Mateus Alves Gonçalves, Renato Mesquita Peixoto, Felipe Barroso de Sousa, Luzianna Macedo Fonseca, Ângela Maria Xavier Eloy
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All animals were submitted to blood collection by puncture of the jugular vein, followed by centrifugation to obtain blood plasma, protein quantification by the Bradford method, one-dimensional electrophoretic separation (1D), and identification of protease activity by zymography and confirmation via reverse zymography in the presence of MMP-2 through the action of tissue inhibitors (TIMP-2). The analysis of protein bands was performed using descriptive statistics and densitometry values for zymography were subjected to the Shapiro-Wilk test to determine normality. Little difference was observed in the occurrence of protein bands between groups. Regarding MMPs, no differences were observed in the expression of proMMP-9, MMP-9, and MMP-2 in animals affected by SRLV. TIMP-2 inhibited proMMP-2 and MMP-2 in all animals. Thus, the profile of protein bands does not change in healthy goats with chronic SRLV infection. 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引用次数: 0
摘要
小反刍慢病毒(Small ruminant lentivirus, SRLV)由于其逃逸机制而难以诊断。因此,蛋白质组学是通过与免疫反应相关的细胞外基质金属蛋白酶(MMPs)寻找生物标志物的一种替代方法。因此,本研究旨在分析巴西东南部慢性SRLV感染的健康和感染的Toggenburg山羊的MMPs特征。采用琼脂凝胶免疫扩散(AGID)微技术、western blot (WB)和巢式聚合酶链反应(nPCR)筛选SRLV阳性山羊和阴性山羊各5只。所有动物均经颈静脉穿刺采血,离心取血浆,Bradford法蛋白定量,一维电泳分离(1D),在MMP-2存在下通过组织抑制剂(TIMP-2)的作用,通过酶谱法鉴定蛋白酶活性,并通过反向酶谱法确认。蛋白带分析采用描述性统计,酶谱分析的密度测定值采用夏皮罗-威尔克检验确定正态性。两组间蛋白带的出现差异不大。关于MMPs,在SRLV影响的动物中,proMMP-9、MMP-9和MMP-2的表达没有差异。TIMP-2在所有动物中均抑制proMMP-2和MMP-2。因此,在慢性SRLV感染的健康山羊中,蛋白质带的特征没有改变。TIMP-2的表达可以通过反向酶谱法证明慢性SRLV感染动物中MMP-2的存在。
Profile of extracellular matrix metalloproteinase in healthy and infected Toggenburg goats with small ruminant lentivirus in Southeast Brazil
Small ruminant lentiviruses (SRLV) are difficult to diagnose due to their escape mechanisms. Therefore, proteomics is an alternative in the search for biomarkers through extracellular matrix metalloproteinases (MMPs), enzymes related to the immune response. In this sense, this study aimed to analyze the profile of MMPs in healthy and infected Toggenburg goats with chronic SRLV infection in Southeast Brazil. Five positive and five negative goats for SRLV were selected using the agar gel immunodiffusion (AGID) microtechnique, western blot (WB), and nested polymerase chain reaction (nPCR). All animals were submitted to blood collection by puncture of the jugular vein, followed by centrifugation to obtain blood plasma, protein quantification by the Bradford method, one-dimensional electrophoretic separation (1D), and identification of protease activity by zymography and confirmation via reverse zymography in the presence of MMP-2 through the action of tissue inhibitors (TIMP-2). The analysis of protein bands was performed using descriptive statistics and densitometry values for zymography were subjected to the Shapiro-Wilk test to determine normality. Little difference was observed in the occurrence of protein bands between groups. Regarding MMPs, no differences were observed in the expression of proMMP-9, MMP-9, and MMP-2 in animals affected by SRLV. TIMP-2 inhibited proMMP-2 and MMP-2 in all animals. Thus, the profile of protein bands does not change in healthy goats with chronic SRLV infection. The TIMP-2 expression allowed proving the existence of MMP-2 in animals chronically infected by SRLV via reverse zymography.