牦牛LC3B蛋白多克隆抗体制备及在生殖器官表达检测中的应用

doi:10.11843/j.issn.0366-6964.2023.06.022

Abstract

Abstract: The purpose of this study was to clone the microtubule associated protein 1 light chain 3B (LC3B) gene of yak and prepare the polyclonal antibody of yak LC3B, so as to lay a foundation for exploring the role of autophagy in the reproductive physiology of yak. The sequence of yak LC3B gene was obtained by gene cloning. The recombinant protein of yak LC3B was purified by prokaryotic expression and affinity chromatography. The purified protein was used as antigen to immunize 10-month-old Japanese big-ear white rabbits. The polyclonal antibody against LC3B was purified and prepared by Sepharose 4B column chromatography, and the antibody titer was determined by enzyme linked immunosorbent assay (ELISA). Western blot (WB), immunohistochemistry (IHC) and immunofluorescence (IF) were used to detect the expressions of LC3B protein in yak reproductive organs. The results showed that the yak LC3B gene (accession number:OP572227) was successfully cloned. The constructed recombinant plasmid pET-32a-LC3B induced the production of recombinant protein and expressed in inclusion bodies and supernatant. The size of purified yak LC3B recombinant protein was 34 ku (containing Trx and His tags). The rabbit anti-yak LC3B protein serum obtained by immunizing Japanese big-ear white rabbits had antibody titers of 1∶640 000 and 1∶320 000, respectively, with good specificity, which showed that the polyclonal antibody of yak LC3B protein was successfully prepared. The purified antibody was used to detect the expressions of LC3B in the reproductive organs of yaks. It showed that LC3B was expressed in the ovaries, oviducts and uterus of the yak, and recognized LC3B-Ⅰ and LC3B-Ⅱ after lipidation, it was located mainly in the cytoplasm and perinuclear. In conclusion, LC3B polyclonal antibody was prepared successfully by cloning LC3B gene in this study, and the expression of LC3B protein was detected in the reproductive organs. It is stated that the antibody prepared has good specificity and it can be applied to detect LC3B protein in yaks and further study on the levels of autophagy.

Key words: yak, LC3B, prokaryotic expression, polyclonal antibody, reproduction

CLC Number:

S823.85

JIAO Zhengxing, PAN Yangyang, WANG Meng, WANG Jinglei, MA Wenbin, GAO Xiang, ZHANG Hui, CUI Yan, YU Sijiu, WANG Libin. Preparation of Polyclonal Antibody against Yak LC3B Protein and Its Application in Detection of Expression in Reproductive Organs[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2436-2447.

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Preparation of Polyclonal Antibody against Yak LC3B Protein and Its Application in Detection of Expression in Reproductive Organs

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