山羊CREBZF蛋白两种亚型过表达载体的构建及其对子宫内膜上皮细胞凋亡的影响

doi:10.11843/j.issn.0366-6964.2022.01.013

摘要/Abstract

摘要： 旨在构建编码山羊CREBZF蛋白2种亚型（长亚型SMILE和短亚型Zhangfei）基因的过表达载体，并初步探究CREBZF对山羊子宫内膜上皮细胞（gEECs）凋亡的影响。本研究针对编码山羊CREBZF不同亚型的基因CDS区分别设计引物，分段进行PCR扩增；运用无缝克隆技术将所获得各目的基因片段与线性化pcDNA3.1（+）载体连接，构建pcDNA3.1（+）-SMILE和pcDNA3.1（+）-Zhangfei重组过表达载体，通过双酶切和测序进行鉴定；转染过表达载体至gEECs，提取总RNA和蛋白，通过qRT-PCR和Western blot验证过表达效率；应用流式细胞术检测过表达SMILE和Zhangfei对gEECs凋亡率的影响。PCR、双酶切显示各目的条带大小正确；测序显示目的片段与NCBI基因库中山羊CREBZF的预测序列一致性为100%；转染过表达载体后，gEECs中SMILE与Zhangfei在转录和翻译水平的表达量均显著增加，并发现SMILE可能在翻译后自身被修饰；空载体组细胞凋亡率为8.45%；转染pcDNA3.1（+）-SMILE组凋亡率显著上升至12.41%（P<0.01）；转染pcDNA3.1（+）-Zhangfei组凋亡率上升至9.83%，但相较于空载体组统计学差异不显著；结果提示，CREBZF可能调控gEECs的凋亡进程。本研究将目的基因分段克隆与无缝克隆技术联用，构建了上述2种过表达载体，为进一步探究CREBZF在反刍动物子宫内膜上皮细胞周期性变化中的作用奠定基础。

关键词: CREBZF, 过表达载体, 无缝克隆, 山羊子宫内膜上皮细胞, 凋亡

Abstract: This study aimed to construct the overexpression vectors encoding two isoforms of goat CREBZF protein (long isoform SMILE and short isoform Zhangfei), and to preliminarily explore the apoptotic effect of CREBZF on goat endometrial epithelial cells (gEECs). In this study, pairs of primers were designed for the CDS region encoding two isoforms of goat CREBZF protein， and the gene fragments were amplified by PCR; Target gene fragments were connected to linear pcDNA3.1(+) by seamless cloning to construct the recombinant overexpression vectors of pcDNA3.1(+)-SMILE and pcDNA3.1(+)-Zhangfei which were identified by double enzyme digestion and sequencing; The overexpression vectors were transfected into gEECs, then total RNA and protein were extracted. Overexpression efficiency was determined by qRT-PCR and Western blot; Flow cytometry was used to investigate the effect of SMILE and Zhangfei overexpression on apoptotic rates of gEECs. PCR and double enzyme digestion results showed that the target bands were in the correct locations;Sequencing results showed that the target gene fragments were 100% consistent with the predicted CREBZF sequence in NCBI GenBank. The results of qRT-PCR and Western blot showed that two isoforms of CREBZF were overexpressed significantly in transcription and translation levels, and SMILE may be modified after translation on gEECs. The apoptotic rate of empty vector group was 8.45%; The apoptotic rate of pcDNA3.1(+)-SMILE group significantly increased to 12.41% (P<0.01); The apoptotic rate of pcDNA3.1(+)-Zhangfei group increased to 9.83%, however, there was no significant statistical difference compared with the empty vector group. It is suggested that CREBZF may regulate the apoptotic process of gEECs. This study combines the segmented cloning of target gene with seamless cloning to construct the above two overexpression vectors, and lays a foundation for further research on the role of CREBZF in ruminant embryo implantation.

Key words: CREBZF, overexpression vector, seamless cloning, goat endometrial epithelial cells, apoptosis

中图分类号:

S827.3

牛宏宇, 张瑞雪, 齐茂振, 马玉洁, 曲栓, 靳亚平, 林鹏飞. 山羊CREBZF蛋白两种亚型过表达载体的构建及其对子宫内膜上皮细胞凋亡的影响[J]. 畜牧兽医学报, 2022, 53(1): 132-140.

NIU Hongyu, ZHANG Ruixue, QI Maozhen, MA Yujie, QU Shuan, JIN Yaping, LIN Pengfei. Construction and Effect of Two CREBZF Isoforms Overexpression Vectors on Goat Endometrial Epithelial Cells Apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 132-140.

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