胶质细胞源性神经营养因子重组腺病毒载体穿梭质粒的构建及鉴定

目的构建含大鼠胶质细胞源性神经营养因子(GDNF)cDNA的重组腺病毒穿梭载体。方法提取新生大鼠纹状体总RNA,用RT-PCR方法克隆大鼠GDNFcDNA,PCR产物回收后经H indⅢ和KpnⅠ双酶切,插入pAdTrack-CMV中,用氯化钙法将其转染入大肠杆菌DH5α中,酶切、PCR及测序分析对重组质粒做进一步鉴定。结果大鼠GDNFcDNA被成功克隆,重组载体的PCR检测、酶切鉴定及测序分析表明,所克隆的GDNFcDNA与基因库注册的相同,大鼠GDNFcDNA被定向插入。结论成功构建了GDNFcDNA重组腺病毒载体。

Construction and Identification of Shuttle Plasmid of Recombinant Adenoviral Vector Carrying Rat GDNF Gene

Objective To construct a recombinant adenoviral vector carrying rat glial cell line-derived neurotrophic factor(GDNE) cDNA.Methods The total RNA was extracted from striatum of the neonatal rat brain,and the gene encoding GDNF was cloned by RT-PCR.The Hind Ⅲ-KpnⅠ fragment of the PCR product was inserted into pAdTrack-CMV.The recombinant plasmid was transfected into E coli DH5α following the calcium chloride-based protocol and identified with restriction analysis,PCR and nucleotide sequence analysis.Results GDNFcDNA was cloned successfully by RT-PCR,the results of restriction analysis,PCR and nuleotide seqence analysis showed that the nuleotide sequence of GDNFcDNA was exactly the same as that registered in the GeneBank,and GDNFcDNA was inserted into the vectors.Conclusion The recombinant adenoviral vector carrying GDNFcDNA is constructed successfully.