Preparation of Bovine (Bos taurus) ARPC3 Polyclonal Antibody and Expression of ARPC3 in Mammary Tissue
BAI Wen-Kai1,2, LIU Pei-Wen1,2, CHEN Yan1,2, WANG An1,2, FANG Bo1,2, YANG Ting-Ji1,2, ZHANG Quan-Wei2,3, WANG Qi1,2, ZHAO Xing-Xu1,2, DONG Wei-Tao1,2*, ZHANG Yong1,2*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China; 3 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Actin associated protein 2/3 complex subunit 3 (ARPC3) is an important component of actin skeleton tissue, which plays an important role in bacterial invasion, inflammation development and apoptosis. To investigate the role of ARPC3 protein in bovine mastitis, in this study, rabbit polyclonal antibody against bovine (Bos taurus) ARPC3 was prepared and its activity was identified. The biological characteristics of bovine ARPC3 (NM 001034271.2) were analyzed by online software. pQE-80L-ARPC3 recombinant plasmid was constructed. The prokaryotic expression system was used to express ARPC3. The concentration of isopropyl-beta-D-thiogalactopyranoside (IPTG) and induction time were optimized. The expressed protein was purified by affinity chromatography, identified by SDS-PAGE and mass spectrometry, and then immunized New Zealand white rabbits (Oryctolagus cuniculus) to prepare polyclonal antibody. The titer of polyclonal antibody was detected by ELISA, and the specificity of polyclonal antibody was identified by Western blot. The results showed that the recombinant plasmid pQE-80L-ARPC3 was successfully constructed, and the recombinant protein (about 20.5 kD) was expressed in the form of inclusion bodies after IPTG induction . The optimal expression conditions were as follows: IPTG concentration was 1 mmol/L for 8 h. The titer of ARPC3 polyclonal antibody was 1∶128 000. Western blot identification of natural protein showed that ARPC3 polyclonal antiserum could specifically recognize ARPC3 in target protein and mammary tissue. Immunohistochemical (IHC) results showed that the polyclonal antibody could be effectively applied for the detection of bovine mastitis, and the expression of ARPC3 protein was positively correlated with the expression of related apoptotic proteins. In this study, the polyclonal antibody of ARPC3 with good specificity was successfully prepared, which can be used to the establishment and application of a new technology for rapid detection of bovine mastitis.
[1] 陈杨慧, 黎源, 王蓓. 2023. 胞内致病菌入侵宿主细胞分子机理研究进展[J]. 微生物学报, 63(08): 2994-3008.(Chen Y H, Li Y, Wang B.2023. Molecular mechanism of intracellular pathogenic bacteria invading host cells[J]. Acta Microbiologica Sinica, 63(08): 2994-3008.) [2] 程晓鸥. 2021. 金黄色葡萄球菌入侵奶牛乳腺上皮细胞并诱导异噬的机制[D]. 博士学位论文, 内蒙古大学, 导师: 王志钢. pp. 56-63.(Cheng X O.2021. Mechanism of Staphylococcus aureus invades bovine mammary epithelial cells and induces xenophagy[D]. Thesis for Ph.D., Inner Mongolia University, Suppervisor: Wang Z G, pp. 56-63.) [3] 郭志新. 2019. 无乳链球菌入侵奶牛乳腺上皮细胞的机制研究[D]. 博士学位论文, 内蒙古大学, 导师: 王志钢, pp. 76-79.(Guo Z X.2019. Study on the mechanism of Streptococcus agalactiae invasion of bovine mammary epithelial cells[D]. Thesis for Ph.D. Inner Mongolia University, Suppervisor: Wang Z G, pp. 76-79.) [4] 井明艳, 孙建义. 2004. 蛋白质的折叠调控与包涵体的形成[J]. 浙江大学学报(农业与生命科学版), (06): 690-696.(Jing M Y, Sun J Y.2004. Protein folding and the formation of inclusion bodies[J]. Journal of Zhejiang University (Agriculture and Life Sciences), (06): 690-696.) [5] 李健, 李庆章. 2007. 小鼠乳腺发育、泌乳和退化的组织形态学(Ⅰ): 一般组织形态学变化[J]. 东北农业大学学报, (02): 196-201.(Li J, Li Q Z.2007. Histomorphology of development, lactation and involution of mice mammary gland(Ⅰ): Changes of common histomorphology[J]. (02): 196-201.) [6] 刘佩雯, 董伟韬, 陈艳, 等. 2024. 奶牛结合珠蛋白生物信息学分析及其多克隆抗体的制备[J]. 中国兽医学报, 44(03): 558-564+594.(Liu P W, Dong W T, Chen Y, et al. 2023. Bioinformatic sanalysis of dairy cattle haptoglobin and its preparation of polyclonal antibody[J]. Chinese Journal of Veterinary Medicine, 44(03): 558-564+594.) [7] 沈霞芬, 卿素珠. 2015. 家畜组织学与胚胎学[M]. 中国农业出版社, 北京. pp. 57-58.(Shen X F, Qing S Z.2015. Livestock Histology and Embryology[M]. China Agricultural Press, Beijing. pp. 57-58.) [8] 王亭玮, 刘转霞, 陆佳, 等. 2023.双峰驼TLR5抗体制备及其在淋巴结中的表达[J]. 西北农林科技大学学报(自然科学版), 51(12): 1-8.