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| Construction of Duck (Anas platyrhynchos) Angiotensin-converting Enzyme 2 (ACE2) Eukaryotic Expression Vector and Screening of Stable Cell Line |
| JI Xiao-Xia, LI Shuai, CAO Xi-Yue, ZHANG Chong-Hao, ZHANG Yuan-Shu* |
| Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin angiotensinogen system (RAS) and a functional receptor of coronavirus such as SARS-CoV-2. To enrich the research content of ACE2 in ducks (Anas platyrhynchos) and explore the relationship between the ACE2 and avian coronavirus, the full-length coding sequence of the duck ACE2 gene was amplified by PCR, and the eukaryotic expression system was constructed by connecting homologous recombination with pcDNA3.1(+ ) vector. The optimal screening concentration of geneticin (G418) was determined for Chinese hamster ovary (CHO) cells, after transfecting of cells, selected monoclonal cell lines for G418. Western blot and immunofluorescence were used to identify the obtained cell lines. The cell line for expressing duck ACE2 protein was established, determined the enzyme activity of ACE2 recombinant protein. The results showed that the duck ACE2 gene was successfully cloned, and its coding region was 2 435 bp; the pcDNA3.1(+ ) -ACE2 plasmid was successfully constructed, and single enzyme digestion verified that a single band appeared at about 7 863 bp. The recombinant plasmid was successfully transfected into CHO cells by Lipofectamine, and exogenous ACE2 recombinant protein was expressed. Three monoclonal cell lines named pcDNA3.1(+ ) -ACE2-1, pcDNA3.1(+ ) -ACE2-2, pcDNA3.1(+ ) -ACE2-3 were successfully obtained, respectively. The recombinant ACE2 protein extracted from the monoclonal cell line showed high enzymatic activity. This study successfully constructed a duck ACE2 eukaryotic expression system and obtained 3 cell lines that can stably express ACE2 recombinant protein. This study provides a basis for the study of duck ACE2 protein and coronavirus and other biological functions and enriches the related theories of the protein.
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Received: 16 September 2021
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Corresponding Authors:
*zhangyuanshu@njau.edu.cn
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[1] 卜乐, 刘志民 . 2010. ACE2 与糖尿病及其并发症[J]. 国际内分泌代谢杂志 , (01): 61-63.
(Bu L, Liu ZM. 2010. ACE2 and diabetes and its complications[J]. Internation‐ al Journal of Endocrinology and Metabolism, (01): 61-63.)
[2] 郭亚琦, 王伟佳, 高智慧, 等 . 2021. 鸡脂肪细胞因子 NRG4 基因的克隆、表达及启动子分析[J]. 农业生物技术学报, 29(11): 2129-2138.
(Guo Y Q, Wang W J, Gao Z H, et al.Cloning, expression and promoter analysis of adi‐ pokine NRG4 gene in chicken[J]. Journal of Agricultural Biotechnology, 29(11): 2129-2138.)
[3] 蒋晓祎, 侯欣妤, 张波, 等 . 2020. ACE2-mCherry 融合基因表达载体的构建及功能验证[J]. 生物技术通讯, 31(06):675-680.
(Jiang X W, Hou X Y, Zhang B, et al.2020. Construction and functional verification of ACE2- mCherry fusion gene expression vector[J]. Bio technolo‐ gy News, 31(06): 675-680.)
[4] 王冲, 谭亚娣, 郭爱珍, 等 . 2005. 家猫 ACE2 基因克隆、测序及生物信息学分析[J]. 武汉大学学报( 理学版), (06):739-743.
(Wang C, Tan Y D, Guo A Z, et al.2005. Clon‐ ing, sequencing and bioinformatics analysis of domestic cat ACE2 gene[J]. Journal of Wuhan University (Natural Science Edition), (06): 739-743.)
[5] 王鹏, 殷晓航, 谢垚垚, 等 . 2021. 鸡 Chemerin 基因的真核表达载体构建与启动子转录效率分析[J]. 农业生物技术学报, 29(11): 2149-2158.
(Wang P, Yin X H, Xie Y Y, et al.2021. Eukaryotic expression vector construction and promoter transcription efficiency analysis of chicken (Gallus gallus) Chemerin gene[J]. Journal of Agricultural Biotechnology, 29(11): 2149-2158.)
[6] 肖航, 王换换, 王凯, 等 . 2019. 猪血管紧张素转化酶 2 重组蛋白诱导表达和纯化条件的优化[J]. 南京农业大学学报 , 42(02): 316-321.
