新西兰白兔BMP15基因的真核表达载体构建、表达模式及其在卵巢组织的表达

doi:10.11843/j.issn.0366-6964.2024.02.014

摘要/Abstract

摘要： 旨在获得新西兰白兔 BMP15基因序列及其组织表达规律,构建其真核表达载体,预测BMP15的生物学功能。本研究选择180日龄性成熟的健康新西兰白兔作为研究对象,采用 RT-PCR 技术扩增BMP15 基因CDS区序列,利用T4连接方法将目的片段连接至线性化的Pmcherry-N1和pCMV-Myc空载体,构建真核表达载体,利用生物信息学分析其编码蛋白的性质及结构。采用实时荧光定量PCR (qRT-PCR) 技术与Western blotting技术检测pCMV-Myc-BMP15过表达情况。接下来,使用qRT-PCR检测BMP15 基因在不同组织中的表达水平。激光共聚焦方法检测BMP15基因的亚细胞定位。利用免疫荧光技术检测内源BMP15在新西兰白兔卵巢组织定位情况。结果表明:克隆得到新西兰白兔BMP15基因CDS区序列长1 182 bp , PCR及测序结果表明pCMV-Myc-BMP15和 Pmcherry-N1-BMP15真核表达载体构建成功。生物信息学分析显示,BMP15基因编码393个氨基酸;BMP15蛋白不稳定系数为55.32,等电点大小为9.69 ,是一种稳定的碱性蛋白质。BMP15蛋白共有26个磷酸化位点,15个糖基化位点,存在信号肽,不存在跨膜结构域。系统进化树表明,新西兰白兔与猪亲缘关系最近,与鸡的亲缘关系最远。二级结构和三级结构分析结果表明,BMP15蛋白主要由α-螺旋(38.68%)、无规则卷曲(37.4%)、延伸链(15.52%)、β-转角(8.40%)组成, 为混合型蛋白。蛋白互作关系预测发现,BMP15蛋白与FSHR、FIGLA、BMPR1B、AMHR2、NOBOX等卵巢生长发育的相关蛋白之间存在相互作用。不同组织表达分析显示,BMP15 基因在卵巢组织中特异性表达;激光共聚焦结果显示,BMP15 主要存在于在细胞质中。将pCMV-Myc-BMP15转染至HEK293T细胞内,BMP15的mRNA水平和蛋白水平均显著上调。卵巢组织免疫荧光检测结果显示,BMP15主要定位在卵巢颗粒细胞的细胞质中。本研究成功构建了BMP15的真核表达载体,对BMP15基因及其编码的蛋白的理化性质和生物学特性进行了预测分析,在HEK293T细胞内成功过表达,并得到了该基因的亚细胞定位情况、组织表达情况及在卵巢组织的分布情况。为后续开展BMP15基因在卵巢生长发育中的功能及机制研究提供了理论依据。

关键词: 新西兰白兔, BMP15基因, 克隆, 组织表达, 细胞定位

Abstract: This study aimed to obtain the gene sequence and expression pattern of bone morphogenetic protein 15 (BMP15) in tissues of New Zealand white rabbits, construct its eukaryotic expression vector, and predict the biological function of BMP15. In this study, 180-day-old healthy New Zealand white rabbits were selected as the study subjects. The CDS region of BMP15 gene was amplified using RT-PCR, the target fragment was ligated to linearized Pmcherry-N1 and pCMV-Myc empty vectors through T4 ligation method, and the eukaryotic expression vector was constructed. The properties and structure of the encoded protein were analyzed by bioinformatics. The overexpression of pCMV-Myc-BMP15 was detected by real-time fluorescence quantitative PCR ( qRT-PCR ) and Western blotting. Subsequently, qRT-PCR was employed to detect the expression level of the BMP15 gene in different tissues. The subcellular localization of BMP15 gene was detected by laser confocal method. The localization of endogenous BMP15 in ovarian tissue of New Zealand white rabbits was determined using immunofluorescence technique. The results revealed that the CDS sequence of BMP15 gene in New Zealand white rabbit was 1 182 bp. PCR and sequencing results confirmed the successful construction of the pCMV-Myc-BMP15 and Pmcherry-N1-BMP15 eukaryotic expression vectors. Bioinformatics analysis indicated that BMP15 gene encoded 393 amino acids. The protein exhibited an instability coefficient of 55.32, with an isoelectric point of 9.69, suggesting stability as a basic protein. BMP15 possessed 26 phosphorylation sites, 15 glycosylation sites, a signal peptides, and lacked transmembrane domains. Phylogenetic analysis revealed that New Zealand white rabbit had a close relationship with Sus Scrofa and a distant relationship with Gallus. Secondary and tertiary structure analysis indicated that BMP15 was a hybrid protein, primarily composed of α-helices, irregular coils, extended strands, and β-turns. Protein interaction prediction suggested that BMP15 protein had interactions with FSHR, FIGLA, BMPR1B, AMHR2, and NOBOX proteins related to ovarian growth and development. Tissue expression analysis demonstrated specific expression in ovarian tissues. Confocal laser scanning revealed cytoplasmic expression of BMP15. Transfection of pCMV-Myc-BMP15 into 293T cells resulted in significant upregulation at both mRNA and protein levels. Immunofluorescence detection of ovarian tissue confirmed cytoplasmic localization of BMP15 in granulosa cells. In this study, the eukaryotic expression vector of BMP15 was successfully constructed, and the physical and chemical properties, as well as biological characteristics of BMP15 gene and its encoded protein, were predicted and analyzed. BMP15 gene overexpression was successfully achieved in HEK293T cells, and information regarding subcellular localization, tissue expression, and distribution in ovarian tissue was obtained. These findings provide a theoretical basis for the subsequent studies on the function and mechanism of BMP15 gene in ovarian growth and development.

Key words: New Zealand white rabbit, BMP15 gene, cloning, tissue expression, cell localization

中图分类号:

S829.1

陈孟娟, 刘雨晴, 王智通, 温佳乐, 许会芬, 于光晴, 李明. 新西兰白兔BMP15基因的真核表达载体构建、表达模式及其在卵巢组织的表达[J]. 畜牧兽医学报, 2024, 55(2): 562-575.

CHEN Mengjuan, LIU Yuqing, WANG Zhitong, WEN Jiale, XU Huifen, YU Guangqing, LI Ming. Construction of Eukaryotic Expression Vector, Expression Pattern of BMP15 Gene, and Its Expression in Ovary of New Zealand White Rabbit[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 562-575.

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