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

摘要/Abstract

摘要： 旨在通过构建PLC-γ1真核过表达载体并制备其多克隆抗体，为探索绵羊PLC-γ1基因对卵母细胞激活作用及早期胚胎发育的影响提供基础数据。本研究以实验室保存的Puc57-P2A-Flag-PLC-γ1菌株为模板设计引物并引入Hind Ⅲ、Age Ⅰ酶切位点，利用PCR扩增并纯化P2A-Flag-PLC-γ1基因，将其连入pcDNA3.1-EGFP载体，进行转化，通过菌液PCR、双酶切及测序鉴定后提取质粒。随后将构建好的重组质粒转染HEK-293T细胞24 h，分为control组、空载组和PLC-γ1过表达组，通过显微镜观察细胞荧光，实时荧光定量PCR （qPCR）及Western blot （WB）检测转染后细胞中PLC-γ1的表达情况；Anti-Flag免疫磁珠纯化PLC-γ1蛋白；以10月龄的新西兰大白兔（健康状态良好，体重约50 kg，n=2）为对象制备多克隆抗体，间接ELISA法测定多克隆抗体效价，WB鉴定其特异性。通过显微注射重组质粒及离子霉素（Ion）结合6-DMAP孤雌激活卵母细胞，qPCR及WB检测PLC-γ1在卵裂后不同时期（2细胞期、4细胞期、8细胞期、桑椹胚期）的绵羊早期胚胎细胞中的表达情况。结果显示，成功构建真核过表达载体；转染24 h后，与空白对照及阴性对照相比，过表达组PLC-γ1表达量明显升高（P<0.01）；WB结果显示成功获得并纯化PLC-γ1蛋白，大小为149 ku；测得PLC-γ1多克隆抗体效价为1：12 800，并可与PLC-γ1蛋白发生免疫反应。将重组质粒注射到绵羊MⅡ期卵母细胞胞质中，qPCR及WB结果显示PLC-γ1在绵羊早期胚胎发育各时期均有表达。综上所述，本研究成功构建了PLC-γ1真核过表达载体并制备了其多克隆抗体，通过显微注射技术将重组质粒注入卵母细胞并实现表达，证明PLC-γ1在绵羊早期胚胎发育各时期均有表达，且具有作为新的卵母细胞激活因子的潜能。既为探索绵羊PLC-γ1基因对卵母细胞激活作用及早期胚胎发育的影响提供了科学依据，也为进一步提升绵羊的繁殖性能奠定基础。

关键词: PLC-γ1, 真核过表达, 蛋白纯化, 多克隆抗体, 早期胚胎

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

中图分类号:

S826.3

刘欣杰, 吴晓雪, 刘素平, 袁利明, 陈宁, 赛务加甫. 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[J]. 畜牧兽医学报, 2022, 53(11): 3842-3855.

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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