本文選題：原核表達(dá)　切入點(diǎn)：質(zhì)粒　出處：《南京醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的： 第一部分：通過構(gòu)建與增生性病變相關(guān)基因NYD-SP15的原核表達(dá)質(zhì)粒，體外誘導(dǎo)NYD-SP15蛋白的表達(dá)，免疫小鼠獲得抗體，并把獲得的多克隆抗體運(yùn)用于增生性玻璃體視網(wǎng)膜病變的免疫組化研究。 第二部分：研究Lims基因在PVR發(fā)生過程中RPE細(xì)胞發(fā)育中的作用。 方法： 第一部分：應(yīng)用PCR技術(shù)擴(kuò)增NYD-SP15全長(zhǎng)開放閱讀框，構(gòu)建PET28a-NYD-SP15載體，再將其轉(zhuǎn)入表達(dá)菌株，IPT6誘導(dǎo)表達(dá)，Ni柱純化，免疫BALB／C小鼠，Western-blot法檢測(cè)純化的蛋白和抗體，，取視網(wǎng)膜增殖膜臨床標(biāo)本，抗體行免疫組化染色。 第二部分：應(yīng)用巢式RT—PCR，以小鼠cDNA為模板，擴(kuò)增Lims基因不同剪切子，構(gòu)入PinPointTM Xa-1P質(zhì)粒，測(cè)序鑒定。 結(jié)果： 第一部分：成功地構(gòu)建了PET28a-NYD-SP15原核表達(dá)質(zhì)粒，并獲得了高效表達(dá)NYD-SP15的BL21菌株，表達(dá)的His標(biāo)簽融合蛋白，分子量為58KD左右，經(jīng)免疫小鼠獲得了抗NYD-SP15抗體。經(jīng)Western-blot法分析，抗體為NYD-SP15特異性抗體。免疫組化染色顯示，NYD-SP15基因表達(dá)于視網(wǎng)膜色素上皮細(xì)胞的胞漿，呈現(xiàn)棕色染色。 第二部分：測(cè)序表明克隆了新的Lims基因變異剪切體Lims E，該變異剪切體編碼區(qū)為1164 bp，編碼387個(gè)氨基酸。 結(jié)論： 第一部分：構(gòu)建的NYD-SP15基因的原核表達(dá)載體，體外高效表達(dá)蛋白，免疫小鼠獲得抗NYD-SP15抗體，在視網(wǎng)膜增殖膜的標(biāo)本中，存在著NYD-SP15基因的表達(dá)，這些將為眼部增殖性玻璃體視網(wǎng)膜病變(PVR)的研究及治療奠定堅(jiān)實(shí)的基礎(chǔ)。 第二部分：比較基因組學(xué)分析顯示，成功地克隆了一新的小鼠Lims基因剪切子Lims E，為進(jìn)一步研究Lims基因可能在RPE細(xì)胞發(fā)育中的功能打下了基礎(chǔ)。
[Abstract]:Objective:The first part: by constructing prokaryotic expression plasmid of NYD-SP15 gene associated with proliferative lesions, the expression of NYD-SP15 protein was induced in vitro, and antibody was obtained by immunizing mice.The obtained polyclonal antibodies were used in immunohistochemical study of proliferative vitreoretinopathy.Part two: to study the role of Lims gene in the development of RPE cells during the development of PVR.Methods:The first part: the full-length open reading frame of NYD-SP15 was amplified by PCR technique, and the PET28a-NYD-SP15 vector was constructed, and then transferred into the expressed strain, which was induced to express Ni column. The purified protein and antibody were detected by Western-blot method in immunized BALB/C mice, and the clinical specimens of retinal proliferating membrane were obtained.The antibody was stained by immunohistochemistry.The second part: using nested RT-PCR, using mouse cDNA as template, amplifying different splices of Lims gene, constructing PinPointTM Xa-1P plasmid and sequencing.Results:In the first part, the prokaryotic expression plasmid of PET28a-NYD-SP15 was successfully constructed, and the BL21 strain expressing NYD-SP15 efficiently was obtained. The fusion protein of His label was expressed with molecular weight of about 58KD. The anti-#en4# antibody was obtained by immunizing mice.Western-blot analysis showed that the antibody was NYD-SP15 specific antibody.Immunohistochemical staining showed that NYD-SP15 gene was expressed in the cytoplasm of retinal pigment epithelial cells and showed brown staining.Part two: sequencing showed that a new variant of Lims gene, Lims E, was cloned. The coding region of the variant was 1164 BP, encoding 387 amino acids.Conclusion:The first part: the prokaryotic expression vector of NYD-SP15 gene was constructed, and the protein was highly expressed in vitro. The anti NYD-SP15 antibody was obtained by immunizing mice. The expression of NYD-SP15 gene was found in the proliferative membrane of retina.These will lay a solid foundation for the study and treatment of ocular proliferative vitreoretinopathy (PVR).Part two: comparative genomics analysis showed that a new mouse Lims gene splitter Lims E was cloned successfully, which laid a foundation for further study on the function of Lims gene in the development of RPE cells.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R774.1;R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 陳燕,周劍鋒,樂蓓蓓,向直富,宋曉華,李崇漁,王辨明;Bcl-2基因及其家族Bax,Bcl-Xl和Fas/Apo-l,P16的檢測(cè)在急性白血病中的臨床意義[J];癌癥;1998年05期

2 杜紅俊,惠延年,王雨生,韓泉洪,馬吉獻(xiàn);柔紅霉素和Bax過表達(dá)對(duì)培養(yǎng)人視網(wǎng)膜色素上皮細(xì)胞凋亡的影響[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2002年19期

3 劉少山,惠延年,王雨生,金明;外源bax基因表達(dá)對(duì)人視網(wǎng)膜色素上皮細(xì)胞bcl-2表達(dá)的影響[J];第四軍醫(yī)大學(xué)學(xué)報(bào);1999年04期

4 趙文韜;Survivin的研究現(xiàn)狀[J];國(guó)外醫(yī)學(xué).臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè);2004年01期

5 杜紅俊,惠延年,王雨生,韓泉洪,馬吉獻(xiàn);柔紅霉素對(duì)培養(yǎng)人視網(wǎng)膜色素上皮細(xì)胞BAK和BCL-XL表達(dá)的影響[J];眼科新進(jìn)展;2003年04期

6 劉少山,惠延年,王雨生;Bax過表達(dá)誘導(dǎo)培養(yǎng)的人視網(wǎng)膜色素上皮細(xì)胞凋亡觀察[J];中華眼底病雜志;2001年02期

7 劉少山,惠延年,馬吉獻(xiàn),王雨生,王劍波;Bcl-2、bax在培養(yǎng)的人視網(wǎng)膜色素上皮細(xì)胞及視網(wǎng)膜前膜中的表達(dá)[J];中華眼底病雜志;1999年01期

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