学校代码： 10385 分类号：
研究生学号：1400416008 密 级：
异丙醇钛
木马检测Xentry-Cytoglobin融合蛋白的原核表达、纯化及活性分析 Prokaryotic Expression, Purification and Activity Analysis of Xentry-Cytoglobin protein
作者姓名： 周立姣
指导教师： 肖卫东教授 许瑞安教授
实践导师： 许福春
专业学位类别/领域： 生物工程
研究方向： 基因工程
所在学院： 生物医学学院
论文提交日期：二零一七年五月十六日
摘 要
细胞球蛋白（Cytoglobin）已被证明有抗氧化、抗肿瘤等多种生物学活性，拥有一定的临床应用潜力。但因为其分子量远超过透过细胞膜的分子量要求，且其易形成多聚体，使其进入细胞大大受限。本实验室前期构建了原核表达载体pET22b-TC，对TAT-Cytoglobin的作用进行了研究。TAT 中最短的转导有效序列为YGRKKRRQRRR 11个氨基酸。而最新报道的Xentry的短肽序列LCLRPVG仅7个氨基酸就具有良好的穿膜效果。所以我们期待利用具有良好穿膜效果的Xentry更短的特性，增加胞内Cytoglobin浓度，促进Cytoglobin生物学效应的发挥。喷胶
成功构建了pET22b-XC，为原核表达载体，乳糖诱导表达，但是可能由于目的蛋白等电点PI过于接近菌体自身一些蛋白的等电点，导致分离纯化目的蛋白困难；随后为了得到利于分离纯化且有生物活性的目的蛋白构建了含有pelB信号肽且含有组氨酸标签的pET22b-pelB-XCH、pET22b-pelB -X LCH的重组表达载体。它们经乳糖诱导和IPTG诱导均无明显表达，且尝试用镍柱层析仍未得到目的蛋白。随后为了获得高表达量尝试胞内表达构建了pET22b-XCH和pET22b-XLCH重组表达载体，依然无明显表达。最后更换载体，构建原核表达载体pET28a-XCH和pET28a-XLCH，结果发现pET 28a-XCH诱导表达明显。用镍柱亲和层析，对XC 分离纯化。利用Cytoglobin的过氧化氢酶性质，通过初步的可见光分析可以证明蛋白有一定的生物学活性。随后又进行了一系列的细胞实验，探索了XC的生物活性。
研究结果说明XC的良好的穿膜效果可能促进了Cytoglobin蛋白的生物学功能在胞内的发挥。且在部分细胞实验中探索发现Xentry-Cytoglobin 比TAT-Cytoglobin有更好的抑制肿瘤细胞增殖的效果。研究为
服务器部署Cytoglobin 蛋白应用于基因工程药物的研发提供了新的渠道。
关键词：细胞珠蛋白 原核表达 穿膜肽 TAT Xentry
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globe7Abstract
It is has been proved that Cytoglobin has biological activity of antioxidant, anti-tumor and others,and may act as clinical potential medicine. But its molecular weight is far more than which can go through the cell membrane whose molecular weight requirements is less than 500Da.More seriously for it has a tendency to form a dimer trimers and other polymers, etc., which greatly limits its ability to enter cells.Our lab has previously constructed prokaryotic expression vector pET22b-TC and purified the targeted protein whose biological activity also studied i vitro and in vivo for further studies. TAT protein transduction domain amino acids sequence are YGRKKRRQRRR, is the shortest transduction effective sequence of TAT.Xentry whose amino acids sequence is LCLRPVG is a newly reported polypeptide with transmembrane ability, which is shorter than TAT.It has a good perforation effect.So we look forward to building fusion protein XC who can help more cytoglobin play its effect due to that more cytoglobin are in a state of inside cells.This study is significant in which prokaryotic
expression vector pET22b-XC was successfully constructed. The recombinant XC protein was well expression by lactose induce. However, it was possible that isoelectric point of the targetd recombinant protein is too close to the isoelectric point of protein in escherichia coli itself.It is hard to isolate and purify the recombinant protein. Then for achieving more biologically active recombinant protein and for easier to isolate and purify,pET22b-pelB-XCHwas constructed who contains the pelB signal peptide and histidine tag.And so was pET22b-pelB-XLCH in which linker is added.But they were not significantly expressed by lactose and IPTG induction.Then try to use nickel column adsorption to collect targeted protein but did not get it.In order to obtain high expression levels Intracellular expression was tried.Then pET22b-XCH and pET22b-XLCH were successfully constructed.However the expression result is similar to before.Finally use pET28a instead of pET22b .
PET28a-XCH and PET28a-XLCH were successfully constructed.It has show that recombinant protein was significantly expressed by IPTG induction with former vector.
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Then to obtain XC protein,Nickel column was used,by which collect protein in cell supernatant after u
ltrasound.Preliminary visible light analysis showed that the protein had biological activity. Then biological activity of XC had been explored in vitro.
In summary,the result of this study demonstrated that significant ability of penetrating cell memberane may promote the intracellular function of cytoglobin.In vitro, XC has showed better biological activity than TAT-Cytoglobin for some studies but some are not. What are the differences between XC and TC whose mechanism into cells are explicit.All need futher studies.
Key Words:Cytoglobin prokaryotic expression Xentry cell-penetrating peptides TAT xentry
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