摘要
利用酸性异硫氰酸胍-酚-氯仿一步法从人胚胎组织中提取总RNA,经Oligo(dT)纤维柱分离纯化出mRNA。用逆转录与聚合酶链反应相结合的RT-PCR法,扩增出人类胰岛素生长因子Ⅱ(IGFⅡ)的cDNA片段,在限制性内切酶Sma Ⅰ存在的连接体系中,将扩增出的cDNA片段克隆进PUC12的Sma Ⅰ位点处。经限制性内切酶EcoR Ⅰ、Sal Ⅰ、Eco47Ⅲ酶切鉴定其方向。以重组质粒的双链DNA为模板,用末端终止法测定其全部核苷酸顺序,证实其核苷酸编码的IGFⅡ在氨基酸顺序上与文献报道的相同。
Total RNA from human fetal liver tissue was extracted using the single-step method of RNA isolation by acid guanidinium thiocyanate phenol-chloroform extraction (AGPC). Poly(A)^+ mRNA was isolated and purified through packed columns containing oligo(dT)cellulose. The cDNA encoding human insulin like growth factor Ⅱ(IGF-Ⅱ) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method with the templates of fetal liver mRNA. The cDNA fragment was cloned into PUC12 plasmid which was digested with SmaI and the ligation system included restriction enzyme SmaI. The direction of cloned cDNA fragment in the resulting plasmid was analysed by EcoR Ⅰ, Sall and Eco47Ⅲ restriction enzyme.Double chain cDNA of recombintant plasmids were presented as templates. Nucleotide sequence of the cDNA fragment was determined by dideoxynucleotide chain-termination method. Though there were three mutated nucleotides: AGC→AGT. CGT→CGA. GAG→GAA, it was concluded that the deduced amino acid sequence for IGF-Ⅱ protein complied with the reported.
关键词
胰岛素类
生长因子Ⅱ
聚合酶链反应
Insulin-like growth factor Ⅱ
RT-PCR
Nucleotide sequence
