光滑鳖甲丝氨酸蛋白酶抑制剂基因ApSerpin-FA72的克隆、表达及功能验证  

Cloning,expression and functional verification of the serine protease inhibitor gene ApSerpin-FA72 from Anatolica polita borealis( Coleoptera:Tenebrionidae)

作　　者：赵钰 刘忠渊[1] ZHAO Yu LIU Zhong-Yuan(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumuqi 830046, Chin)

机构地区：[1]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《昆虫学报》2017年第8期857-864,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31460578)

摘　　要：【目的】本研究旨在对光滑鳖甲Anatolica polita borealis丝氨酸蛋白酶抑制剂基因进行克隆及表达分析,以验证光滑鳖甲丝氨酸蛋白酶抑制剂的功能。【方法】利用PCR和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆获得光滑鳖甲丝氨酸蛋白酶抑制剂基因。采用生物信息学方法对该基因及其编码蛋白的基本性质进行预测和分析,同时构建其编码产物的系统进化树;构建光滑鳖甲丝氨酸蛋白酶抑制剂蛋白重组表达载体,表达、纯化蛋白进行功能验证。【结果】获得光滑鳖甲丝氨酸蛋白酶抑制剂基因Ap Serpin-FA72(Gen Bank登录号:MF188125),其基因编码序列长为1 176 bp,编码由391个氨基酸残基组成的多肽,蛋白理论分子量为43.7 k D,理论等电点为5.14,包含一个由21个氨基酸组成的信号肽。As Serpin-FA72属亲水蛋白,分泌到胞外发挥作用,可能具有胁迫应答的功能,与赤拟谷盗Triboloum castaneum Serpin的同源性最高。纯化得到的融合蛋白Trx A-Ap Serpin-FA72大小约为63.7 k D。功能验证表明,重组蛋白Trx A-Ap SerpinFA72对胰蛋白酶及胰凝乳蛋白酶活性均有抑制作用。【结论】光滑鳖甲丝氨酸蛋白酶抑制剂基因的表达产物对胰蛋白酶及胰凝乳蛋白酶活性具有抑制作用,表明其可能对消化类丝氨酸蛋白酶活性起抑制作用,对其功能活性的验证有助于深入研究Ap Serpin-FA72与丝氨酸蛋白酶之间的关系。【Aim】This study aims to clone and analyze the expression of a serine protease inhibitor gene from Anatolica polita borealis so as to verify its function. 【Methods】A serine protease inhibitor gene from A. polita borealis was cloned using PCR and RACE methods. Bioinformatics approach was applied to analyze the basic properties of the gene and its coded protein,and to make the multiple sequence alignment with its homologous sequences. The recombinant expression vector p ET-32a-Ap Serpin-FA72 was constructed. The expression,purification and functional verification of the recombinant target protein were analyzed. 【Results】A serine protease inhibitor gene was cloned from A. polita borealis and named Ap Serpin-FA72（ Gen Bank accession no.： MF188125）. Its opening reading frame（ ORF） is 1 176 bp in length,encoding a 391 amino-acid residue with the predicted molecular weight of 43. 7 kD and pI of5. 14. Ap Serpin-FA72 is a hydrophilic protein with a signal peptide of 21 amino acids at the terminus. It might play roles in the stress response and immune response,and has the highest homology with Tribolium castaneum Serpin. The purified recombinant protein Trx A-Ap Serpin-FA72 has the molecularweight of 63. 7 k D. The functional verification results revealed that the recombinant protein Trx AAp Serpin-FA72 had an inhibitory effect on the activities of trypsin and chymotrypsin. 【Conclusion】The expression product of Anatolica polita serine protease inhibitor gene has an inhibitory effect on trypsin and chymotrypsin,indicating that it may inhibit the activity of digestive serine protease,and the verification of its functional activity can be helpful to further investigate the relationship between Ap Serpin-FA72 and serine protease.

关 键 词：光滑鳖甲 丝氨酸蛋白酶抑制剂 原核表达 蛋白纯化 功能验证 

分 类 号：Q966[生物学—昆虫学]