猪源分泌性白细胞蛋白酶抑制因子的原核表达及单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.06.030

摘要/Abstract

摘要： 【目的】制备猪源分泌性白细胞蛋白酶抑制因子(SLPI)及其单克隆抗体,为建立猪源SLPI检测方法及其功能研究提供试验材料。【方法】将经串联的猪源SLPI基因克隆至pET-28a(+)、pGEX-6P-1载体构建重组表达载体;利用大肠杆菌表达系统表达重组蛋白,采用SDS-PAGE分析重组蛋白的诱导表达情况;重组菌经超声破碎以镍柱对重组蛋白进行纯化,并进行SDS-PAGE和Western blotting鉴定。用纯化后的重组蛋白免疫BALB/c小鼠,经细胞融合制备杂交瘤细胞,采用间接ELISA筛选和鉴定所制备单克隆抗体和亚类,利用Western blotting及间接免疫荧光(IFA)方法鉴定单克隆抗体的特异性。【结果】试验成功构建了pGEX-6P-1-SLPI、pET-28a-SLPI重组质粒,获得纯度较高的His-SLPI、GST-SLPI重组蛋白,分子质量分别为27和50 ku,前者为包涵体表达,后者为可溶性表达;获得3株可稳定分泌抗猪SLPI单克隆抗体的杂交瘤细胞株,其中,9D10为IgG3亚类,9G11、10E2为IgG2b亚类;3株单克隆抗体均可与猪肠上皮细胞内的SLPI结合,9D10在细胞核中显示较强的荧光信号,而9G11、10E2在细胞浆和细胞核中均有荧光信号。【结论】本研究成功制备了重组His-SLPI和GST-SLPI蛋白,获得3株稳定分泌抗猪源SLPI单克隆抗体,特异性良好,为进一步研究猪源SLPI特性及其在黏膜免疫中的作用搭建技术平台。

关键词: 猪; 分泌性白细胞蛋白酶抑制因子(SLPI); 原核表达; 单克隆抗体

Abstract: 【Objective】 The purpose of this experiment was to prepare porcine derived secretory leukocyte protease inhibitor (SLPI) and its monoclonal antibody,laying the foundation for the establishment of porcine SLPI detection method and function research.【Method】 Series connection of porcine SLPI gene was cloned into pET-28a(+) and pGEX-6P-1 vectors to construct the expression vector.The recombinant protein was expressed in Escherichia coli expression system,and the induced expression of recombinant proteins were analyzed by SDS-PAGE.After recombinant bacteria were ultrasonicated,the purified recombinant proteins were identified by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified recombinant protein,and hybridoma cells were prepared by cell fusion.The monoclonal antibody and subclass were screened and detected by indirect ELISA,and the specificity of monoclonal antibody were identified using Western blotting and indirect immunofluorescence assay (IFA).【Result】 The pGEX-6P-1-SLPI and pET-28a-SLPI recombinant plasmids were successfully constructed,and high purity His-SLPI and GST-SLPI recombinant proteins were obtained with molecular weights of 27 and 50 ku,respectively.His-SLPI was expressed in inclusion body,while GST-SLPI was expressed in soluble form.Three hybridoma cell lines that could stably secrete monoclonal antibodies against porcine SLPI were acquired,namely 9D10 as the IgG3 subclass,9G11 and 10E2 as the IgG2b subclass.All three monoclonal antibodies could bind to SLPI in porcine intestinal epithelial cells.9D10 showed strong fluorescence signal in nucleus,while 9G11 and 10E2 showed fluorescence signals in both cytoplasm and nucleus.【Conclusion】 Recombinant His-SLPI and GST-SLPI proteins were successfully prepared,and three stable secretion monoclonal antibodies against porcine SLPI with good specificity were acquired.The results would provide a technical platform for further studying the characteristics of porcine SLPI and its role in mucosal immunity.

Key words: pigs; secretory leukocyte protease inhibitor (SLPI); prokaryotic expression; monoclonal antibody

中图分类号:

S852.4⁺3

付明智, 李新新, 刘雪威, 李焕荣. 猪源分泌性白细胞蛋白酶抑制因子的原核表达及单克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(6): 2556-2565.

