Bβ448野生型及突变型纤维蛋白原稳定转染细胞系的建立

Establishment of stably transfected cell line expressing BβArg448 or BβLys448 fibrinogen

目的构建野生型(BβArg448)和突变型(BβLys448)纤维蛋白原稳定表达细胞系,为进一步研究BβArg448Lys基因多态性的病理生理学意义及相关蛋白功能研究提供实验基础。方法以含纤维蛋白原野生型Bβ链cDNA全长的表达载体pMLP-FGB(448G)为模板,采用PCR定点突变技术构建BβLys448表达载体,即突变型质粒pMLP-FGB(448A)。应用脂质体转染及药物加压筛选的方法,将纤维蛋白原Aα链、野生型/突变型Bβ链、γ链表达载体共同转染至CHO-K1细胞,RT-PCR检测各条链mRNA的表达。结果野生型及突变型纤维蛋白原稳定转染细胞系在转录水平构建成功。结论野生型及突变型纤维蛋白原稳定转染细胞等在转录水平构建成功,为进一步体外研究BβArg448Lys所致纤维蛋白原的结构与功能异常奠定了基础。

Objective To investigate the effect of BβArg448Lys polymorphism on the function of fibrinogen,and to establish the stably transfected cell line expressing fibrinogen with BβArg448 and BβLys448 polymorphism.Methods Mutant fibrinogen Bβ chain cDNA（BβLys448） pMLP-FGB（448A） was generated from wild type Bβ chain cDNA（BβArg448） pMLP-FGB（448G）,which contains the entire fibrinogen Bβ chain cDNA using site-directed mutagenesis method.The expression vectors encoding the entire fibrinogen Aα chain,wild type/ mutant type Bβ chain and γ chain cDNA were introduced into CHO-K1 cells respectively using Lipofectamine.The expression of all three chains in the transfected cell line was tested by RT-PCR.Results The stably transfected cell line expressing fibrinogen with BβArg448 and BβLys448 polymorphism was successfully established,which provide a useful cell model to the further functional study.Conclusion The stably transfected cell line expressing fibrinogen with BβArg448Lys and BβLys448 polymorphism was successfully established,which provide a useful cell model to the further functional study.