hper1干扰质粒的构建及hper1功能的初步研究

Construction of the hper1 Interference Plasmid and Preliminary Study on Function of hper1.

目的构建高效的针对hper1(人类period1基因)的干扰质粒,并对hper1的功能进行初步研究。方法选择4个hper1的干扰位点,根据位点序列合成构建4个pTER-hper1干扰质粒,并通过测序验证。将4种干扰质粒分别转染至SH-SY5Y细胞,经马血清休克诱导后,提取细胞总RNA,RT-PCR检测hper1mRNA的表达,鉴定干扰效果。提取细胞总蛋白,Western Blot检测P-p44/42丝裂原活化蛋白激酶(mitagen-actinated pratein kinase,MAPK)表达。结果RT-PCR鉴定显示pTER-hper1-Ⅱ干扰质粒干扰效率最高,hper1表达量降低84.9%。Western Blot显示hper1表达受抑制后,P-p44/42MAPK水平升高。结论成功构建并筛选到具有显著干扰效率的pTER-hper1干扰质粒,为进一步研究hper1的功能奠定了基础,并发现hper1对MAPK通路具有负相调控作用。

Objective To construct a high efficient interference plasmid for hper1 （human period, gene） ,and to make a platform for study of function of hperl. Method Four pTER-hper1 interference plasmids according to hper1 sequence were constructed., and tested by sequencing. The 4 constructed plasmids were transferred into SH-SYSY cells, and the SH-SYSY cells were seduced by horse serum. The whole RNA extracted from the SH-SYSY cells was used to test the effect of interference plasmids by RT-PCR. P-p44/42 MAPK was analyzed by western blot technology. Result RTPCR showed that the expression of hperl in SH-SYSY cells transferred pTER- hper1- Ⅱ was decreased by 84.9%, which demonstrated that there was high interference efficiency of pTER- hper1- Ⅱ interference plasmid. Western blot technology showed that P-p44/42 MAPK in SH-SYSY cells was increased. Conclusion The pTER-hperl interference plasmid is successfully constructed, which establishes a basis for studying the function of hperl, hperl may be a negative controller of MAPK pathway.