CAJ | 학술논문

目的：本研究构建精子相关抗原6（SPAG6）基因全长及6个不同长短ARM序列的真核表达载体pEGFP-N2-SPAG6-△ARM，并使其在CHO细胞中表达，观察全长及6个ARM结构缺失后对真核细胞CHO细胞中SPAG6蛋白定位的影响。方法利用NCBI数据库，寻找小鼠SPAG6蛋白的保守功能区，分析全长蛋白序列，检索出SPAG6有7个ARM区域。利用PCR技术扩增出6个缺失不同ARM结构的SPAG6 cDNA，测序后亚克隆至携带绿色荧光蛋白基因的pEGFP-N2真核表达载体中，对阳性克隆进行酶切和测序鉴定，将构建的重组质粒转染到CHO细胞中，分别提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察7个不同SPAG6/GFP融合蛋白在CHO细胞内的定位。结果酶切和测序鉴定表明，全长及6个缺失不同长短ARM结构的真核表达质粒构建成功，转染实验发现重组质粒均能够在CHO细胞中表达，但仅全长表达产物定位于微管，缺失任何一个ARM区域都可影响在细胞中的微管定位。结论SPAG6在哺乳动物细胞内的正确定位依赖于全长。该研究为进一步研究SPAG6蛋白的结构与功能奠定基础。
목적：본연구구건정자상관항원6（SPAG6）기인전장급6개불동장단ARM서렬적진핵표체재체pEGFP-N2-SPAG6-△ARM，병사기재CHO세포중표체，관찰전장급6개ARM결구결실후대진핵세포CHO세포중SPAG6단백정위적영향。방법이용NCBI수거고，심조소서SPAG6단백적보수공능구，분석전장단백서렬，검색출SPAG6유7개ARM구역。이용PCR기술확증출6개결실불동ARM결구적SPAG6 cDNA，측서후아극륭지휴대록색형광단백기인적pEGFP-N2진핵표체재체중，대양성극륭진행매절화측서감정，장구건적중조질립전염도CHO세포중，분별제취세포단백진행Western blot검측。이용공취초격광소묘현미경관찰7개불동SPAG6/GFP융합단백재CHO세포내적정위。결과매절화측서감정표명，전장급6개결실불동장단ARM결구적진핵표체질립구건성공，전염실험발현중조질립균능구재CHO세포중표체，단부전장표체산물정위우미관，결실임하일개ARM구역도가영향재세포중적미관정위。결론SPAG6재포유동물세포내적정학정위의뢰우전장。해연구위진일보연구SPAG6단백적결구여공능전정기출。
Objective To examine the effect of armadillo (ARM)domains on sperm- associated antigen 6 (SPGA6) localization in CHO cells. Methods The sequences coding different ARM domains were amplified by polymerase chain reaction (PCR), and were subcloned into the pEGFP-N2 vector. These plasmids were transfected into CHO cells. The expression of the GFP-tagged SPAG6 proteins was proved by Western blot analysis, and localization of GFP-tagged full length and truncated SPAG6 proteins was observed by using laser scanning confocal microscopy. Results Truncated SPAG6 proteins with series deletion of ARM domains were successfully constructed into the mammalian expressing vector carrying the green fluorescent protein gene, pEGFP-N2. The expression of the fusion protein was confirmed by Western blot analysis. The full length GFP-tagged SPAG6 protein was 1ocalized on the microtubule in the transfected CHO cells, but none of the truncated proteins was localized on the microtybule. Conclusion Microtubule localization of mouse SPAG6 protein is dependent on full length sequence. Construction of the plasmids expressing full length and truncated SPAG6 proteins provides an important tool for further study SPAG6 function.