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

Abstract

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

CLC Number:

S827.3

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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Construction and Effect of Two CREBZF Isoforms Overexpression Vectors on Goat Endometrial Epithelial Cells Apoptosis

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