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本文利用PCR技术,从正常人胎肝染色体DNA库中分离克隆了长度分别为506bp,465bp的中国人促红细胞生成素(EPO)基因组片段。506bp基因片段包括外显子2(信号肽),内含子2和外显子3;465bp片段包括外显子4,内含子4和外显子5。通过在引物中设置的酶切位点将两个克隆片段进行了正确的拼接,从而得到了约1.0kb的EPO次全基因组片段,它包括除信号肽中第2,3,4位氨基酸外的所有编码区及两个内含子序列。 所克隆的次全EPO基因组插入表达载体pSV2-dhfr中的不同克隆位点,构建了3种不同的转移载体质粒,分别转染导入COS-7细胞后,3种转移载体质粒转染的细胞上清液都有明显的EPO活性。本工作证明仅含有内含子2,4序列,去除负调控区和氧敏感区序列的人次全EPO基因组可以在COS-7细胞中获得表达。
In this paper, Chinese human erythropoietin (EPO) genomic fragments of 506bp and 465bp in length were isolated and cloned from normal human fetal liver chromosome DNA by PCR. The 506 bp gene fragment includes exon 2 (signal peptide), intron 2 and exon 3; the 465 bp fragment includes exon 4, intron 4, and exon 5. The two cloned fragments were correctly spliced by restriction sites set in the primers to obtain an EPO sub-complete genome fragment of about 1.0 kb, which contained the first, second, third and fourth amino acid residues All coding regions and two intronic sequences. The cloned sub-EPO genome was inserted into different cloning sites in the expression vector pSV2-dhfr to construct three different transfer vector plasmids, which were transfected into COS-7 cells and transfected with the three kinds of vector plasmids respectively Serum have significant EPO activity. This work proves that only the intron 2 and 4 sequences are included in the sequence, and the full EPO genome can be expressed in COS-7 cells by removing the negative regulatory region and the oxygen sensitive region.