猪抑制素多克隆抗体对C57BL/6J小鼠超数排卵的增效作用

doi:10.12300/j.issn.1674-5817.2025.088

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (2): 271-278.DOI: 10.12300/j.issn.1674-5817.2025.088

猪抑制素多克隆抗体对C57BL/6J小鼠超数排卵的增效作用

赵赫(), 张涛, 李丽, 肖宇宙, 安学芳, 张帆()()

中国科学院武汉病毒研究所, 武汉 430070

收稿日期:2025-06-10 修回日期:2025-09-11 出版日期:2026-04-25 发布日期:2026-04-18
通讯作者:
张帆（1985—），男，硕士，高级实验师，主要从事实验动物质量控制与管理工作。E-mail： zhangf@wh.iov.cn。ORCID：0009-0002-4393-3081
作者简介:赵赫（1987—），男，硕士，实验师，主要从事基因工程小鼠管理工作。E-mail： zhaohe@wh.iov.cn。ORCID：0009-0001-2390-1018
基金资助:
国家重点研发计划“基础科研条件与重大科学仪器设备研发”重点专项“实验动物质量精准监管与安全性评价关键技术研究”(2024YFF0728800);湖北省科技厅项目“基因工程小鼠辅助生殖关键试剂优化及质量控制研究”(2025CFC022)

Enhancement Effect of Porcine Inhibin Polyclonal Antibody on Superovulation in C57BL/6J Mice

ZHAO He(), ZHANG Tao, LI Li, XIAO Yuzhou, AN Xuefang, ZHANG Fan()()

Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430070, China

Received:2025-06-10 Revised:2025-09-11 Published:2026-04-25 Online:2026-04-18
Contact: ZHANG Fan (ORCID: 0009-0002-4393-3081), E-mail: zhangf@wh.iov.cn

摘要/Abstract

摘要：

目的制备兔抗猪抑制素多肽偶联钥孔血蓝蛋白（keyhole limpet hemocyanin，KLH）多克隆抗体，探究其对C57BL/6J小鼠超数排卵的影响。方法将人工合成的猪抑制素多肽偶联KLH蛋白免疫新西兰白兔，制备抗抑制素血清（anti-inhibin serum，AIS，即抑制素多克隆抗体）。将纯化后的AIS联合孕马血清促性腺激素（pregnant mare serum gonadotropin，PMSG）经腹腔注射雌性C57BL/6J小鼠，48 h后再注射人绒毛膜促性腺激素（human chorionic gonadotropin，hCG）诱导超数排卵，15 h后收集并计数超数排卵获得的卵母细胞数；在体外受精24 h后检测2-细胞期胚胎数。结果将KLH免疫新西兰白兔后制备AIS，采用间接ELISA法测定AIS效价，结果显示其效价可达1∶512 000；AIS经硫酸铵沉淀法纯化后进行SDS-聚丙烯酰胺凝胶电泳分析，结果显示在分子量为50 kDa及25 kDa处均出现了清晰可见的条带，符合免疫球蛋白G抗体亚型重链和轻链的理论分子量大小，表明成功制备出猪抑制素多克隆抗体。与常规超数排卵方法相比，AIS 10倍稀释联合PMSG处理可以显著增加小鼠超数排卵产生的卵母细胞数量（P＜0.05），约为常规PMSG处理的1.5倍。结论本研究制备的猪抑制素多克隆抗体作为改良超数排卵试剂，可以提高C57BL/6J小鼠的超数排卵效果，在后续的市场应用中有较大前景。

关键词: 抗猪抑制素血清, C57BL/6J雌性小鼠, 超数排卵, 卵母细胞数, 2-细胞期胚胎数

Abstract:

