Hong Xiao-Wu, Zhang Ya-Ping, Chu Yi-Wei, Gao Hai-Feng, Jiang Zheng-Gang, Xiong Si-Dong
Department of Immunology, Shanghai Medical College, Institute for Immunobiology, Fudan University, Shanghai 200032, China.
Yi Chuan. 2008 May;30(5):595-601. doi: 10.3724/sp.j.1005.2008.00595.
To obtain full-length FKN nucleotide sequences of homonids including human, chimpanzee, gorilla, orangutan and gibbon, and Old World Monkeys including macaque and leaf monkey and make phylogenetic analysis, three exons of FKN were amplified by degenerated PCR using obtained peripheral blood cells DNA as template which was extracted from homonids and Old World Monkeys. After extracting and purifying from agarose gels, PCR products were sequenced and then spliced by using BioEdit. All the FKN sequences were aligned and compared the percent identity by using DNAStar. The phylogenetic tree was constructed using maximum evolution approach in Mega. The negative selection sites were analyzed by using Datamonkey. There is an apparent 30 bp nucleotides deletion mutation in homonids FKN comparing to that of Old World Monkeys besides other point mutations. Homology of nucleotide sequence between human and chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey is 99.2%, 98.4%, 98.1%, 96.5%, 95.9% and 93.8%, respectively. Homology of amino acid sequence of them is 98.5%, 98.0%, 97.7%, 94.7%, 93.7% and 90.5%, respectively. In the same time, the genealogical relationship of human is a lot closer to chimpanzee than it is to gorilla and other apes. It is generally agreed that the evolution rule of FKN gene is in accord with that of the higher primates. In addition, Datamonkey shows that there are 3 negative selection sites 53Q, 84D and 239N in FKN. The full-length FKN gene of human, chimpanzee, gorilla, orangutan, gibbon, macaque and leaf monkey were sequenced successfully, and the FKN sequences analysis lays the foundation for further studying its evolution in immunological function in higher primates and the relation between its structure and function.
为获得包括人类、黑猩猩、大猩猩、猩猩和长臂猿在内的灵长类动物以及包括猕猴和叶猴在内的旧世界猴的全长FKN核苷酸序列并进行系统发育分析,以从灵长类动物和旧世界猴中提取的外周血细胞DNA为模板,通过简并PCR扩增FKN的三个外显子。从琼脂糖凝胶中提取并纯化后,对PCR产物进行测序,然后使用BioEdit进行拼接。使用DNAStar对所有FKN序列进行比对并比较百分比同一性。在Mega中使用最大简约法构建系统发育树。使用Datamonkey分析负选择位点。与旧世界猴相比,灵长类动物的FKN存在明显的30 bp核苷酸缺失突变以及其他点突变。人类与黑猩猩、大猩猩、猩猩、长臂猿、猕猴和叶猴的核苷酸序列同源性分别为99.2%、98.4%、98.1%、96.5%、95.9%和93.8%。它们的氨基酸序列同源性分别为98.5%(黑猩猩)、98.0%(大猩猩)、97.7%(猩猩)、94.7%(长臂猿)、93.7%(猕猴)和90.5%(叶猴)。同时,人类与黑猩猩的亲缘关系比与大猩猩及其他猿类的亲缘关系更为密切。普遍认为FKN基因的进化规律与高等灵长类动物一致。此外,Datamonkey显示FKN中有3个负选择位点53Q、84D和239N。成功测定了人类、黑猩猩、大猩猩、猩猩、长臂猿、猕猴和叶猴的全长FKN基因,FKN序列分析为进一步研究其在高等灵长类动物免疫功能中的进化以及结构与功能的关系奠定了基础。