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

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。提取新生大鼠纹状体总RNA,RTPCR克隆大鼠GDNFcDNA,产物回收后经HindⅢ和KpnⅠ双酶切,插入重组腺病毒穿梭载体pAdTrackCMV中,氯化钙法转染入大肠杆菌DH5α中,酶切、PCR及测序分析对重组质粒做进一步鉴定。结果显示大鼠GDNFcDNA被成功克隆,所克隆的GDNFcDNA与基因库注册的相同,以上结果说明本实验成功构建了GDNFcDNA重组腺病毒载体。

CONSTRUCTION AND IDENTIFICATION OF SHUTTLE PLASMID OF RECOMBINANT ADENOVIRAL VECTOR CARRYING RAT GDNF GENE

To construct a recombinant adenoviral vector contanining rat glial cell line-derived neurotrophic factor (GDNF) cDNA. 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-KpnⅠdouble-digested fragment of the PCR product was inserted into the shuttle vector of adenoviral 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. The results showed that the GDNF cDNA was successfully cloned by RT-PCR and the nuleotide sequence of the GDNF cDNA was exactly the same as that reported in the GeneBank. The above results denonstrate that the recombinant adenoviral vector carrying GDNF cDNA is successfully constructed.