双峰驼Cyp-A的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.07.029

Abstract

Abstract: 【Objective】 The polyclonal antibody of cyclophilin A (Cyp-A) was prepared, in order to provide a basis for revealing the mechanism of mucosal immune response in Bactrian camel.【Method】 The coding region was selected from the Cyp-A gene sequence of Bactrian camel in GenBank (accession No.:XM_010969543.1).The extracellular sequence without signal peptide was intercepted and optimized its base, and the recombinant plasmid pET-28a-Cyp-A was constructed by connecting it with pET-28a (+) vector.The recombinant plasmid pET-28a-Cyp-A was transformed into E.coli BL21 (DE3) competent cell to induce expression.After optimizing the induction conditions, the expression of the recombinant protein was identified by SDS-PAGE.Then the recombinant protein was purified and used to immunize rabbits to prepare rabbit anti-Bactrian camel Cyp-A polyclonal antibody.The antibody titer and specificity were detected by ELISA and Western blotting, respectively.HE and SABC immunohistochemical staining methods were used to observe the location of Cyp-A using self-made polyclonal antibody.【Result】 The recombinant plasmid pET-28a-Cyp-A was mainly expressed in the form of inclusion body.The size of Cyp-A protein was about 17 ku.The optimal induction conditions were 0.7 mmol/L IPTG and 8 h.The optimal elution concentration of the protein was 5 mmol/L imidazole.By ELISA and Western blotting detection, the titer of rabbit anti-Bactrian camel Cyp-A polyclonal antibody was 1:64 000, which could specifically recognize the recombinant protein Cyp-A.HE and SABC immunohistochemical staining results showed that Cyp-A polyclonal antibody could only specifically stain M cells in ileum follicular associated epithelium (FAE).【Conclusion】 The rabbit anti-Bactrian camel Cyp-A polyclonal antibody with high titer and specificity was successfully prepared, which could be used to specifically recognize M cells in ileum of Bactrian camel.

Key words: Bactrian camel; cyclophilin A (Cyp-A); prokaryotic expression; polyclonal antibody; location

CLC Number:

S824

SUO Nanji, LI Peixuan, LIU Zhuanxia, WANG Tingwei, LIU Kejiang, XIE Dongxu, ZENG Weiwei, WANG Wenhui. Prokaryotic Expression and Polyclonal Antibody Preparation of Cyp-A in Bactrian Camel[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2896-2905.

