【作者】 冯云；

【导师】 鲍朗；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2006， 博士

【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抗菌活性分子更多还原

【Abstract】 Human lymphokine-activated killer cells (LAK) have been proved useful to therapy of tumor and intracellular infections, in this study, our objective was to identify new antimicrobial peptides from human LAK cells.Human LAK cells were obtained by culturing human peripheral blood mononuclear cells in the presnence of rIL-2 and PHA for one week. Acid soluble proteins of LAK cells were prepared by homogenizing the cells with 5% acetic acid. Using AU-PAGE and gel overlay assay, three main protein bands that exhibited antimicrobial activity, named HLP-1, HLP-2, HLP-3 were isolated. The three main antimicrobial components were further purified by RP-HPLC. Tricine-SDS-PAGE showed that HPLC fraction 21 ( HLP-3p21) that had potent antimicrobial activity was of a purified single polypeptide with a apparent molecular weight of 14 kDa. The N-terminal amino acid sequence of HLP3p21 was PKRKAGEDAK, which was identical to the N-terminal of human Human high mobility group nucleosomal-binding domain 2 (HMGN2), indicated by National Center for Biotechnoloby Information BLAST search. Mass spectrometric analysis of HLP-3p21 revealed the same molecular mass (m/z=9274.04) as HMGN2. A strong HMGN2 antibody-binding signal at HLP-3p21 migration position was detected by Western blot. These results suggested that HLP-3p21 would be HMGN2.OMIGA protein structure software analysis revealed a transmembrane α-helical structure, the putative antimicrobial domain, located from position 18 to 48 of the更多还原