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目的:探讨PR39对急性缺血心肌的保护作用,构建可分泌表达PR39的神经生长因子4(NT4)-TAT-His-PR39融合基因cDNA。方法:采用互为模板、引物的PCR技术,制备两端含有酶切位点的PR39 cDNA片段。通过常规分子生物学方法,将其与编码NT4信号肽及引导肽、TAT及6×His基因序列用T4 DNA连接酶相连,将NT4-TAT-His-PR39装入质粒pBV220中。结果:融合基因NT4-TAT-His-PR39的长度为421 bp,限制性内切酶切图谱显示,目的带的大小略超过marker的400 bp的位置,证实已经成功地将PR39 cDNA重组到NT4-TAT-His序列的下游。基因测序结果及DNAsis软件分析结果表明,NT4-TAT-His-PR39的全序列与实验设计的序列完全一致。结论:成功地构建能分泌表达PR39、并具穿膜功能和标签的融合基因NT4-TAT-His-PR39。
OBJECTIVE: To investigate the protective effect of PR39 on acute ischemic myocardium and to construct the cDNA of nerve growth factor 4 (NT4) -TAT-His-PR39 fusion gene secreting PR39. Methods: The PR39 cDNA fragment containing the restriction enzyme sites at both ends was prepared by PCR with primers and primers. NT4-TAT-His-PR39 was inserted into plasmid pBV220 by conventional molecular biology methods using T4 DNA ligase linked to the NT4 signal peptide and leader peptide, TAT and 6xHis gene sequences. Results: The length of the fusion gene NT4-TAT-His-PR39 was 421 bp. Restriction endonuclease digestion showed that the size of the target band slightly exceeded the 400 bp position of the marker. It was confirmed that PR39 cDNA was successfully recombined into NT4- Downstream of the TAT-His sequence. The results of DNA sequencing and DNAsis software analysis showed that the sequence of NT4-TAT-His-PR39 was exactly the same as the designed experiment. Conclusion: The fusion gene NT4-TAT-His-PR39 secreting PR39 and transmembrane function and tagging was successfully constructed.