本文關鍵詞： 圓形精子細胞 無標記定量 蛋白組學 mINCA1 RNA干擾　出處：《山西醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究目的： 1.通過無標記定量蛋白質組學策略結合質譜技術鑒定小鼠睪丸組織中圓形精子細胞的蛋白質組表達譜,并利用生物信息學對所鑒定的蛋白進行分析； 2.構建小鼠睪丸組織高表達基因mINCA1的真核表達質粒,并且篩選、鑒定其RNA干擾靶點,為進一步研究mINCA1基因在精子發(fā)生過程中的功能奠定基礎。 研究方法： 1.利用重力沉降法分離純化小鼠睪丸圓形精子細胞,并用免疫熒光染色法鑒定分離的圓形精子細胞； 2.提取圓形精子細胞總蛋白,經全自動2D-Nano-LC-ESI-MS/MS進行質譜分析； 3.利用生物信息學分析質譜檢測到的肽段,確定蛋白的IPI；然后用DAVID分析工具,根據(jù)GeneOntology數(shù)據(jù)庫的注釋對蛋白質的分子功能和參與的生物學過程進行分析；并對蛋白質參與的代謝通路進行分析。 4.提取小鼠睪丸組織總RNA, RT-PCR擴增獲得mINCA1的全長cDNA序列,將其與真核表達載體pMSCVpuro連接,構建重組真核表達質粒pMSCVpuro-mINCA1; 5.將重組質粒pMSCVpuro-mINCA1轉染HEK293T細胞后,分別采用RT-PCR和Western blotting法檢測細胞中mINCAl基因mRNA的轉錄及蛋白表達 6.設計4個針對mINCA1基因的RNA干擾序列,并構建干擾質粒,然后分別與重組真核表達質粒pMSCVpuro-mINCA1共轉染HEK293T細胞,采用Westernblotting篩選mINCA1基因的干擾靶點。 研究結果： 1.重力沉降法分離了小鼠睪丸組織圓形精子細胞,并利用免疫熒光染色對純化的細胞進行鑒定； 2.無標記的定量分析策略結合液質譜聯(lián)用的技術在圓形精子細胞中共鑒定到2331個蛋白并用DAVID分析工具,根據(jù)GeneOntology數(shù)據(jù)庫的注釋對蛋白質的分子功能和參與的生物學過程以及各種蛋白在細胞中的定位進行分析； 3.經PathawayKEGG注釋對蛋白質的代謝通路注釋進行分析,有370種蛋白質涉及能量代謝通路,68種蛋白涉及核糖體代謝,28種參與蛋白酶體途徑,40種參與溶酶體途徑,60種蛋白與mRNA的剪接有關。 4.PCR、雙酶切及測序結果證實重組真核表達質粒pMSCVpuro-mINCA1構建正確； 5.重組質粒轉染HEK293T細胞后,經PCR和Western blotting檢測有mINCA1基因mRNA和蛋白得以表達； 6.干擾質粒PGPU6/GFP/Neo-shRNAl和PGPU6/GFP/Neo-shRNA3可顯著降低mINCA1蛋白的表達量。 研究結論： 1.利用無標記蛋白質組學分析策略獲得了小鼠圓形精子細胞的蛋白質表達譜,為深入研究精子發(fā)生的分子機制提供了豐富的信息。 2.成功構建了mINCA1基因真核表達質粒,并篩選出mINCA1基因的2個有效干擾靶點,為進一步研究mINCA1基因的功能及作用機制奠定了實驗基礎。
[Abstract]:Objectives of the study:. 1. The proteome expression profiles of round sperm cells in mouse testis were identified by unlabeled quantitative proteomics and mass spectrometry, and the identified proteins were analyzed by bioinformatics. 2. The eukaryotic expression plasmid of mouse testis overexpression gene mINCA1 was constructed, and its RNA interference target was screened and identified, which laid a foundation for further study of the function of mINCA1 gene in spermatogenesis. Research methods:. 1. The round sperm cells of mouse testis were isolated by gravity sedimentation method and identified by immunofluorescence staining. 2.The total protein of round sperm cells was extracted and analyzed by MS / MS with automatic 2D-Nano-LC-ESI-MS. 3. Using bioinformatics to analyze the peptides detected by mass spectrometry to determine the IPI of the protein, and then to analyze the molecular function of the protein and the biological process by using the DAVID analysis tool according to the annotations of the GeneOntology database. The metabolic pathway involved in protein was analyzed. 4. The total RNA of mouse testis was extracted, and the full-length cDNA sequence of mINCA1 was amplified by RT-PCR. The full-length cDNA sequence was ligated with the eukaryotic expression vector pMSCVpuro, and the recombinant eukaryotic expression plasmid pMSCVpuro-mINCA1 was constructed. 5. After the recombinant plasmid pMSCVpuro-mINCA1 was transfected into HEK293T cells, the transcription and protein expression of mINCAl gene mRNA were detected by RT-PCR and Western blotting. 6. Four RNA interference sequences targeting mINCA1 gene were designed and constructed, and then co-transfected with recombinant eukaryotic expression plasmid pMSCVpuro-mINCA1 into HEK293T cells. Westernblotting was used to screen the interfering targets of mINCA1 gene. Results of the study:. 1. The round sperm cells of mouse testis were isolated by gravity sedimentation method, and the purified cells were identified by immunofluorescence staining. 2.Unlabeled quantitative analysis strategy combined with liquid-mass spectrometry, 2331 proteins were identified in round sperm cells and analyzed by DAVID. The molecular functions of proteins, the biological processes involved and the localization of proteins in cells were analyzed according to the GeneOntology database. 3. PathawayKEGG annotation showed that there were 370 proteins involved in energy metabolism pathway, 68 proteins involved in ribosomal metabolism, 28 proteins involved in proteasome pathway, 40 proteins involved in lysosomal pathway and 60 proteins related to splicing of mRNA. 4. The construction of recombinant eukaryotic expression plasmid pMSCVpuro-mINCA1 was confirmed by double enzyme digestion and sequencing. 5. After the recombinant plasmid was transfected into HEK293T cells, mINCA1 gene mRNA and protein were detected by PCR and Western blotting. 6. Interference plasmids PGPU6/GFP/Neo-shRNAl and PGPU6/GFP/Neo-shRNA3 significantly reduced the expression of mINCA1 protein. The study concluded that:. 1. The protein expression profiles of mouse round sperm cells were obtained by unlabeled proteomics analysis, which provided abundant information for further study on the molecular mechanism of spermatogenesis. 2. The eukaryotic expression plasmid of mINCA1 gene was successfully constructed, and two effective interference targets of mINCA1 gene were screened, which laid an experimental foundation for further study on the function and mechanism of mINCA1 gene.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698.2

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