Construction of fluorescent human zona pellucida fusion protein expression vectors and expression in CHO cells

Abstract

Objective: To construct expression vectors of the fluorescent fusion proteins of all four human zona pellucida (ZP) genes respectively, and explore their expression in Chinese hamster ovary(CHO) cells and oocytes in vitro. Methods: The desired fluorescent protein sequence was inserted into a specific site of the zona pellucida gene sequence using the Gibson Assembly multi-fragment one-step splicing method to form the desired fusion gene fragment, and both ends of the fragment contained a specific sticky cleavage site of the entry vector. The entry vector pENTR 1A (no ccdB) and the fusion gene fragment were digested with a specific double enzyme, and the target gene fragment was ligated into the entry vector by T4 DNA ligase. Transfer genes from entry vector into destination vector via the LR reaction of the Gateway cloning technology. The plasmids validated by DNA sequencing were amplified and transfected into CHO cells. The expression of desired fusion protein was proved by RT-PCR and Western Blot. The distribution of target proteins in cytoplasm of CHO cells was visualized by laser confocal microscopy. Results: The expression vectors were successfully constructed, and the expression of each ZP protein in CHO cells was detected by RT-PCR and Western blot. The laser confocal microscopy revealed the distribution of ZP proteins. Conclusions: In this study, we successfully constructed fluorescent fusion protein expression plasmids of human zona pellucida genes, enabling the diagnosis of infertility caused by oocyte abnormalities.

Keywords

• |zona pellucida gene|fluorescent fusion protein|expression plasmid|chinese hamster ovary cells