Objective To prepare rabbit anti-porcine inhibin polypeptide-keyhole limpet hemocyanin(KLH) conjugated polyclonal antibody and evaluate its effect on superovulation in C57BL/6J mice. Methods New Zealand white rabbits were immunized with a synthesized porcine inhibin polypeptide conjugated with KLH to produce anti-inhibin serum (AIS, i.e., inhibin polyclonal antibody). Female C57BL/6J mice received intraperitoneal injections of purified AIS in combination with pregnant mare serum gonadotropin (PMSG), followed by human chorionic gonadotropin (hCG) after 48 hours to induce superovulation. Oocytes obtained from superovulation were collected and counted 15 hours post-hCG administration, and the number of 2-cell embryos was assessed 24 hours after in vitro fertilization. Results AIS prepared by immunizing New Zealand White rabbits with KLH-conjugated porcine inhibin polypeptide was subjected to titer determination by indirect ELISA, showing titers reaching 1∶ 512 000. SDS-polyacrylamide gel electrophoresis analysis of ammonium sulfate-purified AIS revealed distinct 50 kDa and 25 kDa bands corresponding to the theoretical molecular weights of IgG antibody heavy and light chains, confirming successful production of porcine inhibin polyclonal antibody. Compared with conventional superovulation methods, AIS diluted 10-fold combined with PMSG significantly increased the number of oocytes obtained from superovulation in mice (P<0.05) by approximately 1.5-fold. Conclusion Porcine inhibin polyclonal antibody, as an improved superovulation reagent, can improve superovulation efficiency in C57BL/6J mice, and shows promising prospects for future applications.

Key words: Porcine anti-inhibin serum, C57BL/6J female mice, Superovulation, Oocyte number, Number of 2-cell embryos

中图分类号:

Q95-33

赵赫,张涛,李丽,等. 猪抑制素多克隆抗体对C57BL/6J小鼠超数排卵的增效作用[J]. 实验动物与比较医学, 2026, 46(2): 271-278. DOI: 10.12300/j.issn.1674-5817.2025.088.

ZHAO He,ZHANG Tao,LI Li,et al. Enhancement Effect of Porcine Inhibin Polyclonal Antibody on Superovulation in C57BL/6J Mice[J]. Laboratory Animal and Comparative Medicine, 2026, 46(2): 271-278. DOI: 10.12300/j.issn.1674-5817.2025.088.

图/表 5

注：Alpha，α-螺旋；Beta，β-折叠；Turn，转角；Coil，无规卷曲。
">

图1 基于DNAStar的抑制素α亚基二级结构与抗原表位的生物信息学分析
注：Alpha，α-螺旋；Beta，β-折叠；Turn，转角；Coil，无规卷曲。

Figure 1 Bioinformatics analysis of secondary structure and antigenic epitopes of inhibin α-subunit based on DNAStar
Note： Alpha， α-helix； Beta， β-sheet； Turn， turn； Coil， random coil.

表1 抑制素抗体效价检测结果 (A_{450 nm})

Table 1 Results of anti-inhibin antibody titer detection

稀释浓度 Dilution ratio	免疫前血清 Pre-immune serum	免疫后血清 Post-immunization rabbit serum	PBST对照 PBST control
1∶1 000	0.086	2.106	0.070
1∶2 000	0.096	2.083	0.068
1∶4 000	0.086	2.033	0.069
1∶8 000	0.087	2.005	0.071
1∶16 000	0.076	1.139	0.069
1∶32 000	0.060	1.187	0.067
1∶64 000	0.075	1.067	0.067
1∶128 000	0.082	1.008	0.066
1∶256 000	0.073	0.761	0.065
1∶512 000	0.071	0.548	0.067
1∶1 024 000	0.073	0.573	0.069
Blank	0.075	0.239	0.067

注：M，蛋白marker；泳道1，AIS纯化蛋白；泳道2，HyperOva高效超数排卵试剂。
">

图2 抑制素抗体的SDS-聚丙烯酰胺凝胶电泳结果
注：M，蛋白marker；泳道1，AIS纯化蛋白；泳道2，HyperOva高效超数排卵试剂。

Figure 2 SDS-polyacrylamide gel electrophoresis of inhibin antibody
Note： M， protein marker； lane 1， the purified protein of AIS； lane 2， HyperOva high-efficiency superovulation reagent.

