人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究
文献类型：期刊论文
| 作者 | 洪晓武1; 张亚平[*]2; 储以微1; 高波1; 邵先安1; 蒋正刚1; 熊思东[*]1 |
| 刊名 | 中国免疫学杂志
 |
| 出版日期 | 2007 |
| 卷号 | 23期号:4页码:326-330 |
| 关键词 | FKN 基因重叠延伸拼接PCR 基因克隆 灵长类 |
| 其他题名 | Comparative study of FKN in genome and protein expression among human,chimpanzee and leaf monkey |
| 通讯作者 | sdxiong@shmu.edu.cn |
| 合作状况 | 其它 |
| 中文摘要 | 克隆人、黑猩猩和叶猴FKN全基因及体外表达,比较研究FKN在进化过程中基因组水平和蛋白表达水平的差异.方法:应用基因重叠延伸拼接PCR法(Gene splicing by overlapping extension PCR,SOE-PCR)将FKN的3个外显子编码序列依次进行前后拼接,然后插入pcDNA3.1/myc-His(-)A真核表达载体中,经酶切、测序鉴定后转染CHO细胞体外表达,RT-PCR、SDS-PAGE和Western blot检测其表达产物.结果:酶切、测序鉴定证实插入的基因片段为完整的FKN,RT-PCR可从转染的CHO细胞中扩增出一条与目的基因大小一致的DNA片段,其表达蛋白能分泌至胞外,SDS-PAGE显示其分子量约为95 000,抗c-myc抗体可与载体上的c-myc蛋白特异性结合.测序显示人、黑猩猩和叶猴相比,FKN基因除了有散在的点突变外,还发现有一明显的30 bp的缺失,但此缺失对FKN蛋白的表达并不影响.结论:成功克隆人、黑猩猩和叶猴FKN全基因,基因组水平和蛋白表达水平的比较研究为后续探讨FKN在高级灵长类物种进化过程中免疫学功能的演变奠定基础. |
| 英文摘要 | Objective：To clone full-length FKN gene of human, chimpanzee and leaf monkey and compare their difference in genome and protein expression level. Methods ：Three exons of FKN were spliced in turn via gene splicing by overlapping extension PCR and inserted into pcDNA3.1/myc-His（ - ）A and then transfected into CHO cells via liposome after enzyme digesting and sequencing. The protein expressed was analyzed with RT-PCR, SDS-PAGE and Western blot. Results： Enzyme digestion and sequence analysis showed that the DNA fragments inserted into eukaryotic vectors were correct full-length FKN gene and the target gene could be amplified from CHO cells transfected with recombinant vectors by RT-PCR. Expressed protein in CHO cells could be secreted into supernatant and combine with c-myc antibody specifically. SDS-PAGE showed the appearance of 95 000 band of FKN protein. Sequence showed that besides other point mutation, there was an apparent 30 nucleotide deletion mutation in human and chimpanzee FKN when compared to that of leaf monkey. However, the deletion mutation didn＇ t after its protein expression level. Conclusion：The full-length FKN gene of human, chimpanzee and leaf monkey have been cloned successfully, the results and their differences in genome and protein expression level lay the foundation for further studying its evolution in immunological function in primates. |
| 收录类别 | 其他 |
| 资助信息 | 上海市学科带头人计划基金资助(04XD14003). |
| 语种 | 中文 |
| 公开日期 | 2010-08-06 |
| 源URL | [http://159.226.149.42:8088/handle/152453/1016]  |
| 专题 | 昆明动物研究所_分子进化基因组学 昆明动物研究所_细胞与分子进化开放实验室 |
| 作者单位 | 1.复旦大学上海医学院免疫学系复旦大学免疫生物学研究所;上海200032 2.中国科学院昆明动物研究所细胞与分子进化开放实验室;昆明650223 |
| 推荐引用方式 GB/T 7714 | 洪晓武,张亚平[*],储以微,等. 人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究[J]. 中国免疫学杂志,2007,23(4):326-330. |
| APA | 洪晓武.,张亚平[*].,储以微.,高波.,邵先安.,...&熊思东[*].(2007).人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究.中国免疫学杂志,23(4),326-330. |
| MLA | 洪晓武,et al."人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究".中国免疫学杂志 23.4(2007):326-330. |
入库方式： OAI收割
来源：昆明动物研究所
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