树鼩CD4及IL-6分子的克隆及分析  

Cloning and analysis of tree shrew CD4 and IL-6

作　　者：任芳芳[1] 徐婧雯[1] 张雪梅[1] 吴忠香[1] 马娜[1] 卢孔杰[1] 董少忠[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2015年第6期579-586,共8页Chinese Journal of Biologicals

基　　金：国家科技支撑计划项目(2014BAI01B01)

摘　　要：目的克隆野生型中缅树鼩的CD4及IL-6分子,并分析其核苷酸、氨基酸序列的多态性及重要的功能位点。方法经野生型树鼩尾静脉采血,分离外周血单核淋巴细胞,经植物血球凝集素(phytohemagglutinin,PHA)刺激24 h后,提取细胞总RNA,以其为模板,进行RT-PCR扩增,扩增产物插入p MD-19T载体,提取阳性重组质粒,经双酶切鉴定及测序;采用Lasergene分析目的基因核苷酸及氨基酸序列,并构建进化树,分析野生型树鼩与已知中缅树鼩的亲缘关系。结果野生型树鼩CD4全长编码序列的开放阅读框长度为1 365 bp,编码454个氨基酸,IL-6全长编码序列的开放阅读框为627 bp,编码208个氨基酸,22只野生型树鼩成功克隆出CD4分子,23只野生型树鼩成功克隆出IL-6分子。不同野生型中缅树鼩的CD4及IL-6分子有不同程度的核苷酸和氨基酸位点的突变,但分别有35%和26.7%的碱基突变为同义突变,且发生非同义突变的位点对树鼩CD4和IL-6分子重要的功能位点无影响。由CD4核苷酸及氨基酸序列构建的系统进化树基本一致;由IL-6核苷酸及氨基酸序列构建的系统进化树中,23只野生树鼩分为2个分支。结论 CD4和IL-6分子具有重要功能的氨基酸位点是保守稳定的,为今后树鼩CD4和IL-6单克隆抗体的制备及树鼩实验动物模型的建立奠定了基础。Objective To clone the CD4 and IL-6 molecules of wild type tree shrew（Tupaia belangeri chinensis） and analyze the polymorphism of nucleotide and amino acid sequences as well as the functional site. Methods The intravenous blood of wild type tree shrew was collected, from which peripheral blood mononuclear cells（PBMCs） were isolated and stimulated with phytohemagglutinin（PHA） for 24 h. Total RNA of PBMCs was extracted and used as a template for amplification by RT-PCR. The amplified product was inserted into vector p MD-19 T, and the positive recombinant plasmids were identified by restriction analysis and sequencing. The nucleotide and amino acid sequences of target gene were analyzed by Lasergene software, based on which a phylogenetic tree was constructed, and the relationship between wild type tree shrew from different sources was analyzed. Results The open reading frame（ORF）of full-length encoding sequence of CD4 of wild type tree shrew, at a length of 1 365 bp, encoded 454 amino acids, while that of IL-6,at a length of 627 bp, encoded 208 amino acids. CD4 molecules were successfully cloned from 22 tree shrews, while IL-6molecules from 23 ones. Mutations at different degrees were observed at nucleotide and amino acid sites of CD4 and IL-6molecules of tree shrew of wild types. However, 35% of base mutations in CD4 molecules and 26. 7% in IL-6 molecules were sense mutations. The sites of nonsense mutations showed no effect on the key function sites of CD4 and IL-6molecules. The phylogenetic trees based on nucleotide and amino acid sequences of CD4 were basically in agreement.However, according to the phylogenetic trees based on nucleotide and amino acid sequences of IL-6, the 23 tree shrews were clustered to two branches. Conclusion The amino acid sites with important functions in CD4 and IL-6 molecules were conserved and stable. It laid a foundation of preparation of monoclonal antibody against CD4 and IL-6 of tree shrew and establishment of tree shrew model.

关 键 词：树鼩 CD4 IL-6 克隆 系统进化树 

分 类 号：Q956[生物学—动物学]