(Wang T W, Liu Z X, Lu J, et al.2023. Preparation of antibody against TLR5 of bactrian camel and its expression in lymph nodes[J]. Journal of Northwest A&F University (Natural Science Edition), 51(12): 1-8.) [9] 张天琦. 2021. LPS处理奶牛乳腺上皮细胞后外泌体中差异蛋白的筛选与鉴定[D]. 硕士学位论文, 黑龙江八一农垦大学, 导师: 王建发, pp. 34-36.(Zhang T Q.2021. Screening and identification of differential proteins in exosomes of bovine mammary epithelial cells treated with LPS[D]. Thesis for M.S., Heilongjiang Bayi Agricultural University, Suppervisor: Wang J F, pp. 34-36.) [10] Bellinvia E, García-González J, Cifrová P, et al.2022. CRISPR-Cas9 Arabidopsis mutants of genes for ARPC1 and ARPC3 subunits of ARP2/3 complex reveal differential roles of complex subunits[J]. Scientific Reports, 12(1): 18205. [11] Campellone K G, Lebek N M, King V L.2023. Branching out in different directions: Emerging cellular functions for the Arp2/3 complex and WASP-family actin nucleation factors[J]. European Journal of Cell Biology, 102(2): 151301. [12] Dhanda A S, Vogl A W, Otey C A, et al.2018. Palladin functionally replaces the ARP2/3 complex and organizes actin-rich structures generated during Listeria monocytogenes infections[J]. The FASEB Journal, 32(S1): 519.2 [13] Dong W, Chen Y, Zhang Q, et al.2022. Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis[J]. Frontiers in Veterinary Science, 9: 984607. [14] Doolittle L K, Rosen M K, Padrick S B.2013. Purification of native Arp2/3 complex from bovine thymus[J]. Methods in Molecular Biology, 1046(3): 231-250. [15] Fäßler F, Dimchev G, Hodirnau V V, et al.2020. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction[J]. Nature Communications, 11(1): 6437. [16] Goley E D, Welch M D.2006. The ARP2/3 complex: An actin nucleator comes of age[J]. Nature Reviews Molecular Cell Biology, 7(10):713-726. [17] Hulse M L, Smith S, Chi E Y, et al.1993. Effect of type III group B streptococcal capsular polysaccharide on invasion of respiratory epithelial cells[J]. Infection and Immunity, 61(11): 4835-4841. [18] Insall R, Müller-Taubenberger A, Machesky L, 2001. et al. Dynamics of the dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis[J]. Cell Motility and the Cytoskeleton, 50(3): 115-128. [19] King V L, Campellone K G.2023. F-actin-rich territories coordinate apoptosome assembly and caspase activation during DNA damage-induced intrinsic apoptosis[J]. Molecular Biology of the Cell, 34(5): ar41. [20] King V L, Leclair N K, Coulter A M, et al.2021. The actin nucleation factors JMY and WHAMM enable a rapid ARP2/3 complex-mediated intrinsic pathway of apoptosis[J]. PLOS Genetics, 17(4): e1009512. [21] Li Q, Liu L J, Tu J C, et al.2021. The ARP2/3 complex, acting cooperatively with class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion[J]. The Plant Cell, 33(9): 3151-3175. [22] Mauricio R P M, Jeffries C M, Svergun D I, et al.2017. The Shigella virulence factor IcsA relieves N-WASP autoinhibition by displacing the verprolin homology/cofilin/acidic (VCA) domain[J]. Journal of Biological Chemistry, 292(1): 134-145. [23] Mullins R D, Stafford W F, Pollard T D.1997. Structure, subunit topology, and actin-binding activity of the ARP2/3 complex from Acanthamoeba[J]. The Journal of Cell Biology, 136(2): 331-343. [24] Pan M H, Xu R, Zhang Y, et al.2023. The impact of ARP2/3 complex inhibition on cytoskeleton dynamics and mitochondrial function during goat oocyte meiosis[J]. Animals, 13(2): 263. [25] Rauch L, Hennings K, Trasak C, et al.2016. Staphylococcus aureus recruits Cdc42GAP via recycling endosomes and exocyst to invade human endothelial cells[J]. Journal of Cell Science, 129(15): 2937-2949. [26] Ronzier E, Rotty J.2020. The role of ARP2/3 complex in inflammatory activation and TLR4 endocytosis[J]. Biophysical Journal, 118(3): 607a. [27] Sun G, Feng C, Guo J, et al.2019. The tomato ARP2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici[J]. Plant Cell and Environment, 42(9): 2664-2680. [28] Tahoun A, Jensen K, El-Sharkawy H, et al.2020. Inflammasome activation in bovine peripheral blood-derived macrophages is associated with actin rearrangement[J]. Animals, 10(4): 655-655. [29] Velle K B, Campellone K G.2018. Enteropathogenic E.coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance[J]. PLOS Pathogens, 14(12): e1007485.
