鸡补体分子C3d的基因克隆及结构分析

Cloning and Structural Analysis of Chicken Complement Component 3d Gene

目的:克隆鸡补体分子C3d基因并解析其结构特点。方法:将已发表的人、小鼠、地鼠、奶牛、野兔、猪、猩猩、绵羊的C3d基因同鸡的C3α链进行序列比较分析,发现在鸡的C3α链上有一段约897bp的序列同上述动物有较高的同源性,在上下端保守区域设计一对引物788bp,应用RT-PCR扩增鸡C3d部分基因,并克隆到pMD18-T载体中,测序正确后再在上下端分别设计一对引物,理论长度分别为378和336bp,最后用3种PCR产物延伸扩出C3d全长序列。结果:获得了鸡C3d基因重组质粒pMD18-C3d,序列分析表明所获的鸡C3dcDNA全长为993bp,编码331个氨基酸残基。鸡与上述人或动物C3d核苷酸的同源性分别为66.6%、66.2%、67.7%、66.2%、67.1%、67.0%、59.6%、67.1%,与其编码的氨基酸的同源性分别为61.5%、61.9%、61.2%、61.9%、56.5%、61.9%、54.5%、61.5%;而哺乳动物间C3d的核苷酸和氨基酸的同源性则分别为74.2%～100%和72.2%～100%。进化树反映出C3d基因具有种的多样性,亲缘关系越近,进化关系也越近。结论:鸡C3d与其他动物的C3d在抗原结合位点上没有氨基酸的变化,而与CR2结合的28肽区氨基酸差异明显,说明鸡C3d结合抗原没有专一性,而结合免疫细胞则有种的特异性,由此可以推测鸡C3d只能增强鸡的特异性免疫反应。

Objective： Chicken complement component 3d（C3d） gene was cloned and it＇s structural characteristic was analyzed. Methods： According to the conservative sequences of announced C3d in human, mouse, hamster, cow, rabbit, pig, chimpanzee and sheep, three pairs of primer were designed. Parts of chicken C3d genes were amplified by RT-PCR. Using these RT-PCR products as template, the complete chicken C3d was amplified by OE-PCR. Results： The product was cloned into pMD18-T vector. Sequence analysis revealed that its length was 993 bp, consisted of 331 amino acids. When nucleotide sequence and encoded amino acid sequence were compared with homologous sequence of human, mouse, hamster, cow, pig, sheep, rabbit and chimpanzee, it displayed that the homologous nucleotide sequences were 66.6%, 66.2%, 67.7%, 66.2%, 67.1%, 67.0%, 59.6%, 67.1% respectively; and the homologous amino acids sequences were 61.5%, 61.9%, 61.2%, 61.9%, 56.5%, 61.9%, 54.5%, 61.5% respectively. However, the homologous nucleotide sequences among other species were from 74.2% to 100%, the homologous amino acids were from 72.2% to 100%. Phylogenetic tree showed C3d had varieties in species, more close relationship, more close evolution. Conclusion： Structural analysis indicated that amino acids of chicken C3d were as same as other species in antigenic combining site. However, in CR2 combining region（a peptide consisted of 28 amino acids）, there were various diversity. It suggested that C3d was conservative in combining antigen, but had specific varieties in amplified B cell activation.