人血小板反应素-1活性片段的克隆与序列分析

Cloning and Sequence Analysis of Active Fragments of Human Thrombospondin-1

目的 为研究抗肿瘤基因工程药物的表达奠定基础。方法 根据 Genbank中人血小板反应素 - 1(TSP- 1) m RNA序列 ,设计和合成了两对特异引物 ,利用逆转录聚合酶反应扩增出人 TSP- 1中的两个活性片段 ,大小分别为 72 8bp和 2 5 7bp,分别命名为 TSP- 1- 和 TSP- 1- 。将此片段纯化后插入 PMDT载体。结果 限制酶切分析表明 :这两个片段已插入载体中 ,其大小与预期值相符。序列分析显示 :TSP- 1- 长 72 8bp,编码的多肽长2 12个氨基酸残基 ,推测其相对分子质量为 2 3.6× 10 3;TSP- 1- 长 2 5 7bp,编码的多肽长 86个氨基酸残基 ,推测其相对分子质量为 9.5× 10 3。结论 同源性分析表明 :TSP- 1- 和 TSP- 1- 与 Genbank中的 TSP-

Objective To lay a foundation for researches on the expression of gene engineering anti tumor drugs. Methods On the basis of mRNA sequence of human thrombospondin 1(TSP 1). two pairs of primers were designed and synthesized. Two active fragments TSP 1, called TSP 1 Ⅰ and TSP 1 Ⅲ, were obtained by using reverse transcription polymerase chain reaction(RT PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purifed and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs. Results Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP 1 Ⅰ is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6×10 3 and the TSP 1 Ⅲ is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5×10 3. Conclusion The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP 1 of Genbank.