摘要
目的初步分析人的细胞黏附蛋白Nectin-3(nectin cell adhesion molecule 3)的序列并研究其亚细胞定位,观察小鼠Nectin-3眼部表达情况。方法分析Nectin-3核苷酸序列并对其编码的3个同源异构体进行氨基酸序列比对分析。构建带eGFP荧光标签的Nectin-33个同源异构体的重组质粒,转染到HEK293T细胞内,Western印迹法实验检测其蛋白表达。分别转染至HeLa细胞内,细胞免疫荧光检测其亚细胞定位。免疫组织化学实验检测Nectin-3在新生小鼠眼球组织的表达情况。结果Nectin-3在目前数据库中共有的转录本有3个,分别编码3个同源异构体iso1(isoform1)、iso2(isoform2)和iso3(isoform3)。序列分析显示它们的N端和/或C端氨基酸序列不同,编码iso3的基因的启动子与iso1和iso2的启动子不同。成功构建Nectin-3的3个同源异构体的荧光融合蛋白表达质粒,Western印迹法显示iso1和iso2的融合蛋白均出现比预测的分子量更小的条带,iso3的融合蛋白与预测的分子量大小基本一致。免疫荧光实验显示iso3表达主要在细胞质中,iso1与iso2表达在细胞核和细胞质均可见;免疫组织化学显示Nectin-3在晶状体上皮细胞和纤维细胞、视网膜上皮细胞和睫状体色素和非色素上皮细胞之间区域均有表达。结论Nectin-3在新生小鼠眼球组织表达,iso1与iso2可能被酶切割,3个同源异构体的亚细胞定位存在差异,这些实验数据将为后续研究Nectin-3的功能奠定实验基础。
Objective To analyze the sequence of Nectin-3(nectin cell adhesion molecule 3)gene,its subcellular localization,and expression in mouse eyes.Methods The nucleotide sequence of Nectin-3 gene was analyzed and the amino acid sequences of three encoded isoforms were compared.The recombinant plasmids of three splice variants of Nectin-3 with eGFP fluorescent tags were constructed and transfected into HEK293T cells;their protein expression was detected with Western blotting.Immunofluorescence technique was used to detect their subcellular localization after transfected into HeLa cells.Immunohistochemistry was performed to detect the expression of Nectin-3 in the eyeball of newborn mice.Results Database showed that Nectin-3 has three splice variants to encode three isoforms.Sequential analysis showed that their N-terminal and/or C-terminal amino acid sequences were different,and the promoter of the gene encoding iso3 was different from the promoters of the other two isoforms.The fluorescent fusion protein recombinant plasmids of the three splice variants of Nectin-3 were successfully constructed.Western blotting showed that the molecular weight of iso1 and iso2 was smaller than which was predicted,and that of iso3 was similar to which was predicted.Immunofluorescence showed that iso3 was mainly expressed in the cytoplasm,iso1 and iso2 were expressed in the nucleus and cytoplasm;immunohistochemistry showed that Nectin-3 was expressed in ciliary non-pigmented epithelial cells,lens epithelial cells and fiber cells,and retinal ganglion cells.Conclusion Nectin-3 is expressed in the eye tissues of newborn mice,Iso1 and Iso2 may be cleaved by enzymes and the subcellular localization of the three isoforms are different.These data provide information for the further study of the function of Nectin-3.
作者
刘向远
王鑫瑶
孙雪娇
陈宇
钟子琳
陈建军
LIU Xiang-yuan;WANG Xin-yao;SUN Xue-jiao;CHEN YU;ZHONG Zi-lin;CHEN Jian-jun(Dept.of Pediatrics,Shanghai Fourth People’s Hospital,School of Medicine,Tongji University,Shanghai 200434,China;Translational Research Institute of Brain and Brain-Like Intelligence,Shanghai Fourth People’s Hospital,School of Medicine,Tongji University,Shanghai 200434,China)
出处
《同济大学学报（医学版）》
2021年第6期760-766,共7页
Journal of Tongji University(Medical Science)
基金
国家自然科学基金项目(81371062)
国家重点研发计划项目(2020YFA0112500)。
