基于噬菌体展示技术筛选抑制素α亚基特异性纳米抗体

doi:10.16431/j.cnki.1671-7236.2023.05.030

摘要/Abstract

摘要： 【目的】通过噬菌体展示技术构建抗抑制素α亚基(INHα)纳米抗体文库,并筛选出针对绵羊INHα的特异性纳米抗体,以期制备出新型抑制素免疫制剂,为提高绵羊外周血促卵泡素(FSH)水平和排卵率做前期准备。【方法】利用pET32a-INHA原核表达载体诱导表达INHα蛋白,并对诱导表达的INHα蛋白进行SDS-PAGE和Western blotting鉴定;用Ni琼脂糖凝胶纯化后的INHα蛋白免疫新疆双峰驼。通过巢式PCR扩增制备骆驼噬菌体展示纳米抗体文库,通过三轮免疫亲和淘选及噬菌体ELISA从该文库中富集和筛选INHα特异性纳米抗体,并通过测序得知纳米抗体的基因序列;用蛋白-蛋白模拟对接初步鉴定该纳米抗体与INHα蛋白的亲和性。【结果】通过原核诱导表达及Ni琼脂糖凝胶纯化得到了大小为36 ku的INHα重组蛋白,通过Western blotting检测到约36 ku的蛋白条带;双峰驼通过6次INHα蛋白免疫抗体效价达到1∶1 024 000;通过巢式PCR获得了400 bp左右的VHH基因,通过电转化等试验构建了库容量为1.05×10¹²CFU的纳米抗体文库,文库阳性率为94%;通过三轮免疫亲和淘选获得3.0×10⁸ CFU的抗INHα纳米抗体文库,利用噬菌体ELISA获得了4株(VHH-4、VHH-29、VHH-89、VHH-108)INHα特异性纳米抗体,且4株INHα特异性纳米抗体基因序列相似度为80.31%;通过蛋白-蛋白模拟对接鉴定发现4株纳米抗体均与INHα具有较强的亲和性。此外,VHH-4与INHα的总自由能为-576.28 kJ/mol,是4种VHH中的最佳抗体。【结论】成功构建了抗INHα纳米抗体库,并从该库中获取了VHH-4、VHH-29、VHH-89和VHH-108 4株INHα特异性纳米抗体。本研究结果可为纳米抗体在生殖免疫学中的应用提供参考。

关键词: 抑制素α亚基(INHα); 纳米抗体; 噬菌体展示技术

Abstract: 【Objective】 The anti-inhibin α subunit (INHα) nanobody library was constructed by phage display technology,and the specific nanobodies against sheep INHα were screened, so as to prepare for the improvement of peripheral blood follicle-stimulating hormone (FSH) level and ovulation rate in sheep.【Method】 The expression of INHα protein was induced by the pET32a-INHA prokaryotic expression vector preserved in the laboratory,and the induced INHα protein was identified by SDS-PAGE and Western blotting.Then the purified INHα protein was used to immunize Xinjiang bactrian camel.The camel phage display nanobody library was prepared by nested PCR amplification.The specific nanobody against INHα was enriched and screened from the library by three rounds of immunoaffinity screening and phage ELISA.The gene sequence of the nanobody was obtained by sequencing. The affinity of the nanobody to INHα protein was preliminarily identified by protein-protein analog docking.【Result】 The results showed that the recombinant protein of INHα with a size of 36 ku was obtained by prokaryotic induction expression and Ni agarose purification,and a protein band of about 36 ku was detected by Western blotting.Bactrian camel immunized with INHα protein for 6 times and the antibody titer reached 1∶1 024 000.The VHH gene of about 400 bp was obtained by nested PCR,and a nanobody library with a capacity of 1.05×10¹²CFU was constructed by electrotransformation and other experiments.The positive rate of the library was 94%.The anti-INHα nanobody library of 3.0×10⁸ CFU was obtained by three rounds of immunoaffinity screening.Phage ELISA was used to obtain four strains of INHα-specific nanobodies (VHH-4,VHH-29,VHH-89 and VHH-108) and the gene sequence similarity of the four INHα-specific nanobodies was 80.31%.Through protein-protein simulation docking,it was found that all the four nanobodies had strong affinity with INHα.In addition,the total free energy of VHH-4 and INHα was -576.28 kJ/mol,which was the best antibody among the four VHH.【Conclusion】 An anti-INHα nanobody library was successfully constructed,and four INHα-specific nanobodies,VHH-4,VHH-29,VHH-89 and VHH-108,were obtained from the library.The results of this study could provide reference for the application of nanobodies in reproductive immunology.

