【摘要】 目的克隆人、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95000,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30bp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。更多还原

【Abstract】 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.更多还原