本文選題：男性不育　切入點：SPAG6　出處：《武漢科技大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：構(gòu)建SPAG6基因全長及六個不同長短ARM序列的真核表達載體pEGFP-N2-SPAG6-△ARM，探討融合蛋白在細胞內(nèi)表達及定位。觀察全長及六個ARM結(jié)構(gòu)缺失后對真核細胞CHO細胞中SPAG6蛋白定位的影響。 方法：1.利用NCBI數(shù)據(jù)庫，尋找小鼠SPAG6蛋白的保守功能區(qū)，分析全長蛋白序列，檢索出SPAG6有七個ARM區(qū)域；2.以健康成年雄性小鼠的睪丸組織為來源，建立小鼠睪丸cDNA文庫；3.以小鼠睪丸cDNA文庫為模板，PCR擴增SPAG6全長及六個不同長短ARM結(jié)構(gòu)的編碼序列；4. TA克隆后測序；5.構(gòu)建攜帶不同長短ARM序列的真核表達載體pEGFP-N2-SPAG6-△ARM，對陽性克隆進行酶切和測序鑒定，將構(gòu)建的重組質(zhì)粒轉(zhuǎn)染到CHO細胞中；6.分別提取細胞蛋白進行Western blot檢測，利用共聚焦激光掃描顯微鏡觀察七個不同SPAG6/GFP融合蛋白在CHO細胞內(nèi)的定位。 結(jié)果：酶切和測序鑒定表明，，全長及六個缺失不同長短ARM結(jié)構(gòu)的真核表達質(zhì)粒構(gòu)建成功，轉(zhuǎn)染實驗發(fā)現(xiàn)重組質(zhì)粒均能夠在CHO細胞中表達，但僅全長表達產(chǎn)物定位于微管，缺失任何一個ARM區(qū)域都可影響在細胞中的微管定位。 結(jié)論：SPAG6在真核細胞內(nèi)的正確定位依賴于全長。該研究為進一步研究SPAG6蛋白的結(jié)構(gòu)與功能奠定基礎(chǔ)。
[Abstract]:Aim: to construct the eukaryotic expression vector pEGFP-N2-SPAG6-ARMof SPAG6 gene and six ARM sequences of different length, to investigate the expression and localization of the fusion protein in cells, and to observe the effect of the full length and six ARM deletion on the localization of SPAG6 protein in eukaryotic CHO cells. Methods 1. By using NCBI database, the conserved functional region of mouse SPAG6 protein was found, and the full-length protein sequence was analyzed. The seven ARM regions of SPAG6 were found, which were derived from testicular tissue of healthy adult male mice. The mouse testis cDNA library was established. The mouse testis cDNA library was used as template to amplify the full length of SPAG6 and the coding sequence of six ARM structures of different length and length. The recombinant plasmid pEGFP-N2-SPAG6- was constructed and cloned by TA and sequenced by sequencing 5. The eukaryotic expression vector pEGFP-N2-SPAG6-. The positive clones were digested and sequenced. The recombinant plasmid was transfected into CHO cells. The proteins were extracted for Western blot detection, and the localization of seven different SPAG6/GFP fusion proteins in CHO cells was observed by confocal laser scanning microscope. Results: restriction endonuclease digestion and sequencing showed that the eukaryotic expression plasmids with full length and six different length ARM structures were successfully constructed. The transfection experiments showed that the recombinant plasmids could be expressed in CHO cells, but only the full-length expression products were located in microtubules. The absence of any ARM region can affect the localization of microtubules in cells. Conclusion the correct localization of SPAG6 6 in eukaryotic cells depends on its full length. This study lays a foundation for further study on the structure and function of SPAG6 protein.
【學位授予單位】：武漢科技大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698.2

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