注：不同处理方法下小鼠超数排卵的卵母细胞数量（A）和2-细胞期胚胎数量（B）。A1-1，AIS原液组；A1-2，AIS 100倍稀释组；A1-3，AIS 10 000倍稀释组；P1，PMSG组；H1，HyperOva组。n=3，^**P＜0.01；^*P＜0.05；^nsP＞0.05。
">

图3 不同超数排卵方法对3~5周龄C57BL/6J小鼠卵母细胞数和2-细胞期胚胎数的影响
注：不同处理方法下小鼠超数排卵的卵母细胞数量（A）和2-细胞期胚胎数量（B）。A1-1，AIS原液组；A1-2，AIS 100倍稀释组；A1-3，AIS 10 000倍稀释组；P1，PMSG组；H1，HyperOva组。n=3，^**P＜0.01；^*P＜0.05；^nsP＞0.05。

Figure 3 Effects of different superovulation methods on the number of oocytes and the number of 2-cell embryos in C57BL/6J mice aged 3 to 5 weeks
Note： Number of oocytes （A） and 2-cell stage embryos （B） in mice with superovulation under different treatments. A1-1， AIS stock solution group； A1-2， AIS 100-fold dilution group； A1-3， AIS 10 000-fold dilution group； P1， PMSG group； H1， HyperOva group. n=3， ^**P＜0.01； ^*P＜0.05； ^nsP＞0.05.

表2 不同超数排卵方法对4周龄C57BL/6J小鼠卵母细胞数以及2-细胞期胚胎数的影响

Table 2 Effects of different superovulation methods on the number of oocytes and 2-cell embryos in 4-week-old C57BL/6J mice

组别 Group	卵母细胞总数 Total number of oocytes	2-细胞期胚胎总数 Total number of 2-cell embryos
A2-1	22.67±3.51	14.00±3.61
A2-2	52.67±3.06^abc	47.67±2.52^abc
P2	36.00±5.29	31.00±5.57
H2	63.00±6.00^a	47.67±5.51^a

