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

摘要/Abstract

摘要： 旨在克隆牦牛微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B, LC3B)基因,制备牦牛LC3B多克隆抗体,为探究自噬在牦牛生殖生理过程中的作用奠定基础。本研究通过基因克隆方法获得牦牛LC3B基因序列,利用原核表达、亲和层析法纯化牦牛LC3B重组蛋白,使用纯化的蛋白为抗原免疫10月龄日本大耳白兔,Sepharose4B柱层析法纯化制备牦牛LC3B多克隆抗体,利用酶联免疫吸附试验(ELISA)测定抗体效价,并使用蛋白质免疫印迹(WB)、免疫组织化学法(IHC)和免疫荧光技术(IF)检测牦牛生殖器官中LC3B蛋白的表达。结果显示,本研究成功克隆了牦牛LC3B基因(登录号:OP572227),构建的重组质粒pET-32a-LC3B能够诱导产生重组蛋白,并且在包涵体和上清中均有表达;所纯化的牦牛LC3B重组蛋白大小为34 ku(含Trx和His标签),符合预期结果。通过免疫日本大耳兔后得到的兔抗牦牛LC3B蛋白血清,抗体效价分别为1∶640 000和1∶320 000,特异性良好,表明牦牛LC3B蛋白多克隆抗体制备成功。纯化后的抗体应用于牦牛生殖器官中LC3B的表达检测,LC3B在牦牛卵巢、输卵管和子宫中表达,并且能识别LC3B-Ⅰ和发生脂化后的LC3B-Ⅱ,主要表达于细胞质及细胞核周。本研究通过成功克隆牦牛LC3B基因制备了牦牛LC3B多克隆抗体,使用抗体检测发现LC3B蛋白在牦牛生殖器官中均有表达,表明所制备的多克隆抗体特异性良好,能够应用于牦牛LC3B蛋白的检测和细胞自噬水平的研究。

关键词: 牦牛, LC3B, 原核表达, 多克隆抗体, 生殖

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

中图分类号:

S823.85

焦正兴, 潘阳阳, 王萌, 王靖雷, 马文斌, 高翔, 张晖, 崔燕, 余四九, 王立斌. 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[J]. 畜牧兽医学报, 2023, 54(6): 2436-2447.

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.