FU Mingzhi, LI Xinxin, LIU Xuewei, LI Huanrong. Prokaryotic Expression of Porcine Secretory Leukocyte Protease Inhibitor and Preparation of Its Monoclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2556-2565.

参考文献

[1] MAJCHRZAK-GORECKA M,MAJEWSKI P,GRYGIER B,et al.Secretory leukocyte protease inhibitor (SLPI),a multifunctional protein in the host defense response[J].Cytokine & Growth Factor Reviews, 2016,28:79-93.
[2] HIEMSTRA P S.Novel roles of protease inhibitors in infection and inflammation[J].Biochemical Society Transactions,2002,30(2):116-120.
[3] SCOTT A,WELDON S,TAGGART C C.SLPI and elafin:Multifunctional antiproteases of the WFDC family[J].Biochemival Society Transactions,2011,39(5):1437-1440.
[4] MUNADZIROH E,PUTRI G A,RISTIANA V,et al.The role of recombinant secretory leukocyte protease inhibitor to CD163,FGF-2,IL-1 and IL-6 expression in skin wound healing[J].Clinical,Cosmetic Investigational Dermatology,2022,15:903-910.
[5] DONALD K,FINLAY B B.Early-life interactions between the microbiota and immune system:Impact on immune system development and atopic disease[J].Nature Reviews Immunology, 2023,23(11):735-748.
[6] NUGTEREN S,SIMONS-OOSTERHUIS Y,MENCKEBERG C L,et al.Endogenous secretory leukocyte protease inhibitor inhibits microbial-induced monocyte activation[J]. European Journal of Immunology,2023,53(2):e2249964.
[7] KAETZEL C S.The polymeric immunoglobulin receptor:Bridging innate and adaptive immune responses at mucosal surfaces[J].Immunological Reviews,2005,206:83-99.
[8] PILETTE C,OUADRHIRI Y,DIMANCHE F,et al.Secretory component is cleaved by neutrophil serine proteinases but its epithelial production is increased by neutrophils through NF-kappa B- and p38 mitogen-activated protein kinase-dependent mechanisms[J]. American Journal Respiratory Cell and Molecular Biology,2003,28:485-498.
[9] MIKAMI Y,IWASE T,KOMIYAMA Y,et al.Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-κB signaling pathway[J].Molecular Immunology,2015,67(2 PtB):568-574.
[10] LEE S,YOO I,CHEON Y,et al.Spatiotemporal expression and regulation of peptidase inhibitor 3 and secretory leukocyte protease inhibitor at the maternal-fetal interface in pigs[J].Animal Bioscience,2023,36(7):1034-1043.
[11] 吴桐.猪伪狂犬病病毒HeN1株gE蛋白单克隆抗体的制备及其抗原表位的鉴定[D].大庆:黑龙江八一农垦大学,2017. WU T.Preparation of the monoclonal antibody against the gE protein and identification of the B-cell epitope in gE of Pseudorabies virus[D].Daqing:Heilongjiang Bayi Agricultural University,2017.(in Chinese)
[12] 陈玉珍,何韶衡,周艳春,等.抗人分泌性白细胞蛋白酶抑制剂单克隆抗体的制备与鉴定[J].细胞与分子免疫学杂志,2006,22(1):54-57. CHEN Y Z,HE S H,ZHOU Y C,et al.Preparation and characterization of monoclonal antibodies against human secretory leukocyte protease inhibitor[J]. Chinese Journal of Cellular and Molecular Immunology,2006,22(1):54-57.(in Chinese)
[13] BARRIOS V E,MIDDLETON S C,KASHEM M A,et al.Tryptase mediates hyperresponsiveness in isolated Guinea pig bronchi[J].Life Sciences,1998,63(26):2295-2303.
