增产物直接与 T载体连接后转化 E.coli DH5 α,用蓝 /白斑试验筛选阳性克隆 ,抽提质粒进行酶切鉴定 ,再行序列分析。结果 :经 RT- PCR获得预期 416bp的目的片段 ,T载体克隆、酶切鉴定及序列分析后证实 ,克隆片段与 Genbank中 Trk A基因序列 10 0 %同源。结论 :成功地构建了含 Trk A基因 c D-NA的 T载体克隆 。Objective:To construct the pGEM-Easy T-vector containing HNGFR (NGF High affinity Receptor,TrkA) gene.Methods:By following total RNA being extracted from dorsal root ganglia (DRG) of rat and RT-PCR being carried out, pGEM-Easy T-vector was linked with PCR products. After E. coli DH5α was transformed with recombinants and screened with blue/ white blot test for positive clones from which plasmids were then extracted, the recombinants were identified by restricted analysis and sequencing. Results: 416 base pairs fragment was obtained through RT-PCR. After cloning by pGEM-Easy T-vector and being identified by restricted analysis and sequencing, it is confirmed that the object fragment has 100% homology with TrkA sequence in Genbank. Conclusion:The T-vector clone with the conservative region of TrkA gene has been constructed successfully. This clone can be used in neuronal toxicological research area such as some metals poisoning.

关 键 词：高亲和力受体 T载体 克隆 神经生长因子 大鼠 DNA 基因 

分 类 号：Q785[生物学—分子生物学]