参考文献 29

[1]	MAKANJI Y, WALTON K L, WILCE M C, et al. Suppression of inhibin A biological activity by alterations in the binding site for betaglycan[J]. J Biol Chem, 2008, 283(24):16743-16751. DOI: 10.1074/jbc.M801045200 .
[2]	MAKANJI Y, ZHU J, MISHRA R, et al. Inhibin at 90: from discovery to clinical application, a historical review[J]. Endocr Rev, 2014, 35(5):747-794. DOI: 10.1210/er.2014-1003 .
[3]	马继福, 巴合提·博代, 贾昊天, 等. 抑制素对促卵泡素作用机制的研究进展[J]. 黑龙江畜牧兽医, 2023(1):37-41, 126-127. DOI: 10.13881/j.cnki.hljxmsy.2022.06.0226 .
	MA J F, Bodai Bakhet, JIA H T, et al. Research progress on the mechanism of inhibin on follicle-stimulating hormone[J]. Heilongjiang Anim Sci Vet Med, 2023(1):37-41, 126-127. DOI: 10.13881/j.cnki.hljxmsy.2022.06.0226 .
[4]	AKAGI S, KANEKO H, NAKANISHI Y, et al. Ovarian response and FSH profile in cows following injection of various doses of inhibin antiserum[J]. J Vet Med Sci, 1997, 59(12):1129-1135. DOI: 10.1292/jvms.59.1129 .
[5]	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. DOI: 10.1016/j.theriogenology.2018.07.031 .
[6]	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. DOI: 10.1016/s0093-691x(98)00160-5 .
[7]	WANG H, HERATH C B, XIA G, et al. Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum[J]. Reproduction, 2001, 122(5):809-816. DOI: 10.1530/rep. 0.122 0809 .
[8]	ISHIGAME H, MEDAN M S, WATANABE G, et al. A new alternative method for superovulation using passive immunization against inhibin in adult rats[J]. Biol Reprod, 2004, 71(1):236-243. DOI: 10.1095/biolreprod.104.027789 .
[9]	MOCHIDA K, MORITA K, SASAOKA Y, et al. Superovulation with an anti-inhibin monoclonal antibody improves the reproductive performance of rat strains by increasing the pregnancy rate and the litter size[J]. Sci Rep, 2024, 14(1):8294. DOI: 10.1038/s41598-024-58611-9 .
[10]	TAKEO T, NAKAGATA N. Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice[J]. PLoS One, 2015, 10(5): e0128330. DOI: 10.1371/journal.pone.0128330 .
[11]	MOCHIDA K, HASEGAWA A, OTAKA N, et al. Devising assisted reproductive technologies for wild-derived strains of mice: 37 strains from five subspecies of Mus musculus [J]. PLoS One, 2014, 9(12): e114305. DOI: 10.1371/journal.pone. 011 4305 .
[12]	顾剑洁, 葛蓉, 徐伟超, 等. 不同超排卵方案对BALB/c小鼠超排卵效果的影响[J]. 实验动物与比较医学, 2017, 37(6): 482-484. DOI: 10.3969/j.issn.1674-5817.2017.06.012 .
	GU J J, GE R, XU W C, et al. Effect of different superovulation schemes on superovulation in BALB/c mice[J]. Lab Anim Comp Med, 2017, 37(6): 482-484. DOI: 10.3969/j.issn.1674-5817.2017.06.012 .
[13]	BYERS S L, PAYSON S J, TAFT R A. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs)[J]. Theriogenology, 2006, 65(9):1716-1726. DOI: 10.1016/j.theriogenology.2005.09.016 .
[14]	姜勋平, 杨利国, 刘桂琼, 等. 抑制素基因免疫对小鼠生殖的影响[J]. 中国兽医学报, 2002, 22(4):368-370. DOI: 10.16303/j.cnki.1005-4545.2002.04.018 .
	JIANG X P, YANG L G, LIU G Q, et al. Effects of inhibin α(1-32) gene immunization on mouse reproductive traits[J]. Chin J Vet Sci, 2002, 22(4):368-370. DOI: 10.16303/j.cnki.1005-4545.2002.04.018 .
[15]	许丹丹, 张敬文, 张健鹏, 等. 驴源马疱疹病毒8型ORF72基因原核表达及多克隆抗体制备[J]. 黑龙江畜牧兽医, 2023(1):117-121. DOI: 10.13881/j.cnki.hljxmsy.2022.03.0196 .
	XU D D, ZHANG J W, ZHANG J P, et al. Prokaryotic expression of donkey-derived Equid alphaherpesvirus type 8 ORF72 gene and polyclonal antibody preparation[J]. Heilongjiang Anim Sci Vet Med, 2023(1):117-121. DOI: 10.13881/j.cnki.hljxmsy.2022.03.0196 .
[16]	韩璐, 宋新辉, 徐佳寅, 等. 基于ferritin纳米载体的抑制素α亚基的多克隆抗体的制备[J]. 基因组学与应用生物学, 2024, 43(5):877-889. DOI: 10.13417/j.gab.043.000877 .
	HAN L, SONG X H, XU J Y, et al. Preparation of polyclonal antibodies based on inhibin α subunits of ferritin nanocarriers[J]. Genom Appl Biol, 2024, 43(5):877-889. DOI: 10.13417/j.gab.043.000877 .
[17]	张成云, 曾宪垠. 抑制素免疫在家畜繁殖中的应用[J]. 畜牧兽医杂志, 2008, 27(2):35-37. DOI: 10.3969/j.issn.1004-6704.2008.02.012 .
	ZHANG C Y, ZENG X Y. Immunization of inhibin and its application in animal reproduction[J]. J Anim Sci Vet Med, 2008, 27(2):35-37. DOI: 10.3969/j.issn.1004-6704.2008.02.012 .
[18]	MUÑOZ I, DEL NIÑO JESUS A, JOSA A, et al. Use of follicle-stimulating hormone (FSH) to increase the in vitro fertilization (IVF) efficiency of mice[J]. J Assist Reprod Genet, 1995, 12(10):738-743. DOI: 10.1007/BF02212903 .
[19]	SCOTT L F, SUNDARAM S G, SMITH S. The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program[J]. Fertil Steril, 1993, 60(3):559-568. DOI: 10.1016/s0015-0282(16)56176-7 .
[20]	LEGGE M, SELLENS M H. Optimization of superovulation in the reproductively mature mouse[J]. J Assist Reprod Genet, 1994, 11(6):312-318. DOI: 10.1007/BF02215719 .
[21]	ERTZEID G, STORENG R, LYBERG T. Treatment with gonadotropins impaired implantation and fetal development in mice[J]. J Assist Reprod Genet, 1993, 10(4):286-291. DOI: 10.1007/BF01204944 .
[22]	VAN DER AUWERA I, D'HOOGHE T. Superovulation of female mice delays embryonic and fetal development[J]. Hum Reprod, 2001, 16(6):1237-1243. DOI: 10.1093/humrep/16.6.1237 .
[23]	EZOE K, DAIKOKU T, YABUUCHI A, et al. Ovarian stimulation using human chorionic gonadotrophin impairs blastocyst implantation and decidualization by altering ovarian hormone levels and downstream signaling in mice[J]. Mol Hum Reprod, 2014, 20(11):1101-1116. DOI: 10.1093/molehr/gau065 .
[24]	DENG S R, XU J, ZENG J W, et al. Ovarian stimulation leads to a severe implantation defect in mice[J]. Reprod BioMedicine Online, 2013, 27(2):172-175. DOI: 10.1016/j.rbmo.2013.03.018 .
[25]	KOLBE T, LANDSBERGER A, MANZ S, et al. Productivity of superovulated C57BL/6J oocyte donors at different ages[J]. Lab Anim, 2015, 44(9):346-349. DOI: 10.1038/laban.746 .
[26]	GATES A H, BOZARTH J L. Ovulation in the PMSG-treated immature mouse: effect of dose, age, weight, puberty, season and strain (BALB/c, 129 and C129F1 hybrid)[J]. Biol Reprod, 1978, 18(3):497-505. DOI: 10.1095/biolreprod18.3.497 .
[27]	TAKEO T, MUKUNOKI A, NAKAGATA N. Ovulation of juvenile, mature, and aged female C57BL/6 mice following coadministration of inhibin antiserum and equine chorionic gonadotropin[J]. Theriogenology, 2019, 135:1-6. DOI: 10.1016/j.theriogenology.2019.05.028 .
[28]	王刚, 刘科, 赵伟健, 等. 我国SPF新西兰兔培育和生物学特性研究方面的进展[J]. 实验动物与比较医学, 2010, 30(3): 229-232. DOI: 10.3969/j.issn.1674-5817.2010.03.019 .
	WANG G, LIU K, ZHAO W J, et al. Research progress on breeding and biological characteristics of SPF rabbit in China[J]. Lab Anim Comp Med, 2010, 30(3): 229-232. DOI: 10.3969/j.issn.1674-5817.2010.03.019 .
[29]	HARRIS J R, MARKL J. Keyhole limpet hemocyanin (KLH): a biomedical review[J]. Micron, 1999, 30(6):597-623. DOI: 10. 1016/s0968-4328(99)00036-0 .

