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目的:克隆人赖氨酸乙酰基转移酶7(KAT7)的2个功能结构域基因,获得其原核表达产物,并纯化蛋白。方法:采用PCR技术从人乳腺文库中扩增人KAT7基因的2个功能结构域(1-330aa)和(331-611aa)编码片段,将其克隆到p ET28a载体中,在大肠杆菌Rossate中表达后,对原核表达产物进行纯化,以SDS-PAGE和Western印迹鉴定表达与纯化产物。结果:从人乳腺文库中分别扩增获得约990和840 bp的DNA片段,并克隆至p ET28a载体,测序结果表明与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量分别约为42 000和36 000的目的蛋白,经纯化后获得了纯度较高的重组蛋白KAT7(1-330aa)和(331-611aa)。结论:获得了重组蛋白KAT7(1-330aa)和(331-611aa),为后续研究KAT7与肿瘤调控奠定了实验基础。
OBJECTIVE: To clone two functional domain genes of human lysine acetyltransferase 7 (KAT7) to obtain the prokaryotic expression product and to purify the protein. METHODS: Two functional domains (1-330aa) and (331-611aa) of human KAT7 gene were amplified from human breast cDNA library by PCR and cloned into pET28a vector and expressed in E. coli Rossate After purification, the prokaryotic expression product was purified and the expressed and purified product was identified by SDS-PAGE and Western blotting. Results: The DNA fragments of about 990 bp and 840 bp in length were amplified from the human breast cDNA library and cloned into pET28a vector. The sequencing results showed that the DNA fragment was completely identical with the target sequence. The relative molecular masses induced in E. coli Rossate were about 42 000 and 36 000 of the target protein purified to obtain higher purity recombinant proteins KAT7 (1-330aa) and (331-611aa). Conclusion: The recombinant proteins KAT7 (1-330aa) and (331-611aa) were obtained, which laid the experimental foundation for further study on the regulation of KAT7 and tumor.