CAJ | 학술논문

目的克隆一个先天性遗传性核性白内障家系致病基因GJA8,研究该基因野生型(wGJA8)和突变型(mGJA8)在体外真核细胞系中的表达.方法以正常人类和家系患者基因组DNA为模板进行PCR扩增,分别调取wGJA8和mGJA8.构建质粒pEGFP-N1-wGJA8和pEGFP-N1-mGJA8,经双酶切测序鉴定后分别转染COS7细胞,用荧光显微镜观察蛋白表达情况,Western印迹和免疫组化法检测蛋白表达.结果目的基因wGJA8和mGJA8克隆成功,经双酶切和DNA测序证实质粒pEGFP-N1-wGJA8和pEGFP-N1-mGJA8构建成功.荧光显微镜观察到野生型蛋白和突变型蛋白在体外培养的COS7细胞内均有表达,Western印迹和免疫组化显示基因wGJA8和mGJA8在COS7细胞中均有蛋白表达.结论成功克隆出该家系致病基因wGJA8和mGJA8,完成了致病基因在真核细胞中的蛋白表达,为进一步研究该家系白内障的确切发病机制奠定了基础.
목적 극륭일개선천성유전성핵성백내장가계치병기인GJA8,연구해기인야생형(wGJA8)화돌변형(mGJA8)재체외진핵세포계중적표체.방법 이정상인류화가계환자기인조DNA위모판진행PCR확증,분별조취wGJA8화mGJA8.구건질립pEGFP-N1-wGJA8화pEGFP-N1-mGJA8,경쌍매절측서감정후분별전염COS7세포,용형광현미경관찰단백표체정황,Western인적화면역조화법검측단백표체.결과 목적 기인wGJA8화mGJA8극륭성공,경쌍매절화DNA측서증실질립pEGFP-N1-wGJA8화pEGFP-N1-mGJA8구건성공.형광현미경관찰도야생형단백화돌변형단백재체외배양적COS7세포내균유표체,Western인적화면역조화현시기인wGJA8화mGJA8재COS7세포중균유단백표체.결론 성공극륭출해가계치병기인wGJA8화mGJA8,완성료치병기인재진핵세포중적단백표체,위진일보연구해가계백내장적학절발병궤제전정료기출.
Objective To clone the sequence of mutation type GJA8 gene(mGJA8)and wild type GJA8 gene(wGJA8)of a congenital inherited nuclear cataract family and study their expression in eukaryotic cell lines in vitro.Methods The mGJA8 and wGJA8 were amplified from this family's DNA and healthy people's DNA by PCR respectively.The mGJA8 and wGJA8 were recombined with plasmid pEGFP-N1 respectively.The accuracy of pEGFP-N1-GJA8 was confirmed by restriction enzyme digestion and DNA sequencing.Finally pEGFP-N1- mGJA8 and pEGFP-N1- wGJA8 and GFP protein were transfected into COS7 cells by lipofectin.The expression of pEGFP-N1-GJA8 and GFP fusion protein were to observe under fluorescence microscope,and to detecte by Western-blotting and Immunohistochemical staining.Results The mCJA8 and wGJA8 were cloned successfully.With restricting enzyme digestion analysis and DNA sequencing,recombinant plasmid pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 were constructed correctly and their GFP fusions were expressed in transfected COS7 cells.The expression of pEGFP-N1-mGJA8 and pEGFP-N1-wGJA8 fusion protein were observed under fluorescence microscope,and detected by Westernblotting and lmmunohistochemical staining successfully.Conclusions The mGJA8 gene and wGJA8 gene are cloned successfully,and pEGFP-N1-mGJA8 and pEGFP-N1-mGJA8 fusion protein Can be expressed in COS7cells,which establish the foundation for further studying the mechanism of this congenital inherited nuclear cataract family.