中文摘要：

目的利用基因工程方法克隆SD大鼠Atohl基因CDS区序列，构建大鼠核转录因子Atohl的真核表达载体并在真核细胞中表达。方法从两只SD大鼠结肠黏膜提取总RNA，采用逆转录PCR法扩增Atohl基因CDS区序列并亚克隆于PMD一19T载体中。测序鉴定后将Atohl基因连接于含有EGFP和内部核糖体转入位点（IRES）的真核细胞表达载体pIRES2-EGFP中，对重组质粒进行酶切鉴定和测序鉴定后，以脂质体介导法转染至293T细胞，荧光显微镜和Westernblot检测其在293T细胞中的表达。结果扩增得到大鼠AtohlCDS区长1056bp，编码351个氨基酸，与GeneBank公布的参考序列对比，有两处碱基发生突变，但克隆序列编码的氨基酸序列与参考序列完全一致，两处碱基应为单核苷酸多态性（SNP），突变为无义突变，不影响蛋白表达。双酶切和测序结果证明Atohl已正确地克隆到真核表达载体pIRES2一EGFP中，荧光显微镜和Westernblot证实Atohl目的蛋白能在293T细胞中稳定表达。结论基因工程方法可成功克隆出Atohl编码序列，真核表达载体pAtohl—IREg2-EGFP构建成功并可以在293T细胞中表达。

英文摘要：

Objective Useing gene engineering technique to clone SD rats Atohl cDNA coding sequence and construct the Eukaryotic expression vector for its expression in eukaryotic cells. Methods Total RNA was extracted from colon of SD rats, by means of asymmetrical primer/template , double stranded cDNA of Atohl was gained , then the cDNA coding sequence was cloned into PMD--19T vector and sequenced. The purified digested fragment was connected into Eukaryotic expression vector plRES2--EGFP with the EGFP gene and the internal ribosomes site （IRES）. Recombinant plasmid was identified by enzyme digestion and sequence analysis,and thea was transfect- ed into 293T cells with Lipofectamine and the expression of protein was detected by fluorescence microscope and Western blotting. Results DNA sequence analysis showed that rat Atohl gene amplified length of CDS area 1056bp, encoding 351 amino acids, and contrast the reference sequences published in GenetMnk, there were two base mutation in the sequence, it may be Single nucleotide polymorphisms in the nueleotide to induce nonsense mutation, deduced amino acid of cloning sequences as the same as reference sequences. Two bases were single nucleotide polymorphism （SNP）, and mutation was a nonsense mutation, did not affect protein expression. Enzyme digestion and sequence analysis of recombinant plasmid demonstrated that Atohl gene was correctly inserted into euearyotic expression vector plRES2- EGFP. The expres- sion of the green fluorescent protein in the 293T cells was observed by fluorescence microscope after recombinant plasmid into 293T cells 48 h. The mRNA and protein expression of Atohl in infected 293T had been cornformed by Western blotting. Conclusion Atohl CDS region was cloned and recombinant plasmid has been constructed successfully, and the Atohl of pIRES2--EGFP--Atohl was successfully expressed in the 293T cells, which will guide further research on gene therapy for sensorineural hearing loss.