小鼠圆形精子细胞蛋白组学鉴定及mINCA1基因真核表达载体的构建

发布时间：2018-02-25 10:05

本文关键词： 圆形精子细胞无标记定量蛋白组学 mINCA1 RNA干扰　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究目的： 1.通过无标记定量蛋白质组学策略结合质谱技术鉴定小鼠睾丸组织中圆形精子细胞的蛋白质组表达谱,并利用生物信息学对所鉴定的蛋白进行分析； 2.构建小鼠睾丸组织高表达基因mINCA1的真核表达质粒,并且筛选、鉴定其RNA干扰靶点,为进一步研究mINCA1基因在精子发生过程中的功能奠定基础。研究方法： 1.利用重力沉降法分离纯化小鼠睾丸圆形精子细胞,并用免疫荧光染色法鉴定分离的圆形精子细胞； 2.提取圆形精子细胞总蛋白,经全自动2D-Nano-LC-ESI-MS/MS进行质谱分析； 3.利用生物信息学分析质谱检测到的肽段,确定蛋白的IPI；然后用DAVID分析工具,根据GeneOntology数据库的注释对蛋白质的分子功能和参与的生物学过程进行分析；并对蛋白质参与的代谢通路进行分析。 4.提取小鼠睾丸组织总RNA, RT-PCR扩增获得mINCA1的全长cDNA序列,将其与真核表达载体pMSCVpuro连接,构建重组真核表达质粒pMSCVpuro-mINCA1; 5.将重组质粒pMSCVpuro-mINCA1转染HEK293T细胞后,分别采用RT-PCR和Western blotting法检测细胞中mINCAl基因mRNA的转录及蛋白表达 6.设计4个针对mINCA1基因的RNA干扰序列,并构建干扰质粒,然后分别与重组真核表达质粒pMSCVpuro-mINCA1共转染HEK293T细胞,采用Westernblotting筛选mINCA1基因的干扰靶点。研究结果： 1.重力沉降法分离了小鼠睾丸组织圆形精子细胞,并利用免疫荧光染色对纯化的细胞进行鉴定； 2.无标记的定量分析策略结合液质谱联用的技术在圆形精子细胞中共鉴定到2331个蛋白并用DAVID分析工具,根据GeneOntology数据库的注释对蛋白质的分子功能和参与的生物学过程以及各种蛋白在细胞中的定位进行分析； 3.经PathawayKEGG注释对蛋白质的代谢通路注释进行分析,有370种蛋白质涉及能量代谢通路,68种蛋白涉及核糖体代谢,28种参与蛋白酶体途径,40种参与溶酶体途径,60种蛋白与mRNA的剪接有关。 4.PCR、双酶切及测序结果证实重组真核表达质粒pMSCVpuro-mINCA1构建正确； 5.重组质粒转染HEK293T细胞后,经PCR和Western blotting检测有mINCA1基因mRNA和蛋白得以表达； 6.干扰质粒PGPU6/GFP/Neo-shRNAl和PGPU6/GFP/Neo-shRNA3可显著降低mINCA1蛋白的表达量。研究结论： 1.利用无标记蛋白质组学分析策略获得了小鼠圆形精子细胞的蛋白质表达谱,为深入研究精子发生的分子机制提供了丰富的信息。 2.成功构建了mINCA1基因真核表达质粒,并筛选出mINCA1基因的2个有效干扰靶点,为进一步研究mINCA1基因的功能及作用机制奠定了实验基础。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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