参考文献 32

[1]	DERETIC V.Autophagy in inflammation,infection,and immunometabolism[J].Immunity,2021,54(3):437-453.
[2]	KUMA A,KOMATSU M,MIZUSHIMA N.Autophagy-monitoring and autophagy-deficient mice[J].Autophagy, 2017,13(10): 1619-1628.
[3]	MARUYAMA T,NODA N N.Autophagy-regulating protease Atg4:structure,function,regulation and inhibition[J].J Antibiot,2018,71(1):72-78.
[4]	HWANG H J,HA H,LEE B S,et al.LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy[J].Nat Commun,2022,13(1):1436.
[5]	YOSHII S R,MIZUSHIMA N.Monitoring and measuring autophagy[J].Int J Mol Sci,2017,18(9):1865.
[6]	GAO H,KHAWAR M B,LI W.Essential role of autophagy in resource allocation during sexual reproduction[J]. Autophagy,2020,16(1):18-27.
[7]	GAO F Y,LI G P,LIU C,et al.Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells[J].J Cell Biol,2018,217(6):2103-2119.
[8]	SUN Y C,WANG Y Y,SUN X F,et al.The role of autophagy during murine primordial follicle assembly[J].Aging (Albany NY),2018,10(2):197-211.
[9]	MA L Z,TANG X R,GUO S,et al.miRNA-21-3p targeting of FGF2 suppresses autophagy of bovine ovarian granulosa cells through AKT/mTOR pathway[J].Theriogenology,2020,157:226-237.
[10]	O’DONNELL L,SMITH L B,REBOURCET D.Sertoli cells as key drivers of testis function[J].Semin Cell Dev Biol,2022, 121:2-9.
[11]	LIU C,WANG H N,SHANG Y L,et al.Autophagy is required for ectoplasmic specialization assembly in sertoli cells[J]. Autophagy,2016,12(5):814-832.
[12]	WANG H N,WAN H F,LI X X,et al.Atg7 is required for acrosome biogenesis during spermatogenesis in mice[J].Cell Res,2014,24(7):852-869.
[13]	GAWRILUK T R,KO C M,HONG X M,et al.Beclin-1 deficiency in the murine ovary results in the reduction of progesterone production to promote preterm labor[J].Proc Natl Acad Sci U S A,2014,111(40):E4194-E4203.
[14]	ZHOU Z H,WANG H Y,ZHANG X W,et al.Defective autophagy contributes to endometrial epithelial-mesenchymal transition in intrauterine adhesions[J].Autophagy,2022,18(10):2427-2442.
[15]	SU Y,ZHANG J J,HE J L,et al.Endometrial autophagy is essential for embryo implantation during early pregnancy[J].J Mol Med,2020,98(4):555-567.
[16]	OESTREICH A K,CHADCHAN S B,POPLI P,et al.The autophagy gene Atg16L1 is necessary for endometrial decidualization[J]. Endocrinology,2020,161(1):bqz039.
[17]	LIU W W,CHEN M,LIU C,et al.Epg5 deficiency leads to primary ovarian insufficiency due to WT1 accumulation in mouse granulosa cells[J].Autophagy,2023,19(2):644-659.
[18]	TANG Z H,ZHANG Z H,LIN Q Q,et al.HIF-1α/BNIP3-mediated autophagy contributes to the luteinization of granulosa cells during the formation of corpus luteum[J].Front Cell Dev Biol,2021,8:619924.
[19]	TSUKAMOTO S,KUMA A,MURAKAMI M,et al.Autophagy is essential for preimplantation development of mouse embryos[J].Science,2008,321(5885):117-120.
[20]	BAEKEN M W,WECKMANN K,DIEFENTHÄLER P,et al.Novel insights into the cellular localization and regulation of the autophagosomal proteins LC3A,LC3B and LC3C[J].Cells,2020,9(10):2315.
[21]	WANG W,CHEN Z X,BILLIAR T R,et al.The carboxyl-terminal amino acids render pro-human LC3B migration similar to lipidated LC3B in SDS-PAGE[J].PLoS One,2013,8(9):e74222.
[22]	NIETO-TORRES J L,ENCALADA S E,HANSEN M.LC3B phosphorylation:autophagosome’s ticket for a ride toward the cell nucleus[J].Autophagy,2021,17(10):3266-3268.
[23]	于慧敏,吴鹏杰,李南南,等.中华蜜蜂GABARAP基因克隆、生物信息学分析及原核表达载体的构建[J].中国畜牧兽医, 2022,49(1):60-69.YU H M,WU P J,LI N N,et al.Cloning,bioinformatics analysis and prokaryotic expression of GABARAP gene in Apis cerana cerana[J].China Animal Husbandry & Veterinary Medicine,2022,49(1):60-69.(in Chinese)
[24]	张加姿,李开心,于宝佳,等.小麦自噬相关因子ATG5的原核表达和抗血清制备[J].生物技术通报,2017,33(7):69-74.ZHANG J Z,LI K X,YU B J,et al.Prokaryotic expression of wheat autophagy-related factor ATG5 and preparation of its antiserum in rabbits[J].Biotechnology Bulletin,2017,33(7):69-74.(in Chinese)
[25]	余振兴,朱倩,姚翠鸾.紫贻贝LC3B蛋白的原核表达及抗血清制备[J].水产学报,2017,41(4):498-505.YU Z X,ZHU Q,YAO C L.Recombinant expression and polyclonal antibody preparation of Mytilus galloprovincialis LC3B[J].Journal of Fisheries of China,2017,41(4):498-505.(in Chinese)
[26]	崔海英.DGAT1调控自噬流影响前列腺癌生长的作用及机制研究[D].长春:吉林大学,2022.CUI H Y.The effects and mechanisms of DGAT1 on the growth of prostate cancer by regulating autophagy flux[D].Changchun:Jilin University,2022.(in Chinese)
[27]	LUCAS-HERALD A K,MITCHELL R T.Testicular sertoli cell hormones in differences in sex development[J].Front Endocrinol, 2022,13:919670.
[28]	YIN J,NI B,YANG Y D,et al.Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes[J].Reproduction,2018,156(6):545-558.
[29]	梁丙坤,肖婷婷,陈美洁,等.LC3B和JAK2/STAT3通路分子在山羊卵巢黄体中的表达研究[J].畜牧与兽医,2022,54(5):14-18.LING B K,XIAO T T,CHEN M J,et al.Expression of LC3B and JAK2/STAT3 pathway molecules in the ovarian corpus luteum of goats[J].Animal Husbandry &Veterinary Medicine,2022,54(5):14-18.(in Chinese)
[30]	ULLAH S,ZHANG M D,YU H,et al.Heat exposure affected the reproductive performance of pregnant mice:Enhancement of autophagy and alteration of subcellular structure in the corpus luteum[J].Reprod Biol,2019,19(3):261-269.
[31]	周吉隆.let-7g调控卵泡颗粒细胞凋亡与自噬及卵泡发育过程中自噬的机制研究[D].南京:南京农业大学,2017.ZHOU J L.The regulation of let-7g in ovarian granulosa cell apoptosis and autophagy and regulatory mechanisms during follicular development[D].Nanjing:Nanjing Agricultural University,2017.(in Chinese)
[32]	ZHENG Y X,MA L Z,LIU N,et al.Autophagy and apoptosis of porcine ovarian granulosa cells during follicular development[J].Animals,2019,9(12):1111.