人PROKR2蛋白274位缬氨酸突变质粒的构建及表达

Construction and Expression of the pKR5-mGlu-Flag-hPROKR2(V274D/R/T/A)

目的:构建人前动力蛋白受体2(hPROKR2)274位缬氨酸残基突变的真核表达质粒pKR5-mGlu-FlaghPROKR2(V274D或V274R或V274T或V274A),并观察其蛋白表达。方法:将pKR5-mGlu-Flag-mPKR2质粒和PCR扩增的hPROKR2编码序列用MluⅠ、XbaⅠ酶切后连接,构建pKR5-mGlu-Flag-hPROKR2质粒;以后者为模板,利用定点突变技术分别构建pKR5-mGlu-Flag-hPROKR2(V274D)、(V274R)、(V274T)、(V274A)真核表达质粒;用Western印迹验证Flag-hPROKR2(V274D)、(V274A)、(V274T)、(V274R)在真核细胞中的表达。结果:构建了pKR5-mGlu-Flag-hPROKR2(V274D)、(V274R)、(V274T)、(V274A)真核表达质粒,并观察到相应的蛋白表达。结论:pKR5-mGlu-Flag-hPROKR2(V274D)、(V274R)、(V274T)、(V274A)真核表达质粒的构建,为后期检测第274位缬氨酸对hPROKR2蛋白分子空间结构及功能的影响创造了条件。

Objective：To construct eukaryotic expression plasmid pKR5-mGlu-Flag-hPROKR2（V274D/R/T/A）and verify their expression. Methods：The coding sequence of hPROKR2 was amplified by PCR and digested by XbaⅠ and MluⅠ,and was inserted into the same enzyeme cutting pKR5-mGlu-Flag-mPKR2 to construct pKR5-mGlu-Flag-hPROKR2. The eukaryotic expression plasmid pKR5-mGlu-Flag-hPROKR2（V274D/R/T/A） were obtained by the site directed mutagenesis technique. The expression of Flag-hPROKR2（V274D/R/T/A） protein was verified by Werstern blot. Results：The four resulting plasmids were confirmed by DNA sequencing and the corresponding protein was expressed withthe Mr over 250×10-3. Conclusion：The pKR5-mGlu-Flag-hPROKR2（V274D/R/T/A） have been consturcted and they will be used in the researchof the effects of 274 valine on the spatial structure and function of hPROKR2 protein.