光滑鳖甲丝氨酸蛋白酶抑制剂基因ApSerpin-FA72的克
发布时间：2018-08-20 08:39
【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。
[Abstract]:[objective] to clone and express the Anatolica polita borealis serine protease inhibitor gene, [methods] the (rapid amplification of cDNA endsrace gene was cloned by PCR and cDNA terminal amplification. The basic properties of the gene and its encoded protein were predicted and analyzed by bioinformatics, and the phylogenetic tree of the encoding product was constructed. The purified protein was verified. [results] A Serpin-FA72 (Gen Bank inhibitor gene (p Serpin-FA72 (Gen Bank accession number: MF188125) was obtained. Its coding sequence was 1 176 BP, encoding a peptide composed of 391 amino acid residues. The theoretical molecular weight of the protein is 43.7 KD and the theoretical isoelectric point is 5.14. The protein contains a 21 amino acid signal peptide. As Serpin-FA72 is a hydrophilic protein, secreting to extracellular proteins, which may have the function of stress response. The homology with Triboloum castaneum Serpin was the highest. The Trx A-Ap Serpin-FA72 size of the purified fusion protein was about 63.7 KD. Functional verification shows that, The recombinant protein Trx A-Ap SerpinFA72 can inhibit the activity of trypsin and chymotrypsin. [conclusion] the expression product of the inhibitor of trypsin and chymotrypsin can inhibit the activity of trypsin and chymotrypsin. The results suggested that it might inhibit the activity of digestive serine-like protease, and the verification of its functional activity would be helpful to further study the relationship between AP Serpin-FA72 and serine protease.
【作者单位】： 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;
【基金】：国家自然科学基金项目(31460578)
【分类号】：Q78
，
本文编号：2193040
[Abstract]:[objective] to clone and express the Anatolica polita borealis serine protease inhibitor gene, [methods] the (rapid amplification of cDNA endsrace gene was cloned by PCR and cDNA terminal amplification. The basic properties of the gene and its encoded protein were predicted and analyzed by bioinformatics, and the phylogenetic tree of the encoding product was constructed. The purified protein was verified. [results] A Serpin-FA72 (Gen Bank inhibitor gene (p Serpin-FA72 (Gen Bank accession number: MF188125) was obtained. Its coding sequence was 1 176 BP, encoding a peptide composed of 391 amino acid residues. The theoretical molecular weight of the protein is 43.7 KD and the theoretical isoelectric point is 5.14. The protein contains a 21 amino acid signal peptide. As Serpin-FA72 is a hydrophilic protein, secreting to extracellular proteins, which may have the function of stress response. The homology with Triboloum castaneum Serpin was the highest. The Trx A-Ap Serpin-FA72 size of the purified fusion protein was about 63.7 KD. Functional verification shows that, The recombinant protein Trx A-Ap SerpinFA72 can inhibit the activity of trypsin and chymotrypsin. [conclusion] the expression product of the inhibitor of trypsin and chymotrypsin can inhibit the activity of trypsin and chymotrypsin. The results suggested that it might inhibit the activity of digestive serine-like protease, and the verification of its functional activity would be helpful to further study the relationship between AP Serpin-FA72 and serine protease.
【作者单位】： 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;
【基金】：国家自然科学基金项目(31460578)
【分类号】：Q78
，
本文编号：2193040
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