本文選題：HPSE2 + EGFP��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：研究背景：Urofacial （Ochoa)綜合征（UFS）是一種罕見的常染色體隱性遺傳病。通過家系連鎖分析及染色體外顯子測序，HPSE2缺陷被證明是UFS的致病基因，但其機(jī)制還不完全清楚，對(duì)該基因的功能也多局限于生物信息學(xué)預(yù)測。 目的：利用生物信息學(xué)預(yù)測，HPSE2為分泌型蛋白可能性大，其N端38個(gè)氨基酸為分泌信號(hào)肽。本研究以EGFP為表達(dá)及鑒定標(biāo)簽，驗(yàn)證HPSE2基因在哺乳動(dòng)物細(xì)胞內(nèi)是否為分泌表達(dá)。 方法：利用重組PCR技術(shù)，在EGFP基因5′端加入包含有HPSE2分泌信號(hào)肽的CDS區(qū)(1~588bp)，構(gòu)建為HPSE2-EGFP-partial融合基因；構(gòu)建5′端融合IgGκ分泌信號(hào)肽的EGFP融合基因IgGκ-EGFP，將兩種融合基因克隆到哺乳動(dòng)物表達(dá)載體pcDNA3.1(+)上，構(gòu)建成pcDNA3.1(+)-HPSE2 EGFP-partial和pcDNA3.1(+)-IgGκ-EGFP表達(dá)質(zhì)粒。以pEGFP-C3作為胞漿表達(dá)對(duì)照，pcDNA3.1(+)-IgGκ-EGFP作為分泌表達(dá)對(duì)照，pEGFP-N1-HPSE2為陽性對(duì)照，并利用脂質(zhì)體轉(zhuǎn)染人胚腎Hek293T細(xì)胞，通過熒光顯微鏡及Western檢測重組基因表達(dá)產(chǎn)物在細(xì)胞內(nèi)外的分布。 結(jié)果：經(jīng)酶切、PCR及測序鑒定，重組表達(dá)質(zhì)粒pcDNA3.1(+)-HPSE2-EGFP-partial和pcDNA3.1(+)-IgGκ-EGFP構(gòu)建成功，經(jīng)熒光顯微鏡及Western blot檢測，證實(shí)HPSE2基因的表達(dá)產(chǎn)物分布在細(xì)胞內(nèi)，，所以此蛋白并非分泌型蛋白，這與2010年Levy-Adam,F提出的HPSE2是一種分泌型蛋白的結(jié)論完全相反。 結(jié)論：HPSE2蛋白的N端不包含分泌信號(hào)肽，其分布于胞漿中，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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R3416

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張水軍;郭文治;趙永福;史冀華;鄭守華;宋燕;翟文龍;;乙酰肝素酶2蛋白的表達(dá)與胰腺癌浸潤、轉(zhuǎn)移的關(guān)系及其意義[J];中國普通外科雜志;2006年06期

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