国人ICA69基因cDNA的克隆及序列分析

Cloning and Sequencing of cDNA Encoding Islet Cell Autoantigen 69kD Protein from Chinese

目的 :克隆国人胰岛细胞自身抗原69kD蛋白基因(hiICA69)并经序列分析予以确证。方法 :采用聚合酶链式反应技术 ,从中国人胰岛细胞瘤cDNA文库中扩增出hiICA69编码序列cDNA ,将基因片段插入 pSPORT1质粒 ,进一步亚克隆到 pUC18载体中 ,经蓝白斑和限制性酶谱分析得以初步筛选后 ,双脱氧末端终止法对其全部核苷酸序列予以确定。结果 :证实了hiICA69基因全长为1449bp、编码483个氨基酸。与Pietropaolo等报道的序列比较 ,仅在编码第139位氨基酸的密码子由CAA→CTA ,即由谷氨酰胺→亮氨酸 ,其余均与文献报道和EMBL核酸数据库提供的序列相同。结论 :这一基因的获得和定征 ,为后续研究打下基础。

Objective: It is reported that cDNA encoding human islet cell autoantigen 69kD protein (hiICA69) has been cloned,so to confirm the nucleotide sequences from the insulinoma cells of Chinese. Methods: cDNA encoding hiICA69 has been amplificated by PCR,from the cDNA library of Chinese insulinoma cells. The PCR product was inserted into the pSPORT 1 vector, and was subcloned into the pUC18 plasmid. After the positive colony was screened by the blue/ white colony and the restriction analysis, the nucleotide sequences of the full-length cDNA were analysed by means of the dideoxy chain termination method. Results: The results showed that the amplified fragment contained 1449bp, encoded 483-amino acids. For the sequencing analysis of ICA69 gene from the insulinoma in Mongolian race, the nucleotide sequence of the recombinant was coincident with that reported by Miyazaki and that from EMBL data's bank in addition to one difference of only base on the codon. The change located in the 416th base (A→T), which led to the change of one amino acid (Gln→Leu). Conclusion: The gene obtained by the method of gene engineering and identified by means of sequence analysis would be able to lay a foundation for follow-up research.