References

[1] KNOOP K A, KUMAR N, BUTLER B R, et al.RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium[J].Journal of Immunology, 2009, 183(9):5738-5747.
[2] WANG J, GUSTI V, SARASWATI A, et al.Convergent and divergent development among M cell lineages in mouse mucosal epithelium[J]. Journal of Immunology, 2011, 187(10):5277-5285.
[3] NAOKI K, AKITO TA, KENGO H, et al.Circadian expression of glycoprotein 2(Gp2) gene is controlled by a molecular clock in mouse Peyer's patches[J]. Genes Cells, 2020, 25:270-278.
[4] RAFAŁ K, MICHAŁ B, JULIANE S, et al.Salmonella adhesion of to pancreatic secretory granule membrane major glycoprotein GP2 of human and porcine origin depends on FimH sequence variation[J].Frontiers in Microbiology, 2018, 9:1905.
[5] TAMAS T, DAVID T, NORA S, et al.Loss of tolerance to gut immunity protein, glycoprotein 2(GP2) is associated with progressive disease course in primary sclerosing cholangitis[J].Scientific Reports, 2018, 8(1):399.
[6] SCOLARI F, IZZI C, GHIGGERI G M.Uromodulin:From monogenic to multifactorial diseases[J].Nephrology Dialysis Transplantation, 2015, 30(8):1250-1256.
[7] KIMURA S, NIO-KOBAYASHI J, KISHIMOTO A, et al.The broad distribution of GP2 in mucous glands and secretory products[J].Biomed Research, 2016, 37(6):351-358.
[8] RAJIV C, DAVIS T L.Structural and functional insights into human nuclear cyclophilins[J].Biomolecules, 2018, 8(4):161.
[9] ANANDAN V, THULASEEDHARAN T, SURESH K A, et al.Cyclophilin A impairs efferocytosis and accelerates atherosclerosis by overexpressing CD47 and down-regulating calreticulin[J].Cells, 2021, 10(12):3598.
[10] 刘丽萍, 火兴民, 谢飞, 等.双峰驼FCGRT基因的原核表达及多克隆抗体的制备[J].动物医学进展, 2021, 42(4):39-43. LIU L P, HUO X M, XIE F, et al.Prokaryotic expression of FCGRT gene of Bactrian camel and preparation of its polyconal antibodies[J].Progress in Veterinary Medicine, 2021, 42(4):39-43.(in Chinese)
[11] 李敏, 吴秀萍, 张旺东, 等.双峰驼IgM部分重链的原核表达及其多克隆抗体的制备[J].动物医学进展, 2020, 41(4):33-37. LI M, WU X P, ZHANG W D, et al.Prokaryotic expression of IgM heavy chain gene and polyclonal antibody preparation from Bactrian camel[J].Progress in Veterinary Medicine, 2020, 41(4):33-37.(in Chinese)
[12] 吴秀萍, 陆佳, 张亮, 等.双峰驼IgA基因的原核表达及抗体制备[J].动物医学进展, 2019, 40(12):30-34. WU X P, LU J, ZHANG L, et al.Prokaryotic expression and polyclonal antibody prepartion of Bactrian camel IgA gene[J].Progress in Veterinary Medicine, 2019, 40(12):30-34.(in Chinese)
[13] 张学锋, 李淑娴, 李航, 等.IgA和IgG分泌细胞在双峰驼CALT中的分布规律[J].中国兽医学报, 2016, 36(2):353-356. ZHANG X F, LI S X, LI H, et al.IgA and IgG secreting cells in CALT of Bactrian camel[J]. Chinese Journal of Veterinary Science, 2016, 36(2):353-356.(in Chinese)
[14] 刘晓霞, 曹婷婷, 李沛轩, 等.IgM分泌细胞在不同年龄家兔胃肠道黏膜中的分布[J].畜牧与兽医, 2021, 53(10):48-53. LIU X X, CAO T T, LI P X, et al.Distribution of IgM secreting cells in the gastrointestinal mucosa of rabbits at different ages[J].Animal Husbandry & Veterinary Medicine, 2021, 53(10):48-53.(in Chinese)
[15] 谢飞, 李淑娴, 火兴民, 等.CD8⁺ T细胞在不同年龄家兔胃肠道的分布规律[J].中国兽医学报, 2021, 41(2):279-283. XIE F, LI S X, HUO X M, et al.Distribution of CD8⁺ T cells in the astrointestinal tract of rabbits in different ages[J].Chinese Journal of Veterinary Science, 2021, 41(2):279-283.(in Chinese)
[16] 火兴民, 李淑贤, 王雯慧.年龄差异对家兔CD4⁺ T细胞在胃肠分布的影响[J].中国兽医学报, 2021, 41(2):273-278. HUO X M, LI S X, WANG W H.Effects of age difference on the distribution of CD4⁺ T cells in gastrointestinal of rabbits[J]. Chinese Journal of Veterinary Science, 2021, 41(2):273-278.(in Chinese)
[17] 李淑娴, 郭明刚, 张学锋, 等.CD11c+DC和CD103+DC及CD68+Mφ在双峰驼肠系膜淋巴结中的分布特点[J].中国兽医科学, 2015, 45(8):843-847. LI S X, GUO M G, ZHANG X F, et al.Distribution characteristics of CD11c+DC, CD103+Dc and CD68+Mφ in the mesenteric lymph nodes of Bactrian camel[J].Chinese Veterinary Science, 2015, 45(8):843-847.(in Chinese)
[18] 李沛轩, 刘晓霞, 刘丽萍, 等.双峰驼FcαR基因的原核表达、抗体制备及其在脾脏中的表达模式[J].农业生物技术学报, 2022, 30(10):1944-1953. LI P X, LIU X X, LIU L P, et al.Prokaryotic expression, antibody preparation of FcαR gene in Bactrin camel (Camelus bactrianus) and its expression pattern in spleen[J].Journal of Agricultural Biotechnology, 2022, 30(10):1944-1953.(in Chinese)
[19] 李东海, 张旺东, 程翠翠, 等.双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备[J].兽类学报, 2018, 38(5):486-490. LI D H, ZHANG W D, CHENG C C, et al.Isolation and purification of IgG subclasses in Bactrian camel serum and preparation of polyclonal antibodies against IgG subclasses[J].Acta Theriologica Since, 2018, 38(5):486-490.(in Chinese)
[20] GALIGNIANA M D, MORISHIMA Y, GALLAY P A, et al.Cyclophilin-A is bound through its peptidylprolyl isomerase domain to the cytoplasmic dynein motor protein complex[J].Journal of Biological Chemistry, 2004, 279(53):55754-55759.
[21] TRIVEDI D K, ANSARI M W, DUTTA T, et al.Molecular characterization of cyclophilin A-like protein from Piriformospora indica for its potential role to abiotic stress tolerance in E.coli[J].BMC Research Notes, 2013, 6:555.
[22] ARAI M, TSUKIYAMA-KOHARA K, TAKAGI A, et al.Resistance to cyclosporin A derives from mutations in Hepatitis C virus nonstructural proteins[J]. Biochemical and Biophysical Research Communications, 2014, 448:56-62.
[23] MAMATIS J E, PELLIZZARI-DELANO I E, GALLARDO-FLORES C E, et al.Emerging roles of cyclophilin A in regulating viral cloaking[J].Frontiers in Microbiology, 2022, 13:828078.
[24] HONDO T, SOMEYA S, NAGASAWA Y, et al.Cyclophilin A is a new M cell marker of bovine intestinal epithelium[J].Cell and Tissue Research, 2016, 364(3):585-597.
[25] REZZANI R, FAVERO G, STACCHIOTTI A, et al.Endothelial and vascular smooth muscle cell dysfunction mediated by cyclophylin A and the atheroprotective effects of melatonin[J].Life Sciences, 2013, 92:875-882.

Prokaryotic Expression and Polyclonal Antibody Preparation of Cyp-A in Bactrian Camel

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