Key words: inhibin α subunit (INHα); nanobody; phage display technology

中图分类号:

马继福, 阿德力·克坦, 解津刚, 巴合提·博代, 李振伟, 吾热力哈孜·哈孜汗. 基于噬菌体展示技术筛选抑制素α亚基特异性纳米抗体[J]. 中国畜牧兽医, 2023, 50(5): 2032-2043.

MA Jifu, Adil·Ketan, XIE Jingang, Bakhet·Bodai, LI Zhenwei, Oralhazi·Hazikhan. Screening of Nanobody Specific to Inhibin α Subunit Based on Phage Display Technology[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2032-2043.

参考文献

[1] MCCULLAGH D R.Dual endocrine activity of the testes[J].Science,1932,76(1957):19-20.
[2] MASON A J,HAYFLICK J S,LING N,et al.Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-β[J].Nature,1985,318(6047):659-663.
[3] VALE W,RIVIER C,HSUEH A,et al.Chemical and biological characterization of the inhibin family of protein hormones[J].Recent Progress in Hormone Research,1988,44:1-34.
[4] FINDLAY J K,CLARKE I J,ROBERTSON D M.Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrous cycle[J].Endocrinology,1990,126(1):528-535.
[5] ROSER J F,MCCUE P M,HOYE E.Inhibin activity in the mare and stallion[J].Domestic Animal Endocrinology,1994,11(1):87-100.
[6] KANEKO H,NAKANISHI Y,AKAGI S,et al.Immunoneutralization of inhibin and estradiol during the follicular phase of the estrous cycle in cows1[J].Biology of Reproduction,1995,53(4):931-939.
[7] NAMBO Y,KANEKO H,NAGATA S,et al.Effect of passive immunization against inhibin on FSH secretion,folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares[J].Theriogenology,1998,50(4):545-557.
[8] SHI F,MOCHIDA K,SUZUKI O,et al.Development of embryos in superovulated guinea pigs following active immunization against the inhibin alpha-subunit[J].Endocrine Journal,2000,47(4):451-459.
[9] SAMIR H,NAGAOKA K,WATANABE G.The stimulatory effect of subluteal progesterone environment on the superovulatory response of passive immunization against inhibin in goats[J].Theriogenology,2018,121:188-195.
[10] 丽娜·沙肯,巴合提·博代,杨华,等.抑制素α主动和被动免疫对哈萨克羊生殖激素含量的影响[J].中国畜牧兽医,2021,48(2):658-667. LINA S,BAHETI B,YANG H,et al.Effects of inhibinαon reproductive hormone content in kazakh sheep by active and passive immunity[J].Chineseanimal Husbandry&Veterinary Medicine,2021,48(2):658-667.(in Chinese)
[11] CATHERINE R,WYLIE V.Immunoneutralization of endogenous inhibin modifies hormone secretion and ovulation rate in the rat[J].Endocrinology,1989,1:152-157.
[12] MEDAN M S,AKAGI S,KANEKO H,et al.Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate[J].Reproduction,2004,128(4):475-482.
[13] ISHIGAME H,MEDAN M S,WATANABE G,et al.A new alternative method for superovulation using passive immunization against inhibin in adult rats1[J].Biology of Reproduction,2004,71(1):236-243.
[14] ZHAO A Z,TOHIDKIA M,SIEGEL D,et al.Phage antibody display libraries:A powerful antibody discovery platform for immunotherapy[J].Critical Reviews in Biotechnology,2016,36(2):276-289.
[15] HAMERS-CASTERMAN C,ATARHOUCH T,MUYLDERMANS S,et al.Naturally occurring antibodies devoid of light chains[J].Nature,1993,363(6428):446-448.
[16] ROTHBAUER U,ZOLGHADR K,TILLIB S,et al.Targeting and tracing antigens in live cells with fluorescent nanobodies[J].Nature Methods,2006,3(11):887-889.
