日本七鳃鳗L-STAT3的克隆及体内外表达分析

Cloning and Expression Analysis of Lampetra japonica L-STAT3 in Vitro and in Vivo

作为信号转导和转录激活子(STAT)蛋白家族的成员之一,信号转导和转录激活因子3(STAT3)在细胞分化、免疫等众多过程都发挥着重要的作用。该研究在克隆获得日本七鳃鳗STAT3全长的基础上,对其氨基酸序列与其他物种来源的STAT3氨基酸序列进行了同源性比对,将日本七鳃鳗STAT3命名为L-STAT3。对L-STAT3基因分别进行了原核和真核表达,其中原核表达呈包涵体形式存在。根据毕赤酵母密码子偏好性将L-STAT3核苷酸序列进行了密码子优化,实现了L-STAT3在毕赤酵母中的分泌型表达。进一步在转录和蛋白质水平检测了L-STAT3基因在七鳃鳗不同组织中的表达情况。该研究构建了适用于日本七鳃鳗功能基因表达的毕赤酵母表达系统,为后续验证基因功能奠定了基础。对于七鳃鳗L-STAT3基因表达模式的分析可为研究七鳃鳗适应性免疫系统发育和调控的过程提供理论基础。

As a member of the STAT(signal transduction and transcriptional activator)protein family,STAT3 plays important roles in many physiological processes,including cell differentiation and immunity.In this study,the full-length of STAT3 was cloned from Lampetra japonica and its amino acid sequence was homologous with those of other species,and then was named as L-STAT3.The prokaryotic expression and eukaryotic expression of L-STAT3 gene were performed respectively,while expression of L-STAT3 gene in E.coli was in the form of inclusion body.The nucleotide sequence of L-STAT3 was optimized according to the codon preference of Komagataella phaffii and the secretion expression of L-STAT3 could be achieved.The expression of L-STAT3 gene in different tissues of L.japonica was further examined at transcription and protein levels.These attempts provided technical support for obtaining important functional proteins of lamprey and laid a foundation for further verification of lamprey protein functions.In addition,the analysis of the expression pattern of L-STAT3 can provide a theoretical basis for studying the adaptive immune process of lamprey.