Zhang Yanping, Zhang Yuanding, Li Lina, Ma Lei, Sun Yurui, Zhang Zonglin, Liu Jinwei, Deng Huiyan, Zhu Wei
Department of Otolaryngology, the Second Affiliated Hospital of General Hospital of Chinese PLA, Beijing, 100091, China.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2009 Aug;23(16):724-7.
To construct GJB2 gene mutations common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutations in GJB2 gene.
Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutation 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 mutations were inserted into pEGFP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method.
The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence signals were distributed uniformly in cytoplasm.
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.
构建中国人常见的GJB2基因突变的增强绿色荧光蛋白（EGFP）融合蛋白载体，寻找研究GJB2基因缺失突变机制的更好方法。
首先通过体外点突变方法构建235delC、299 - 300delAT和176del16bp的非融合蛋白载体。然后通过聚合酶链反应（PCR）扩增上述3种突变的表达部分，并将PCR产物克隆到TA克隆载体中。经EcoRI/BamHI限制性内切酶酶切后，将3种缺失突变插入pEGFP - N1载体。采用测序验证融合蛋白载体的有效性。通过脂质体复合物法将重组DNA样本转染人胚肾293（HEK293）细胞。
重组质粒在HEK293细胞中高表达。绿色荧光信号均匀分布于细胞质中。
成功构建了中国人常见的GJB2基因突变的EGFP融合蛋白载体。这可能为探索中国人常见的非综合征性听力损失的原因提供更好的方法。