[17] HASSANZADEH-GHASSABEH G,DEVOOGDT N,DE PAUW P,et al.Nanobodies and their potential applications[J].Nanomedicine (London,England),2013,8(6):1013-1026.
[18] ZHANG J R,WANG Y,DONG J X,et al.Development of a simple pretreatment immunoassay based on an organic solvent-tolerant nanobody for the detection of carbofuran in vegetable and fruit samples[J].Biomolecules,2019,9(10):576.
[19] LIU H,WANG Y,DUAN H,et al.An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits Porcine reproductive and respiratory syndrome virus replication[J].Veterinary Microbiology,2015,181(3):252-260.
[20] QIU L C,FENG Y,MA X,et al.A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor[J].Acta Biochim Biophys Sin,2017,49(6):513-519.
[21] 李宁娟,合木巴提·卡马勒汗,马晓玲,等.采用免疫亲和色谱方法从骆驼血清中纯化抗CD47特异性抗体[J].基因组学与应用生物学,2019,38(3):1012-1017. LI N J,HEMBAT K,MA X L,et al.Purification of anti-CD47 specific antibody from camel serum by immunoaffinity chromatography[J].Genomics and Applied Biology,2019,38(3):1012-1017.(in Chinese)
[22] 刘清艳,王欢,丁铲,等.传染性支气管炎病毒N蛋白的原核表达及其多克隆抗体的制备与鉴定[J/OL].中国动物传染病学报,2022:1-10.Doi:10.19958/j.cnki.cn31-2031/S.20220602.001. LIU Q Y,WANG H,DING C,et al.Preparation and validation of infectious bronchitis virus nucleocapsid protein polyclonal antibody[J/OL].Chinese Journal of Animal Infectious Diseases,2022:1-10.Doi:10.19958/j.cnki.cn31-2031/S.20220602.001.(in Chinese)
[23] 蒋柏勇,阿来·沙力塔那,美丽排提·玉素甫,等.古尔图病毒核蛋白的原核表达纯化及ELISA检测方法的建立[J].中华预防医学杂志,2022,56(6):824-830. JIANG B Y,ALAI S,MEILIPAITI Y,et al.Prokaryotic expression and purification of Gultu virus nucleoprotein and establishment of ELISA detection method[J].Chinese Journal of Preventive Medicine,2022,56(6):824-830.(in Chinese)
[24] 姜顺传.动物免疫失败的原因及对策[J].现代农业科技,2008,9:189-190. JIANG S C.Causes and countermeasures of animal immune failure[J].Modern Agricultural Science and Technology,2008,9:189-190.(in Chinese)
[25] WYNES J,KAIKIS A C.Current advancements in Ankle arthrodiastasis[J].Clinics in Podiatric Medicine and Surgery,2018,35(4):467-479.
[26] 李全允,伊丽,吉日木图.骆驼总RNA的提取方法选择及TRIzol与脾脏比例对RNA质量的影响[J].黑龙江畜牧兽医,2020,22:138-143. LI Q Y,YI L,JIMUTURI.Selection of camel total RNA extraction method and the effect of TRIzol to spleen ratio on RNA quality[J].Heilongjiang Animal Science and Veterinary Medicine,2020,22:138-143.(in Chinese)
[27] 杜东霞,张冉.辅助噬菌体在噬菌体展示中的应用及研究进展[J].微生物学通报,2009,36(2):261-266. DU D X,ZHANG R.Application and progress of helper phage in phage display[J].Microbiology China,2009,36(2):261-266.(in Chinese)
[28] 侯伟.噬菌体展示系统的构建及其在疾病防控领域应用研究进展[J].中国畜牧兽医,2022,49(5):1688-1696. HOU W.Research progresson construction of phage display system and its application in the field of disease prevention and control[J].Chineseanimal Husbandry and Veterinary Medicine,2022,49(5):1688-1696.(in Chinese)
[29] 武荣飞,杨溢,王鹏志,等.单克隆抗体制备技术最新研究进展[J].中国预防兽医学报,2022,44(3):333-337. WU R F,YANG Y,WANG P Z,et al.The latest research progress of monoclonal antibody preparation technology[J].ChineseJournal of Preventive Veterinary Medicine,2022,44(3):333-337.(in Chinese)
[30] 秦鑫.人血管内皮生长因子受体3结合肽的筛选和鉴定[D].西安:第四军医大学,2007. QIN X.Screening and identification of a novel peptide ligand of human vascular endothelia growth factor receptor 3[D].Xi'an:Fourth Military Medical,2007.(in Chinese)