Expression of recombinant canine perilipin3 (PLIN3) and its assessment for immunoreactivity

IF 1 Q4 GENETICS & HEREDITY

Gene Reports Pub Date : 2025-02-25 DOI:10.1016/j.genrep.2025.102157

Amrita Behera , Ghanshyam Sahu , Vineet Kumar Pandey , Franco PS , Pawan Kumar , Mukesh Kumar , Mohini Saini , Karuna Irungbam

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引用次数: 0

Abstract

Perilipins are the lipid droplet associated proteins (LDAPs) involved in the biogenesis and stabilization of lipid droplets. LDAPs contribute significantly to the pathogenesis of several diseases stemming from dysregulated lipid metabolism including cancers. Several studies indicate lipid droplet (LD) accumulation in cancer cells, emphasizing the critical importance of comprehending the role of perilipins in cancers. This study seeks to generate hyperimmune sera directed against canine perilipin3 (PLIN3), with the aim of investigating the expression of perilipin3 in canine mammary tumors (CMT). The limited availability of canine specific commercial perilipin antibodies substantiate the necessity of developing canine specific antibodies to better understand the role of perilipins in CMT pathogenesis. A fragment of the canine perilipin3 gene was amplified effectively from total RNA extracted from canine mammary tumor samples and it was cloned and expressed as a recombinant protein in pET32a+ prokaryotic system. The recombinant canine PLIN3 protein was purified using Ni- NTA (Nickel-nitroacetic acid) affinity chromatography and used to generate hyperimmune sera in chickens. The generated antisera showed strong reactivity to the recombinant canine PLIN3 protein in dot blot assays. Immunohistochemistry analysis revealed specific immunoreactivity to PLIN3 in CMT tissue samples, with elevated expression levels compared to control tissues. These data indicate that PLIN3 may be differentially expressed in canine mammary tumors, highlighting its potential role in tumor progression. The study provides a valuable tool for advancing the investigation of lipid droplet-associated proteins in canine cancers, particularly CMT, and offers insights into lipid metabolism as a possible therapeutic target in veterinary oncology.

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重组犬外周血蛋白3 （PLIN3）的表达及免疫反应性评价

脂滴相关蛋白（LDAPs）参与脂滴的生物形成和稳定。LDAPs在包括癌症在内的脂质代谢失调引起的几种疾病的发病机制中起着重要作用。一些研究表明，脂滴（LD）在癌细胞中积累，强调了理解periilipins在癌症中的作用的关键重要性。本研究旨在生成针对犬periilipin3 （PLIN3）的高免疫血清，目的是研究periilipin3在犬乳腺肿瘤（CMT）中的表达。犬类特异的periilipin商用抗体的有限可用性证实了开发犬类特异抗体的必要性，以更好地了解periilipin在CMT发病机制中的作用。从犬乳腺肿瘤样品中提取的总RNA中有效扩增出犬perilipin3基因片段，并克隆出重组蛋白，在pET32a+原核系统中表达。采用Ni- NTA（镍-硝基乙酸）亲和层析纯化重组犬PLIN3蛋白，并用于鸡高免疫血清的制备。抗血清对重组犬PLIN3蛋白表现出较强的反应性。免疫组织化学分析显示CMT组织样本中对PLIN3具有特异性免疫反应性，与对照组织相比表达水平升高。这些数据表明，PLIN3可能在犬乳腺肿瘤中存在差异表达，突出了其在肿瘤进展中的潜在作用。该研究为推进犬类癌症（特别是CMT）中脂滴相关蛋白的研究提供了有价值的工具，并为脂代谢作为兽医肿瘤学可能的治疗靶点提供了见解。

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

Gene Reports Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

3.30

自引率

7.70%

发文量

246

审稿时长

49 days

期刊介绍： Gene Reports publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses. Gene Reports strives to be a very diverse journal and topics in all fields will be considered for publication. Although not limited to the following, some general topics include: DNA Organization, Replication & Evolution -Focus on genomic DNA (chromosomal organization, comparative genomics, DNA replication, DNA repair, mobile DNA, mitochondrial DNA, chloroplast DNA). Expression & Function - Focus on functional RNAs (microRNAs, tRNAs, rRNAs, mRNA splicing, alternative polyadenylation) Regulation - Focus on processes that mediate gene-read out (epigenetics, chromatin, histone code, transcription, translation, protein degradation). Cell Signaling - Focus on mechanisms that control information flow into the nucleus to control gene expression (kinase and phosphatase pathways controlled by extra-cellular ligands, Wnt, Notch, TGFbeta/BMPs, FGFs, IGFs etc.) Profiling of gene expression and genetic variation - Focus on high throughput approaches (e.g., DeepSeq, ChIP-Seq, Affymetrix microarrays, proteomics) that define gene regulatory circuitry, molecular pathways and protein/protein networks. Genetics - Focus on development in model organisms (e.g., mouse, frog, fruit fly, worm), human genetic variation, population genetics, as well as agricultural and veterinary genetics. Molecular Pathology & Regenerative Medicine - Focus on the deregulation of molecular processes in human diseases and mechanisms supporting regeneration of tissues through pluripotent or multipotent stem cells.