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目的:探索利用DNA改组(DNA shuffling)技术,获得更高活性tPA的可能性。方法:以人、恒河猴及大白鼠tPA cDNA为一组基因,进行tPA的DNA改组(DNA family shuffling)。以改组后构建的tPA多样性文库转染CHO细胞并进行克隆和筛选。结果:得到了两株有意义的克隆:t9和t17,其中t9克隆表达的tPA活性略高于人tPA,初步的比活性测定结果表明,活性约提高4倍。t17克隆表达的tPA虽然有88个氨基酸的缺失,但仍表现出与人tPA相同的活性。两株克隆经测序证明,为改组后的基因,其序列以人和恒河猴的tPA cDNA序列为主,少数序列来源于大白鼠tPA cDNA。结论:这一探索性结果将为后续几轮的tPA DNA改组探明道路,为最终从改 组后tPA多样性基因库中筛选到比较理想的重组体打下基础。
Objective: To explore the possibility of using DNA shuffling technology to obtain higher activity of tPA. Methods: TPA DNA shuffling was performed using tPA cDNA from human, rhesus monkey and rat as a group. CHO cells were transfected with tPA diversity libraries constructed after shuffling and cloned and screened. RESULTS: Two interesting clones, t9 and t17, were obtained. The tPA activity of t9 clone was slightly higher than that of human tPA. The preliminary assay of specific activity showed that the activity of tPA was increased about 4 times. Although the tPA cloned by t17 showed a deletion of 88 amino acids, it still showed the same activity as human tPA. The two clones were sequenced and proved to be shuffled. The sequences of the cloned genes were mainly from tPA cDNA sequences from humans and rhesus monkeys, while few were from rat white tPA cDNAs. CONCLUSIONS: This exploratory result will prove the way for tPA DNA shuffling in subsequent rounds, laying the foundation for the final screening of the ideal recombinant from tPA diversity gene pool after shuffling.