(Xiao H, Wang H H, Wang K, et al.2019. The expression and optimization of purifying recombinant conditions of the recombinant ACE2 pro‐ tein of porcine[J]. Journal of Nanjing Agricultural Uni‐ versity, 42(02): 316-321.)
[7] 肖航, 王凯, 王换换, 等 . 2018. 真核表达质粒 pcDNA.3.1(+)- ACE2 的构建及其在 CHO 细胞中的表达[J]. 畜牧与兽医, 390(01): 54-58.
(Xiao H, Wang K, Wang H H, et al.2018. Construction of eukaryotic expression plasmid pcDNA.3.1(+)-ACE2 and its expression in CHO cells[J]. Animal Husbandry and Veterinary Medicine, 50(01): 54-58.)
[8] 谢旭东, 陈君柱, 王兴祥, 等 . 2005. 小鼠 ACE2 基因的克隆和体外表达以及序列分析与组织分布[J]. 浙江大学学报(医学版), (01): 51-57.
(Xie X D, Chen J Z, Wang X X, et al.2005. Cloning and in vitro expression, sequence analysis and tissue distribution of mouse ACE2 gene[J]. Journal of Zhejiang University (Health Sciences),(01):51-57).
[9] 杨维维, 许媛媛, 荣超, 等 . 2015. 山羊血管紧张素转换酶 2 基因的克隆表达与生物信息学分析[J]. 南京农业大学学报,38(01): 120-126.
(Yang W W, Xu Y Y, Rong C, et al.2015. Cloning, expression and bioinformatics analysis of goat angiotensin converting enzyme 2 gene[J]. Journal of Nanjing Agricultural University, 38(01): 120-126.)
[10] Dhara V G, Naik H M, Majewska N I, et al.2018. Recombi‐ nant antibody production in CHO and NS0 cells: differ‐ ences and similarities[J]. Bio Drugs Clinical Immuno‐ therapeutics Biopharmaceuticals & Gene Therapy, 32:571-584.
[11] Donoghue M, Hsieh F, Baronas E, et al.2000. A novel angio‐ tensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9[J]. Circulation Research, 87(5): e1-e9.
[12] Duan Y, Prasad R, Feng D, et al.2019. Bone marrow-derived cells restore functional integrity of the gut epithelial and vascular barriers in a model of diabetes and ACE2 defi‐ ciency[J]. Circulation Research, 125(11): 969-988.
[13] Fischer S, Handrick R, Otte K, et al.2015. The art of CHO cell engineering: A comprehensive retrospect and future perspectives[J]. Biotechnology Advances, 33: 1878-1896.
[14] Jia H.2016. Pulmonary angiotensin-converting enzyme 2 (ACE2) and inflammatory lung disease[J]. Shock, 46(3):239-248.
[15] Hoffmann M, Kleine-Weber H, Schroeder S, et al.2020. SARS-CoV-2 cell entry depends on ACE2 and TM‐ PRSS2 and is blocked by a clinically proven protease in‐ hibitor[J]. Cell, 181(2): 271-280.
[16] Guan W J, Ni Z Y, Hu Y, et al.2020. Clinical characteristics of coronavirus disease 2019 in China[J]. New England Journal of Medicine, 382(18): 1708-1720.
[17] Lan J, Ge J, Yu J, et al.2020. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 re‐ ceptor[J]. Nature, 581(7807): 215-220.
[18] Li W, Moore MJ, Vasilleva N, et al.2003. Angiotensin-con‐verting enzyme 2 is a functional receptor for the SARS coronavitus[J]. Nature, 426(6965): 450-454.
[19] Patel V B, Takawale A, Ramprasath T, et al.2015.Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2[J]. Journal of Molecular Medi‐ cine-JVM, 93(9): 1003-1013.
[20] Ren X Ff, Glende J, Falah M A, et al.2006. Analysis of ACE2 in polarized epithelial cells: Surface expression and function as receptor for severe acute respiratory syn‐ drome-associated coronavirus[J]. Journal of General Vi‐ rology, 87(6): 1691-1695.
[21] Wang K, Liu X Q, Xiao H, et al.2017. The correlation be‐ tween inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J]. International Immunopharmacology,46(Complete): 23-30.
[22] Xiao HL, Zhao L X, Yang J, et al.2018. Association between ACE2/ACE balance and pneumocyte apoptosis in a porcine model of acute pulmonary thromboembolism with cardiac arrest[J]. Molecular Medicine Reports, 17(3):4221-4228.
[23] Zhou P, Yang X L, Wang X G.2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin[J]. Nature, 579(7798): 270-273. |
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