家蚕BmLITAF基因的克隆表达及生物信息学分析

Cloning and Bioinformatic Analyses of BmLITAF Gene from Bombyx mori

在对家蚕蛹cDNA文库进行分析时，发现一条与脂多糖诱导肿瘤坏死因子a的转录激活因子LITAF的保守结构域同源的保守序列。基因序列全长为981bp，由97bp的5’端非翻译区序列（5’UTR）、390bp的开放读码框（ORF）和494bp的3’端非翻译区序列（3’UTR）组成。利用生物信息学方法对BmLITAF进行基因结构分析发现，此基因由3个外显子和2个内含子组成，编码129个氨基酸残基；其预测理论分子量为13．9kDa，预测等电点6．47；在90～100位氨基酸残基区域具有较高的疏水性，具有跨膜结构域；定位细胞内的可能性较大。根据BmLITAF的cDNA序列进行PCR扩增获得目的基因，将其克隆到质粒pGEX-4T-3中，测序验证克隆正确。经IPTG诱导，结果发现目的蛋白主要以包涵体的形式存在。

A homologous sequence of LPS-induced TNF-α factor（LITAF）is found in silkworm pupae when analysing its cDNA library, so the authors named it as BrnLITAF. The total cDNA length of BmL- ITAF is 981 bp, which consists of a 5＇ untranslated region （UTR） of 97 bp, a 3＇ UTR of 494 bp, and an open reading frame （ORF） of 390 bp. The analysis of electronic cloning method shows that the BmLITAF includes three exons and two introns, which can encode a protein of 129 amino acids with an estimated molecular mass of 13. 9 kD and theoretical isoelectric point of 6. 77. Moreover, the 90-100 region of BmLI- TAF has the high hydrophobic property and a transmembrane structure domain. According to the cDNA sequence of BmLITAF, the target gene is cloned by PCR amplification, and then the target gene is transferred into pGEX--4T-3 vector. The sequencing result is identical with that of the electronic clone. After the IPTG induction, the target protein of recombinant BrnLITAF is existed as inclusion body in Escherichia coli.