Biosynthesis and Cytotoxic Activity of In Vitro Expressed Scygonadin Protein

Q3 Environmental Science
Nurfarhana Rosli, S. C. Zainathan, S. N. K. Addis
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引用次数: 0

Abstract

Antimicrobial peptides (AMP) are key components of an innate immune response which represent immediate action of the defence mechanism of an organism.  It is considered a novel therapeutic agent due to its abundance in nature and a broad range of defence activity against microbial. Preceding research has shown that scygonadin AMPs isolated from seminal plasma of mud crab had the potential as a novel antimicrobial agent. However, its cytotoxicity properties on cultured cells have never been experimentally addressed. In this study, the scygonadin protein was expressed in vitro, followed by cytotoxicity assessment via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A full-length sequence of the scygonadin gene of 387 bp was cloned into pBAD/Myc-His A vector and expressed in TOP10 cells. The protein expression was induced, purified and quantified before being subjected to cytotoxicity analysis. Next, an African green monkey kidney (Vero) cell was chosen to evaluate the cytotoxicity level of scygonadin in vitro. A total of 1x104 cells/mL were seeded into a 96-well plate before being treated to various concentrations of scygonadin protein and hydrogen peroxide as a positive control for the toxicity test.  The cells’ viability treated with scygonadin AMP and hydrogen peroxide was also verified with fluorescent analysis. The result demonstrated that the scygonadin did not cause any cytotoxicity effects while hydrogen peroxide showed an IC50 value at 0.003mM and this was further confirmed by fluorescent staining analysis. The absence of scygonadin toxicity in cells indicates its potential for biopharmaceutical use. 
体外表达的促性腺素蛋白的生物合成及细胞毒活性
抗菌肽(AMP)是先天免疫反应的关键组成部分,代表了生物体防御机制的直接作用。由于其丰富的性质和广泛的防御微生物活性,被认为是一种新型的治疗剂。前期研究表明,从泥蟹精浆中分离得到的卵腺素AMPs具有作为新型抗菌药物的潜力。然而,其对培养细胞的细胞毒性尚未得到实验证实。本研究通过体外表达促性腺素蛋白,并通过MTT(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑)法评价细胞毒性。在pBAD/Myc-His - A载体中克隆了387 bp的scgonadin基因全长序列,并在TOP10细胞中表达。在进行细胞毒性分析之前,诱导、纯化和定量表达蛋白。其次,选择非洲绿猴肾(Vero)细胞,体外评价促性腺素的细胞毒性水平。将1x104个细胞/mL接种到96孔板中,然后用不同浓度的scgonadin蛋白和过氧化氢作为阳性对照进行毒性试验。用荧光分析验证了scgonadin AMP和过氧化氢处理后的细胞活力。结果表明,scygonadin不产生任何细胞毒性作用,而过氧化氢的IC50值为0.003mM,荧光染色分析进一步证实了这一点。在细胞中不存在促性腺素毒性表明其在生物制药方面的应用潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
1.40
自引率
0.00%
发文量
10
审稿时长
16 weeks
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