CAJ | 학술논문

目的:构建中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与增强型绿色荧光蛋白(EGFP)的融合蛋白表达载体,寻找体外研究GJB2基因缺失突变致聋机制的有效途径.方法:用体外定点突变法构建235 de1C、299-300 delAT和176 de1 16 bp突变全序列与EGFP表达载体,以此为模板PCR扩增突变有效表达序列,将PCR产物连接到pMD19-T载体中,EcoRI/BamHI双酶切克隆载体,测序鉴定序列正确性后,将酶切产物插入pEGFP-N1载体中,脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白.结果:GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp在HEK293细胞中高效表达,表达主要位于细胞质中.结论:成功构建了中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与EGFP的融合蛋白表达载体,为进一步研究其致聋机制奠定了基础.
목적:구건중국인상견GJB2기인돌변235 de1C、299-300 delAT화176 de1 16 bp여증강형록색형광단백(EGFP)적융합단백표체재체,심조체외연구GJB2기인결실돌변치롱궤제적유효도경.방법:용체외정점돌변법구건235 de1C、299-300 delAT화176 de1 16 bp돌변전서렬여EGFP표체재체,이차위모판PCR확증돌변유효표체서렬,장PCR산물련접도pMD19-T재체중,EcoRI/BamHI쌍매절극륭재체,측서감정서렬정학성후,장매절산물삽입pEGFP-N1재체중,지질체전염HEK293세포,형광현미경관찰표체적융합단백.결과:GJB2기인돌변235 de1C、299-300 delAT화176 de1 16 bp재HEK293세포중고효표체,표체주요위우세포질중.결론:성공구건료중국인상견GJB2기인돌변235 de1C、299-300 delAT화176 de1 16 bp여EGFP적융합단백표체재체,위진일보연구기치롱궤제전정료기출.
Objective:To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method: Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA cloning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG-FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were trans-fected with the recombinant DNA samples by the liposome complex method. Results The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion; GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.