摘要
用改进的氯化锂沉淀沉法和寡聚(dT)一纤维素亲和层析法由猪垂体制得总mRNA。以此总mRNA为模板,合成cDNA。钝端连接重组到质粒pUC19的SmaI位点,转化E.coli JM107,筛选出阳性克隆。用限制性内切酶酶切签定及5’端部分核苷酸序列分析证明:克隆了全长猪生长激素cDNA,其长度约为896bp。
The porcine pituitary total mRNA was made by the modified methods of LiCl precipitation and Oligo (dT) -cellulose affinity chromatography and was used as template for cDNA synthesis. The first-strand of cDNA was synthesized by reverse transcriptase and the Second -strand was Synthesized by RNase H and E. coli DNA polymerase Ⅰ.Through gel filtration on Sepharose CL 4Bcolumn,the longer ds-cDNA was selected and was inserted into Smal site of pUC19 by blunt-end ligation. After transformation of E.coli JM107 by above recombinant plasmids, positive clones were screened out by colony and dot hybridizations with 32p-labelled two synthetic probes.By the analysis of one of the positive clones with six restriction endonucleases and the DNA sequencing of its 5' end, it was concluded that full-length porcine growth hormone cDNA had been cloned. Its lngth is about 896bp.
关键词
猪
生长激素
CDNA
分子克隆
Porcine Growth hormone cDNA Molecular cloning
