靶向人CPNE7可变剪接异构体的鼠源抗血清制备与应用鉴定

Preparation and characterization of mouse antiserum targeting human CPNE7 alternative splice isoforms

摘要

目的 钙依赖性膜结合蛋白7（CPNE7）的关键可变剪接异构体在多种肿瘤中异常表达，为深入研究其功能，本研究拟制备一种能特异性同时识别CPNE7-FL与CPNE7-S两种主要可变剪接异构体的鼠源抗血清。方法 基于生物信息学筛选CPNE7异构体的共同保守序列，设计并合成肽段免疫8只BALB/c小鼠制备抗血清，通过间接ELISA测定抗血清效价。随后，构建CPNE7原核表达载体并建立A549细胞过表达体系，采用Western blot验证抗血清的特异性。结果 成功获得效价达1:32000的抗血清。Western blot结果表明，该抗血清能有效识别原核表达的GST-CPNE7融合蛋白，并能在真核细胞中检测到过表达的CPNE7-FL与CPNE7-S异构体。结论 本研究成功制备了高效价、高特异性的抗人CPNE7抗血清，为后续开展CPNE7异构体表达谱分析、功能研究及其在肿瘤诊疗中的作用提供有效的研究工具。

Abstract

Objective Aberrant expression of key alternative splice isoforms of calcium-dependent membrane-binding protein 7 (CPNE7) has been observed in various cancers. To facilitate functional studies, this study aims to generate a mouse-derived antiserum capable of specifically recognizing both major splice variants, CPNE7-FL and CPNE7-S. Methods Conserved sequences shared among CPNE7 isoforms were identified by bioinformatical analysis, and synthetic peptides were designed and used to immunize eight BALB/c mice to prepare antiserum. The antiserum titer was determined by indirect ELISA. Subsequently, a prokaryotic expression vector for CPNE7 was constructed and an A549 overexpression system was established, and the specificity of the antiserum was verified by Western blot. Results A high-titer antiserum with a dilution of up to 1:32000 was successfully obtained. Western blot results showed that the antiserum effectively recognized recombinant GST-CPNE7 fusion proteins in E. coli and detected the overexpressed CPNE7-FL and CPNE7-S isoforms in mammalian cells. Conclusion A high-titer and high-specificity mouse antiserum against human CPNE7 was successfully prepared. This antiserum provides a valuable tool for future studies on the expression patterns and biological functions of CPNE7 isoforms, laying a foundation for exploring their potential roles in cancer diagnosis and therapy.

关键词

钙依赖性膜结合蛋白7 / 可变剪接 / 抗血清 / 抗体制备

Key words

calcium-dependent membrane-binding protein 7 / alternative splicing / antiserum / antibody production

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基金资助

广西自然科学基金重点项目(2018GXNSFDA050009)