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本文重点介绍利用分子杂交技术,通过用(POMC,Pro-Opiolnelancortin)阿片肽-黑色素-肾上腺皮质激素前体的 DNA 探针从人脑的 cDNA 基因库中筛选出12个特异性基因克隆。重组 POMC 的质粒经一系列的培养、扩增、裂解、氯化铯梯度超速离心、限制性内切酶消化、低熔点琼脂糖电泳回收特异性插入的 DNA 片段(～600~bp),所得的特异性 DNA 片段经~(32)P-[dCTP]同位素的标记,制备成放射性比活7×10~8cpm 的DNA 探针;通过 DNA 杂交原理,从人脑 cDNA 文库中筛选出与 POMC 相关的12个信号基因克隆,经 X-gal 的复筛得到纯的 POMC 重组体。所得的重组体对今后神经肽类激素基因在体外复制、表达分泌具有重要意义,而且为今后利用遗传工程手段生产稀有的神经肽激素药物奠定基础。
This article highlights the use of molecular hybridization to screen 12 cDNA clones from the human brain cDNA library by DNA probes using the opioid peptide-melanocortin precursor (POMC, Pro-Opiolnelancortin). The recombinant POMC plasmid was subjected to a series of steps of culture, amplification, lysis, cesium chloride gradient ultracentrifugation, restriction endonuclease digestion and low melting point agarose electrophoresis to recover specifically inserted DNA fragments (~600 bp) Specific DNA fragments were labeled with ~ (32) P- [dCTP], and DNA probes with specific radioactivity of 7 × 10 ~ 8cpm were prepared. According to the DNA hybridization principle, POMC-related Twelve signal genes were cloned and recombined with X-gal to obtain pure POMC recombinants. The obtained recombinants are of great significance for the future replication and secretion of neuropeptide hormone genes in vitro and lay a foundation for the future production of rare neuropeptide hormone drugs by means of genetic engineering.