本文關(guān)鍵詞： 人Ermin(hErmin) 少突膠質(zhì)細(xì)胞 細(xì)胞骨架 膠質(zhì)瘤 多克隆抗體　出處：《第四軍醫(yī)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的制備抗hErmin氨基端160個氨基酸(hErmin160)多克隆抗體,鑒定hErmin分子在正常成人腦組織中的表達(dá),以及研究hErmin對細(xì)胞形態(tài)的影響,為深入研究hErmin分子的功能奠定基礎(chǔ)。方法PCR擴(kuò)增編碼hErmin160的核苷酸序列,克隆入原核表達(dá)載體pET41-b(+),轉(zhuǎn)化大腸桿菌BL21(DE3)pLysS,IPTG誘導(dǎo)表達(dá)hErmin160融合蛋白,表達(dá)產(chǎn)物用SDS-PAGE和Western-blot進(jìn)行鑒定,經(jīng)鎳柱純化后,免疫家兔,制備其抗血清,并對免疫后抗血清進(jìn)行親和純化,用Western-blot檢測抗體純化的效果;用純化的抗hErmin160多克隆抗體為一抗,分別用Western-blot法和免疫組織化學(xué)法對hErmin分子在正常成人腦組織中的表達(dá)進(jìn)行鑒定;通過轉(zhuǎn)染,在體外培養(yǎng)的細(xì)胞中過表達(dá)hErmin分子,用細(xì)胞免疫熒光法觀察hErmin分子對細(xì)胞形態(tài)的影響。結(jié)果測序證實(shí)獲得hErmin氨基端前160個氨基酸的編碼序列;SDS-PAGE分析表明,表達(dá)產(chǎn)物的相對分子質(zhì)量為51ku,與理論值相符;灰度掃描分析hErmin160融合蛋白的表達(dá)量占上清中菌體蛋白總量的10%,純化產(chǎn)物純度達(dá)92%,Western-blot鑒定所表達(dá)的融合蛋白;抗血清和純化后的多克隆抗體Western-blot反應(yīng)均為陽性;純化所得的抗體能與真核表達(dá)系統(tǒng)表達(dá)的hErmin分子結(jié)合,并且hErmin分子在正常成人腦組織中的表達(dá)得到確認(rèn);轉(zhuǎn)染過表達(dá)hErmin分子的細(xì)胞長出了更多突起。結(jié)論:利用大腸桿菌原核表達(dá)系統(tǒng),獲得了較高純度的可溶形式hErmin160融合蛋白,并通過親和純化成功制備hErmin160多克隆抗體;我們鑒定了一個新的ERM家族成員hErmin分子在人腦中的表達(dá);并且在體外培養(yǎng)的細(xì)胞中,此分子的表達(dá)可以促進(jìn)細(xì)胞長出更多突起。
[Abstract]:Objective to prepare a polyclonal antibody against 160 amino acid amino acids of hErmin, to identify the expression of hErmin in normal adult brain and to study the effect of hErmin on cell morphology. Methods the nucleotide sequence encoding hErmin160 was amplified by PCR and cloned into the prokaryotic expression vector pET41-b1, and transformed into E. coli BL21DE3 pLysSIPTG to induce the expression of hErmin160 fusion protein. The expression product was identified by SDS-PAGE and Western-blot. After purified by nickel column, rabbits were immunized to prepare their antiserum, and the antiserum was purified by affinity. The purification effect of antibody was detected by Western-blot, and the purified polyclonal antibody against hErmin160 was first antibody. The expression of hErmin molecules in normal adult brain tissues was identified by Western-blot and immunohistochemistry, and hErmin molecules were overexpressed in cultured cells by transfection. Results the coding sequence of 160 amino acids before amino terminal of hErmin was obtained by sequencing and SDS-PAGE analysis showed that the relative molecular weight of the expressed product was 51 ku. which was consistent with the theoretical value. The expression of hErmin160 fusion protein accounted for 10% of the total bacterial protein in the supernatant, the purity of the purified protein reached 92% Western-blot, and the Western-blot reaction of anti-serum and purified polyclonal antibody was positive. The purified antibody could bind to the hErmin molecule expressed in eukaryotic expression system, and the expression of hErmin molecule in normal adult brain tissue was confirmed. Conclusion: high purity soluble hErmin160 fusion protein was obtained by E. coli prokaryotic expression system and hErmin160 polyclonal antibody was successfully prepared by affinity purification. We have identified the expression of a new member of ERM family hErmin in human brain, and in vitro cultured cells, the expression of this molecule can promote the cell to grow more protrusions.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.1

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 鄭華;少突膠質(zhì)系細(xì)胞的生物學(xué)特性及神經(jīng)保護(hù)作用研究[D];復(fù)旦大學(xué);2006年

相關(guān)碩士學(xué)位論文 前1條

1 王小娣;前部缺血性視神經(jīng)病變的動物模型制作[D];陜西中醫(yī)學(xué)院;2006年

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本文編號：1542392