重组早孕因子的原核表达及多克隆抗体制备

doi:10.14088/j.cnki.issn0439-8114.2023.05.023

湖北农业科学 ›› 2023, Vol. 62 ›› Issue (5): 129-134.doi: 10.14088/j.cnki.issn0439-8114.2023.05.023

重组早孕因子的原核表达及多克隆抗体制备

王聪¹, 王菡¹, 张海玲², 肖毅然¹, 郭禹汐¹, 刘梦迪¹, 李浩菘¹, 吴宗澄¹, 段芙春¹, 卢士英¹

1.吉林大学动物医学学院/人兽共患病研究教育部重点实验室,长春 130112;
2.中国农业科学院特产研究所,长春 130112

收稿日期:2023-02-16 出版日期:2023-05-25 发布日期:2023-06-12
通讯作者: 卢士英（1973-）,女,教授,博士,主要从事动物性食品安全研究,（电子信箱）lushiying1129@163.com。
作者简介:王聪（1997-）,男,辽宁大连人,在读硕士研究生,研究方向为动物性食品安全,（电话）16688378807（电子信箱）865948244@qq.com。
基金资助:
国家自然科学基金面上项目（32072943）

Prokaryotic expression of recombinant early pregnancy factor and preparation of polyclonal antibody

WANG Cong¹, WANG Han¹, ZHANG Hai-ling², XIAO Yi-ran¹, GUO Yu-xi¹, LIU Meng-di¹, LI Hao-song¹, WU Zong-cheng¹, DUAN Fu-chun¹, LU Shi-ying¹

1. College of Veterinary Medicine/Key Laboratory of Zoonosis Research,Ministry of Education,Jilin University,Changchun 130112,China;
2. Institute of Specialty, Chinese Academy of Agricultural Sciences,Changchun 130112,China

Received:2023-02-16 Online:2023-05-25 Published:2023-06-12

摘要/Abstract

摘要： 对牛源早孕因子进行生物信息学分析,构建了含有促溶标签的原核表达载体pET-28a-srEPF,经IPTG成功诱导表达,亲和层析纯化早孕因子,再以重组蛋白为免疫原,免疫动物制备多克隆抗体, Western blotting对表达蛋白及其多抗进行检测。结果表明,成功制备早孕因子重组蛋白,分子质量为 26 ku,纯化后BCA法测定其浓度为1.1 mg/mL,早孕因子重组蛋白和去除抑制区的EPF蛋白均可与制备的多抗特异性结合,间接ELISA测定其效价为1:256 000。

关键词: 早孕因子, 原核表达, 多克隆抗体

Abstract: Bioinformatics analysis of bovine early pregnancy factor was conducted, and prokaryotic expression vector pET-28a-srEPF containing pro-soluble labels was constructed. The prokaryotic expression vector was successfully induced by IPTG, and the early pregnancy factor was purified by affinity chromatography. Then, recombinant protein was used as immunogen to immunized animals to prepare polyclonal antibodies. The expressed proteins and their polyantibodies were detected by Western blotting. The results showed that the recombinant protein of early pregnancy factor had been successfully prepared, and its molecular weight was about 26 ku. After purification, the concentration was determined by BCA method to be 1.1 mg/mL. Both the recombinant protein of early pregnancy factor and EPF protein after enzyme digestion could be specifically bound to the prepared polyclonal antibody, and the titer determined by indirect ELISA was 1:256 000.

Key words: early pregnancy factor, prokaryotic expression, polyclonal antibodies

中图分类号:

王聪, 王菡, 张海玲, 肖毅然, 郭禹汐, 刘梦迪, 李浩菘, 吴宗澄, 段芙春, 卢士英. 重组早孕因子的原核表达及多克隆抗体制备[J]. 湖北农业科学, 2023, 62(5): 129-134.

WANG Cong, WANG Han, ZHANG Hai-ling, XIAO Yi-ran, GUO Yu-xi, LIU Meng-di, LI Hao-song, WU Zong-cheng, DUAN Fu-chun, LU Shi-ying. Prokaryotic expression of recombinant early pregnancy factor and preparation of polyclonal antibody[J]. HUBEI AGRICULTURAL SCIENCES, 2023, 62(5): 129-134.

参考文献

[1] 权凯,张改平.早孕因子的生物学特性及其在动物繁殖领域中的应用[J].中国畜牧兽医,2009,36(4):122-125.
[2] MORTON H,HEGH V,CLUNIE G J.Immunosuppression detectde in pregnant mice by rosette inhibition test[J].Nature,1974,249:459-460.
[3] CAVANAGH A C.Identification of early pregnancy factor as chaperonin10: Implications for understanding its role[J].Review of reproduction,1996,1:28-32.
[4] ROLFE B E,MORTON H,CAVANAGH A C,et al.Detection of an early pregnancy factor like substance in sera of patients with testicular germ cell tumors[J]. Am J Rep rod Immuno1, 1983, 3(2): 97-100.
[5] SOMODEVILA-TORRES M J, MORTON H, ZHANG B, et al. Purification and characterisation of functiona1 early pregnancy factor expressed in Sf9 insect cels and in Escherichia coli[J]. Protein Expr Purif, 2003,32(2):276-287.
[6] CHENG S J, MA A Y, QIAO C X, et al.Early pregnancy factor activity in serum of patients with surgical abortion[J]. Am J Reprod Immunol, 2000, 44(4): 211-213.
[7] 杜晓明,张改平,王丽,等.重组人早孕因子的表达与纯化[J].中国农学通报,2011,27(14):40-44.
[8] CHENG S J, ZHENG Z Q.Early pregnancy factor in cervical mucus of pregnant women[J].American journal of reproductive immunology, 2004, 51(2): 102-105.
[9] JIA H,HALILOU AI, HU L,et al.Heat shock protein 10 (Hsp10) in immune-related diseases:Onecoin,twosides[J].International journal of biochemistry and molecular biology,2011,2(1):47-57.
[10] MORTON H,ROLFE B,CLUNIE G J.An early pregnancy factor detected in human serum by the rosette inhibition test[J].Lancet,1977,309(8008):394-397.
[11] ZHANG N B,WALSH M D,NGUYEN K B, et al.Early pregnancy factor treatment suppresses the inflammatory response and adhesion moleculeexpression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice[J]. J Neurol Sci,2003, 212(1-2): 37-46.
[12] ATHANASAS-PLATSIS S, HOSKIN M J, ROLFE B E, et al.A monoclonal antibody to an early pregnancy factor-induced suppressor factor (EPF-S1) disrupts implantation in mice[J].Am J Reprod Immunol,1995,33(3):259-266.
[13] FAN X G,ZHENG Z Q.A study of early pregnancy factor activity in preimplantation[J]. Am J Reprod Immunol,1997,37(5):359-364.
[14] HAQ A, MOT H I, AL-HUSSEIN K.Isolation, purification and partial characterization of early pregnancy factor(EPF)from sera of pregnant women[J]. Eur J Med Res, 2001, 6(5):209-214.
[15] FAN X,YAN L,JIA S,et al.A study of early pregnancy factor activity in the sera of women with trophoblastic tumor[J].Am J Reprod Immunol,1999,41(3):204-208.

重组早孕因子的原核表达及多克隆抗体制备

Prokaryotic expression of recombinant early pregnancy factor and preparation of polyclonal antibody

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