[14] NUGTEREN S,SAMSOM J N.Secretory leukocyte protease inhibitor (SLPI) in mucosal tissues:Protects against inflammation,but promotes cancer[J].Cytokine & Growth Factor Reviews,2021,59:22-35.
[15] SHIN D,CHANG S Y,BOGERE P,et al.Beneficial roles of probiotics on the modulation of gut microbiota and immune response in pigs[J]. PLoS One,2019,14(8):e0220843.
[16] 邢燕子.密码子偏好在翻译前水平对基因表达的影响研究[D].上海:华东理工大学,2020. XING Y Z.Study the role of codon bias in regulating gene expression pretranslationally[D].Shanghai:East China University of Science and Technology,2020.(in Chinese)
[17] 吉学桥,张利清,陆睿,等.人白细胞介素-2原核表达条件优化和表达产物纯化[J].畜牧与兽医,2013,45(10):21-25. JI X Q,ZHANG L Q,LU R,et al.Condition optimization of prokaryotic expression of human interleukin-2 and protein purification[J]. Animal Husbandry & Veterinary Medicine,2013,45(10):21-25.(in Chinese)
[18] 朱丽霖.IFIT5的原核表达及其对ALV-J复制的影响[D].荆州:长江大学,2022. ZHU L L.Prokaryotic expression of IFIT5 and its effect on ALV-J replication[D].Jingzhou:Yangtze University,2022.(in Chinese)
[19] 程凯慧,沈付娆,邹积振,等.牛冠状病毒S蛋白抗原表位基因的串联表达及抗血清制备[J].中国兽医杂志,2018,54(10):59-61. CHENG K H,SHEN F R,ZOU J Z,et al.Preparation of antisera and tandem expression of the antigen epitopes of Bovine coronavirus S protein[J]. Chinese Journal of Veterinary Medicine,2018,54(10):59-61.(in Chinese)
[20] 罗凯明,赵虎,高向东.透明质酸单克隆抗体的制备及免疫分析方法的建立[J].中国药科大学学报,2015,46(6):740-744. LUO K M,ZHAO H,GAO X D.Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay[J].Journal of China Pharmaceutical University,2015,46(6):740-744.(in Chinese)
[21] 王云龙,翟晋豫,王继创,等.糖类抗原19-9单抗制备及DAS-ELISA的建立[J].中国免疫学杂志,2014,30(8):1088-1092. WANG Y L,ZHAI J Y,WANG J C,et al.Preparation against carbohydrate antigen 19-9 monoclonal antibodies and establishment of DAS-ELISA[J]. Chinese Journal of Immunology,2014,30(8):1088-1092.(in Chinese)
[22] 梁骥.脾内免疫快速制备单克隆抗体及化疗药联合肿瘤疫苗治疗结肠癌[D].长春:吉林大学,2013. LIANG J.Rapid preparation of monoclonal antibodies by intrasplenic immunization and treatment of colon cancer by tumor vaccine combined with chemotherapeutics[D].Changchun:Jilin University,2013.(in Chinese)
[23] 许道艳,刘磊,刘仁沿,等.麻痹性贝毒单克隆抗体的制备和酶联免疫检测方法的建立[J].中国免疫学杂志,2013,29(1):69-73. XU D Y,LIU L,LIU R Y,et al.Development of paraltic shellfish poisoning (PSP) monoclonal antibody and establishment of enzyme linked immolunosorbent assay (ELISA) detection method[J].Chinese Journal of Immunology,2013,29(1):69-73.(in Chinese)
[24] 林金香,王心睿.单克隆抗体制备技术的最新进展[J].中国免疫学杂志,2021,37(20):2544-2548. LIN J X,WANG X R.Recent advances on preparation technology of monoclonal antibody[J].Chinese Journal of Immunology,2021,37(20):2544-2548.(in Chinese)
[25] 徐田丽.诺氟沙星单克隆抗体的制备和胶体金试纸条的研制[D].厦门:集美大学,2016. XU T L.Production of monoclone antibody and colloidalgold immunchromatographic test strip against norfloxacin[D].Xiamen:Jimei University,2016.(in Chinese)
[26] RAMOS N,SIBILA M,NEIRA V.Editorial:Porcine respiratory disease complex:Dynamics of polymicrobial infections,synergistic effects and management strategies[J].Frontiers in Veterinary Science,2023,10:1329073.