小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

The Construction of Eukaryotic Expression Plasmids of Containing a Sense and an Antisense Resistin Gene Respectively

目的构建载有小鼠抵抗素(resistin)基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中,通过RT-PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3.1(+)或pcDNA3.1(-)真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363bp相符;重组pGEM-T被EcoRⅠ和XbaⅠ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3.1(+)和pcDNA3.1(-)测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。

Abstract Objective The aim is to construct respectively the eukaryotic expression plasmids containing a sense and an antisense resistin gene, which laid a foundation for studing the biological functions of resistin. Methods Primers were designed from the published mouse resistin mR NA sequence （AF323080）. Synthesis of resistin cDNA with RT-PCR. The purified PCR product was ligated to pGEM-T vector by T4 DNA ligase. The pGEM-T- resistin was verified by using EcoR I and Xba I digestion and an analysis of the nucleotide sequences. Resistin cDNA subcloned into PcDNA3.1 ^（＋） or PcDNA3.1 ^（-） eukaryotic expression vector. The recombination expression plasmids were analyzed nucleotide se quences. Results The RT-PCR product was showed only one band between 250 bp and 500 bp, and was consistent with theoretic value 363 bp. The recombinant pGEM-T- resistin plasmid was digested into two fragments by EcoR I and Xba I . They are consistent with theoretic values 3 000 bp and 355 bp. The nucleotide sequence analysis was consistent with the database of the result resistin mRNA codingsequence. DNA fragment nucleotide sequence inserted pcDNA3.1 ^（＋）or pcDNA3.1 ^（-） was consistent with the resistin gene mRNA codingsequence or with the re sistin gene anitsense mRNA codingsequence. Conclusion The sense and antisenses resistin gene expression plasmids have been successfully cloned.