HPSE2基因表达载体的构建及其表达产物的分布研究

发布时间：2018-06-16 22:59

本文选题：HPSE2 + EGFP　；参考：《华中科技大学》2012年硕士论文

【摘要】：研究背景：Urofacial （Ochoa)综合征（UFS）是一种罕见的常染色体隐性遗传病。通过家系连锁分析及染色体外显子测序，HPSE2缺陷被证明是UFS的致病基因，但其机制还不完全清楚，对该基因的功能也多局限于生物信息学预测。目的：利用生物信息学预测，HPSE2为分泌型蛋白可能性大，其N端38个氨基酸为分泌信号肽。本研究以EGFP为表达及鉴定标签，验证HPSE2基因在哺乳动物细胞内是否为分泌表达。方法：利用重组PCR技术，在EGFP基因5′端加入包含有HPSE2分泌信号肽的CDS区(1~588bp)，构建为HPSE2-EGFP-partial融合基因；构建5′端融合IgGκ分泌信号肽的EGFP融合基因IgGκ-EGFP，将两种融合基因克隆到哺乳动物表达载体pcDNA3.1(+)上，构建成pcDNA3.1(+)-HPSE2 EGFP-partial和pcDNA3.1(+)-IgGκ-EGFP表达质粒。以pEGFP-C3作为胞浆表达对照，pcDNA3.1(+)-IgGκ-EGFP作为分泌表达对照，pEGFP-N1-HPSE2为阳性对照，并利用脂质体转染人胚肾Hek293T细胞，通过荧光显微镜及Western检测重组基因表达产物在细胞内外的分布。结果：经酶切、PCR及测序鉴定，重组表达质粒pcDNA3.1(+)-HPSE2-EGFP-partial和pcDNA3.1(+)-IgGκ-EGFP构建成功，经荧光显微镜及Western blot检测，证实HPSE2基因的表达产物分布在细胞内，，所以此蛋白并非分泌型蛋白，这与2010年Levy-Adam,F提出的HPSE2是一种分泌型蛋白的结论完全相反。结论：HPSE2蛋白的N端不包含分泌信号肽，其分布于胞浆中，且主要聚集于核周。
[Abstract]:Background: Urofacial Ochoa syndrome (UFS) is a rare autosomal recessive disease. The HPSE2 gene was proved to be a pathogenic gene of UFS by pedigree linkage analysis and chromosome exon sequencing. However, the mechanism of HPSE2 was not fully understood, and the function of the gene was limited to bioinformatics prediction. Objective: bioinformatics was used to predict that HPSE2 is a secretory protein and its N-terminal 38 amino acids are secretory signal peptides. In this study, EGFP was used to identify whether HPSE2 gene was secreted in mammalian cells. Methods: HPSE2-EGFP-partial fusion gene was constructed by adding a CDS region containing HPSE2 secreting signal peptide into the 5'terminal of EGFP gene by recombinant PCR technique. An EGFP fusion gene, IgG 魏 -EGFP, was constructed and cloned into mammalian expression vector pcDNA3.1 (). The expression plasmids of pcDNA3.1 (hPSE2) EGFP-partial and pcDNA3.1 (pDNA-IgG 魏 -EGFP) were constructed. PEGFP-C3 was used as cytoplasmic expression control pcDNA3.1 (pEGFP-N1-HPSE2) was used as secretory expression control, and human embryonic kidney Hek293T cells were transfected with liposome. The distribution of recombinant gene expression products was detected by fluorescence microscopy and Western blot. Results: the recombinant expression plasmids pcDNA3.1 (hPSE2-EGFP-partial and pcDNA3.1 (-IgG 魏 -EGFP) were successfully constructed by restriction endonuclease digestion and sequencing. Fluorescence microscopy and Western blot analysis showed that the HPSE2 gene was distributed in the cells, so the HPSE2 gene was not a secretory protein. This is contrary to the conclusion that HPSE2 is a secretory protein proposed by Levy-Adamer F in 2010. Conclusion the N terminal of the protein contains no secretory signal peptide. It is distributed in the cytoplasm and mainly clustered around the nucleus.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416

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