小鼠淋巴细胞趋化因子重组腺病毒载体的构建及其在骨髓树突状细胞中的表达  

CONSTRUCTION OF THE RECOMBINANT ADENOVIRUS VECTOR HARBORING MOUSE LYMPHOTACTIN AND ITS EXPRESSION IN BONE MARROWDERIVED DENDRITIC CELLS

作　　者：章卫平[1] 曹雪涛[1] 马施华[1] 何龙[1] 袁正隆 陈国友[1] 

机构地区：[1]第二军医大学免疫学教研室

出　　处：《中国免疫学杂志》1998年第1期2-6,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金

摘　　要：经ＲＴ－ＰＣＲ克隆小鼠淋巴细胞趋化因子（Ｌｙｍｐｈｏｔａｃｔｉｎ，Ｌｔｎ）的全长编码ｃＤＮＡ，将其置于ＣＭＶ启动子下游，插入Ｅ１区替代的腺病毒载体ｐＡｘ１ｃｗ。Ｌｔｎ重组腺病毒载体ｐＡｘ１ｃｗ．ＣＩｍＬｔｎ与经ＥｃｏＴ２２Ｉ酶切的Ａｄ５腺病毒ＤＮＡ－末端肽复合物共转染２９３细胞，制备Ｌｔｎ重组腺病毒，扩增到的病毒滴度为３．６×１０９ｐｆｕ／ｍｌ。用重组ＧＭ－ＣＳＦ从小鼠骨髓中扩增到的树突状细胞本身并不表达Ｌｔｎ；体外感染Ｌｔｎ重组腺病毒后４ｈ即可经ＲＴ－ＰＣＲ检测到Ｌｔｎ的表达，其２４ｈ的细胞培养上清体外对ＣＤ４＋Ｔ细胞和ＣＤ８＋细胞具有显著的趋化活性，表明所制备的Ｌｔｎ重组腺病毒能有效介导Ｌｔｎ在树突状细胞中的表达，为研究Ｌｔｎ基因修饰的树突状细胞诱导Ｔ细胞免疫应答奠定基础。he full-length cDNA encoding mouse lymphotactin was cloned by RTPCR, and inserted into E1substituted adenovirus vector pAx1cw. Subsequently, Ltn recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22Idigested Ad5TPC, and Ltn recombinant adenoviruses were generated efficiently by homologous recombination, with the titers of 36×109 pfu/ml. Mouse bonemarrow dendritic cells propagated with GMCSF don't express any detectable Ltn mRNA by RTPCR. 4 h after infection with Ltn recombinant adenoviruses in vitro, mRNA expression of Ltn can be detected by RTPCR. 24 h after infection, culture supernatants from Ltn genetransfected dendritic cells could attract CD4+T cells and CD8+T cells effectively in vitro. These data suggested that the Ltn adenovirus vector constructed was capable of mediating Ltn expression in mouse dendritic cells efficiently.

关 键 词：趋化因子 淋巴细胞 树突状细胞 腺病毒 载体 

分 类 号：R392.11[医药卫生—免疫学]