一个新的ERM家族分子的鉴定及对细胞形态的影响

发布时间：2018-02-27 11:02

本文关键词： 人Ermin(hErmin) 少突胶质细胞细胞骨架胶质瘤多克隆抗体　出处：《第四军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的制备抗hErmin氨基端160个氨基酸(hErmin160)多克隆抗体,鉴定hErmin分子在正常成人脑组织中的表达,以及研究hErmin对细胞形态的影响,为深入研究hErmin分子的功能奠定基础。方法PCR扩增编码hErmin160的核苷酸序列,克隆入原核表达载体pET41-b(+),转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达hErmin160融合蛋白,表达产物用SDS-PAGE和Western-blot进行鉴定,经镍柱纯化后,免疫家兔,制备其抗血清,并对免疫后抗血清进行亲和纯化,用Western-blot检测抗体纯化的效果;用纯化的抗hErmin160多克隆抗体为一抗,分别用Western-blot法和免疫组织化学法对hErmin分子在正常成人脑组织中的表达进行鉴定;通过转染,在体外培养的细胞中过表达hErmin分子,用细胞免疫荧光法观察hErmin分子对细胞形态的影响。结果测序证实获得hErmin氨基端前160个氨基酸的编码序列;SDS-PAGE分析表明,表达产物的相对分子质量为51ku,与理论值相符;灰度扫描分析hErmin160融合蛋白的表达量占上清中菌体蛋白总量的10%,纯化产物纯度达92%,Western-blot鉴定所表达的融合蛋白;抗血清和纯化后的多克隆抗体Western-blot反应均为阳性;纯化所得的抗体能与真核表达系统表达的hErmin分子结合,并且hErmin分子在正常成人脑组织中的表达得到确认;转染过表达hErmin分子的细胞长出了更多突起。结论:利用大肠杆菌原核表达系统,获得了较高纯度的可溶形式hErmin160融合蛋白,并通过亲和纯化成功制备hErmin160多克隆抗体;我们鉴定了一个新的ERM家族成员hErmin分子在人脑中的表达;并且在体外培养的细胞中,此分子的表达可以促进细胞长出更多突起。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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