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目的构建癌基因p28GANK缺失不同ankyrin序列的突变体,进一步研究p28GANK的功能及在肝癌发生发展中的作用。方法利用QuikChangSite-DirectedMutagenesisKit构建p28GANK5个ankyrin缺失突变体并检测各个突变体对p53的影响。结果构建出分别缺失第1个ankyrin(39～71aa)、第2个ankyrin(72～104aa)、第3个ankyrin(105～137aa)、第4个ankyrin(138～170aa)和第5个ankyrin(171～203aa)的突变体,琼脂糖凝胶电泳及蛋白免疫印迹法验证正确。野生型p28GANK可促进p53降解,其他突变体对p53的表达无明显影响。结论成功构建了p28GANK的5个突变体,证实p28GANK对p53的调控需要全部的ankyrin序列,为ankyrin的研究和p28GANK对肝癌的调控作用研究奠定了基础。
Objective To construct a mutant of oncogene p28GANK with different ankyrin sequences and further study the function of p28GANK and its role in the development of hepatocellular carcinoma. Methods Five Ankyrin deletion mutants of p28GANK were constructed by QuikChangSite-Directed Mutagenesis Kit and the effect of each mutant on p53 was tested. Results The first ankyrin (39 ~ 71aa), the second ankyrin (72 ~ 104aa), the third ankyrin (105 ~ 137aa), the fourth ankyrin (138 ~ 170aa) and the fifth ankyrin 171 ~ 203aa) mutant, agarose gel electrophoresis and Western blot method to verify the correct. Wild-type p28GANK can promote the degradation of p53, while other mutants have no significant effect on the expression of p53. Conclusion Five mutants of p28GANK were successfully constructed and confirmed that all the ankyrin sequences were required for the regulation of p53 by p28GANK, which laid the foundation for the study of ankyrin and the regulation of p28GANK on liver cancer.