人LAK细胞免疫效应分子HMGN2的初步研究

发布时间：2018-12-15 08:04

【摘要】：人LAK细胞(lymphocyte-activated killer cells，淋巴因子激活的杀伤细胞)／NK细胞、CTL细胞被认为是机体抗瘤细胞系统和抗胞内微生物感染的主要免疫细胞群体。LAK中存在多种具有杀菌作用的活性物质，已经在LAK细胞中发现了穿孔素、颗粒酶、颗粒溶素、LL-37、α-防御素等抗菌分子。LAK细胞中是否还有其它内源性抗菌肽尚不清楚。将rIL-2、PHA刺激培养一周的人LAK细胞用5％的乙酸匀浆，制备酸溶性粗提物。AU-PAGE及电泳凝胶琼脂糖弥散法检测出其中含有三组杀菌多肽成分，分别命名为HLP-1、HLP-2、HLP-3。利用制备性AU-PAGE电泳技术初步分离获得这三个组分。将这三个组分用反向高效液相色谱技术进一步分离纯化，琼脂糖弥散法筛选纯化各组分的抗菌活性，较强抗菌活性的组分进行Tricine-SDS-PAGE电泳初步测定分子量及纯度。选择高度纯化的蛋白组分HLP-3p21，采取Edman降解法测定其氮端氨基酸序列，得到HLP-3p21的N-端10个氨基酸序列，分别为：脯氨酸(Pro，，P)，赖氨酸(Lys，K)，精氨酸(Arg，R)，赖氨酸(Lys，K)，丙氨酸(Ala，A)，谷氨酸(Glu，E)，甘氨酸(Gly，G)，天冬氨酸(Asp，D)丙氨酸(Ala，A)，赖氨酸(Lys，K)。将该段氨基酸序列登录美国国立医学图书馆(NCBI)，应用Blast检索工具进行短序列匹配的人蛋白质数据库检索发现：这段序列与人非组蛋白HMGN2的氮端氨基酸序列完全相同。对HLP-3p21进行质谱精确分子量分析，其分子量为9274.04 Da，也与HMGN2相同。使用本实验室自制的兔抗HMGN2多克隆抗体对HLP-3p21用做Western blot，结果显示HLP-3p21蛋白条带处有较强的杂交信号。根据以上试验结果，我们判断HLP-3p21抗菌活性分子
[Abstract]:Human LAK cells (lymphocyte-activated killer cells, lymphokine-activated killer cells) / NK cells, CTL cells are considered to be the main immune cell population of anti-tumor cell system and anti-microorganism infection. There are many bactericidal active substances in LAK. Perforin, granzyme and granolysin have been found in LAK cells. LL-37, 伪-defensin and other antimicrobial molecules. It is not clear whether there are other endogenous antimicrobial peptides in LAK cells. Human LAK cells stimulated by rIL-2,PHA for one week were incubated with 5% acetic acid homogenate to prepare acid soluble crude extracts. Three groups of bactericidal polypeptides were detected by AU-PAGE and electrophoresis gel agarose dispersion, respectively named HLP-1,. HLP-2,HLP-3. The three components were preliminarily separated by AU-PAGE electrophoresis. The three components were further separated and purified by reverse performance liquid chromatography. The antimicrobial activity of the purified components was screened by agarose dispersion method. The molecular weight and purity of the components with strong antibacterial activity were determined by Tricine-SDS-PAGE electrophoresis. The N-terminal amino acid sequence of highly purified protein component HLP-3p21, was determined by Edman degradation method. Ten N-terminal amino acid sequences of HLP-3p21 were obtained, namely, proline (Pro,P), lysine (Lys,K), arginine (Arg,). R), lysine (Lys,K), alanine (Ala,A), glutamic acid (Glu,E), glycine (Gly,G), aspartic acid (Asp,D) alanine (Ala,A), lysine (Lys,K). The amino acid sequence was recorded in the (NCBI), of the National Library of Medicine of the United States for short sequence matching human protein database retrieval using Blast search tool. It was found that the sequence was identical to the N-terminal amino acid sequence of human non-histone HMGN2. The accurate molecular weight analysis of HLP-3p21 by mass spectrometry showed that the molecular weight of 9274.04 Da, was the same as that of HMGN2. Using rabbit anti HMGN2 polyclonal antibody made in our laboratory as Western blot, for HLP-3p21, the results showed that there was a strong hybridization signal in the band of HLP-3p21 protein. Based on the above results, we judge the antibacterial activity of HLP-3p21.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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