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角蛋白18是角蛋白的一种,属于S型角蛋白,是在哺乳动物和两栖类动物胚胎发育中第一个表达的中间纤维蛋白。根据已发表的人、爪蟾及斑马鱼角蛋白18 DNA序列,通过DNAman同源性比较获得保守序列,并从中设计二对引物。从稀有鲫基因组中PCR扩增得到2 779 bp的角蛋白18的DNA序列片段;通过基因组步移,分别获得该基因的上游和下游序列片段;最后将获得的全基因片段克隆至质粒载体pMD19-T进行序列测定检验。结果显示所克隆的稀有鲫角蛋白18基因全长3 765 bp。对该基因进行NCBI的Blastn同源性比较分析,表明稀有鲫同斑马鱼比较角蛋白18 DNA序列存在73.6%同源相似性;同时对该基因进行生物学分析,开放阅读框(Open ReadingFrame,ORF)预测表明稀有鲫角蛋白18是含有7个外显子和6个内含子,共编码367个氨基酸的蛋白质。研究亮点:首次成功克隆了稀有鲫角蛋白18基因全长序列3 765 bp,并且通过软件预测分析得出氨基酸序列,把氨基酸序列在DNA序列中进行标记,显示稀有鲫角蛋白18基因全长序列含有7个外显子和6个内含子,共编码367个氨基酸。另外,没有采用常用的从RNA反转录为cDNA为模板获得基因全长,而是直接以DNA为模板出发,利用基因组步移的方法获得基因全长。
Keratin 18, a type of keratin, belongs to the S-type keratin and is the first intermediate fibrin expressed in embryos of mammals and amphibians. According to published human, Xenopus and zebrafish keratin 18 DNA sequences, conserved sequences were obtained by DNAman homology and two pairs of primers were designed. The DNA sequence fragment of 2 779 bp of keratin 18 was obtained by PCR amplification from the genome of Rana nigromaculata giganticola. The upstream and downstream sequence fragments of the gene were obtained by genome walking. Finally, the whole gene fragment was cloned into the plasmid vector pMD19 -T for sequencing assays. The results showed that the length of the cloned rabbit keratin 18 gene was 3 765 bp. The NCBI Blastn homology analysis of the gene showed that the rare crucian carp and zebrafish compared with keratin 18 DNA sequence 73.6% homologous similarity; at the same time the gene for biological analysis, open reading frame (Open ReadingFrame, ORF) predicted that Rana krylovus is a protein that contains 7 exons and 6 introns and encodes a protein of 367 amino acids. Research Highlights: The first successful cloning of the 7 765 bp full-length sequence of the keratin 18 gene of Rana urticae and prediction of the amino acid sequence by software, the amino acid sequence tagged in the DNA sequence, The long sequence contains seven exons and six introns, encoding a total of 367 amino acids. In addition, instead of using the commonly used RNA reverse transcriptase cDNA as a template to obtain the full length of a gene, DNA is directly used as a template to obtain the full length of the gene by using a genome walking method.