PLC-γ1的多克隆抗体制备及对绵羊早期胚胎发育作用的初步研究

doi:10.11843/j.issn.0366-6964.2022.11.011

Abstract

Abstract: The study aimed to construct PLC-γ1 eukaryotic overexpression vector and preparing its polyclonal antibody, and provide basic data for exploring the effect of sheep PLC-γ1 gene on oocyte activation and early embryonic development. In this study, the Puc57-P2A-Flag-PLC-γ1 strain preserved in the laboratory was used as a template to design primers and introduce Hind III and Age I restriction sites. PCR was used to amplify and purify the P2A-Flag-PLC-γ1 gene. It was connected into pcDNA3.1-EGFP vector for transformation, and the plasmid was extracted by bacterial liquid PCR, double restriction digestion and sequencing identification. Then HEK-293T cells were transfected with the constructed recombinant plasmid for 24 h, and were divided into the control group, no-load group and PLC-γ1 overexpression group. The cell fluorescence was observed under a microscope, and the expression of PLC-γ1 in the transfected cells was detected by real-time quantitative PCR (qPCR) and Western blot (WB). PLC-γ1 protein was purified by Anti-Flag immunomagnetic beads; Polyclonal antibodies were prepared from 10-month-old New Zealand white rabbits (healthy condition, about 50 kg, n=2). The titer of polyclonal antibodies was determined by indirect ELISA, and its specificity was identified by WB. The oocytes were parthenogenetically activated by microinjection of recombinant plasmids and ionomycin (Ion) combined with 6-DMAP. The expression of PLC-γ1 was detected by qPCR and WB at different stages (2 cell stage, 4 cell stage, 8 cell stage and the morula stage) after cleavage in early embryo cells of sheep. The results showed that eukaryotic overexpression vector was successfully constructed. After transfection 24 h, the expression level of PLC-γ1 in overexpression group was significantly increased compared with blank control and negative control (P<0.01). WB results showed that the PLC-γ1 protein was successfully obtained and purified, for 149 ku. The PLC-γ1 polyclonal antibody titer was 1:12 800, and it could react with PLC-γ1 protein. The recombinant plasmid was injected into the cytoplasm of sheep MⅡ oocytes, and the results of qPCR and WB showed that PLC-γ1 was expressed in all stages of early embryo development of sheep. In conclusion, in this study, PLC-γ1 eukaryotic overexpression vector was successfully constructed and polyclonal antibody was prepared. The recombinant plasmid was injected into oocytes by the microinjection technique and realized its expression. It proved that PLC-γ1 was expressed in all stages of early embryo development of sheep and has the potential as a new oocyte activator. It not only provides a scientific basis for exploring the effects of PLC-γ1 gene on the activation of sheep oocytes and early embryo development, but also lays a foundation for further improving the reproductive performance of sheep.

Key words: PLC-γ1, eukaryotic overexpression, protein purification, polyclonal antibody, early embryo

CLC Number:

S826.3

LIU Xinjie, WU Xiaoxue, LIU Suping, YUAN Liming, CHEN Ning, SAIWU Jiafu. Preparation of Polyclonal Antibody against PLC-γ1 and Preliminary Study on Its Effect on Early Embryo Development in Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3842-3855.

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Preparation of Polyclonal Antibody against PLC-γ1 and Preliminary Study on Its Effect on Early Embryo Development in Sheep

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