發(fā)布時(shí)間：2018-08-20 08:39

【摘要】：【目的】本研究旨在對(duì)光滑鱉甲Anatolica polita borealis絲氨酸蛋白酶抑制劑基因進(jìn)行克隆及表達(dá)分析,以驗(yàn)證光滑鱉甲絲氨酸蛋白酶抑制劑的功能�！痉椒ā坷肞CR和cDNA末端快速擴(kuò)增(rapid amplification of cDNA ends,RACE)技術(shù)克隆獲得光滑鱉甲絲氨酸蛋白酶抑制劑基因。采用生物信息學(xué)方法對(duì)該基因及其編碼蛋白的基本性質(zhì)進(jìn)行預(yù)測(cè)和分析,同時(shí)構(gòu)建其編碼產(chǎn)物的系統(tǒng)進(jìn)化樹;構(gòu)建光滑鱉甲絲氨酸蛋白酶抑制劑蛋白重組表達(dá)載體,表達(dá)、純化蛋白進(jìn)行功能驗(yàn)證。【結(jié)果】獲得光滑鱉甲絲氨酸蛋白酶抑制劑基因Ap Serpin-FA72(Gen Bank登錄號(hào):MF188125),其基因編碼序列長為1 176 bp,編碼由391個(gè)氨基酸殘基組成的多肽,蛋白理論分子量為43.7 k D,理論等電點(diǎn)為5.14,包含一個(gè)由21個(gè)氨基酸組成的信號(hào)肽。As Serpin-FA72屬親水蛋白,分泌到胞外發(fā)揮作用,可能具有脅迫應(yīng)答的功能,與赤擬谷盜Triboloum castaneum Serpin的同源性最高。純化得到的融合蛋白Trx A-Ap Serpin-FA72大小約為63.7 k D。功能驗(yàn)證表明,重組蛋白Trx A-Ap SerpinFA72對(duì)胰蛋白酶及胰凝乳蛋白酶活性均有抑制作用�！窘Y(jié)論】光滑鱉甲絲氨酸蛋白酶抑制劑基因的表達(dá)產(chǎn)物對(duì)胰蛋白酶及胰凝乳蛋白酶活性具有抑制作用,表明其可能對(duì)消化類絲氨酸蛋白酶活性起抑制作用,對(duì)其功能活性的驗(yàn)證有助于深入研究Ap Serpin-FA72與絲氨酸蛋白酶之間的關(guān)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.
【作者單位】： 新疆大學(xué)生命科學(xué)與技術(shù)學(xué)院新疆生物資源基因工程重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金項(xiàng)目(31460578)
【分類號(hào)】：Q78
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本文編號(hào)：2193040