猪抑制素多克隆抗体对C57BL/6J小鼠超数排卵的增效作用

Enhancement Effect of Porcine Inhibin Polyclonal Antibody on Superovulation in C57BL/6J Mice

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 29

相关文章 7

编辑推荐

Metrics

本文评价

[1]	张婀娜, 郎雪楠, 赵文, 陈雪婷, 董婉维, 周生来, 于洋, 王惟, 张梅英, 郑志红. SD大鼠超数排卵的优化[J]. 实验动物与比较医学, 2015, 35(4): 316-317.
[2]	刘青青, 牛玉娜, 周正宇, 王婧, 卢明康, 薛智谋. 小鼠生物净化中的动情周期判定及超数排卵[J]. 实验动物与比较医学, 2014, 34(1): 56-58.
[3]	李海滨, 田江天, 宋军, 赖良学, 顾为望. 转基因兔与克隆兔高效胚胎移植体系的建立[J]. 实验动物与比较医学, 2011, 31(3): 172-177.
[4]	王新蕾1，邵义祥1，王鹏2. 动物超数排卵技术研究进展[J]. 实验动物与比较医学, 2007, 27(4): 280-284.
[5]	岳惠芬1，翟丹梅2，苏建家1，李媛1，黄右军3，杨春1，班克臣1，欧超1，曹骥1. 树鼩超数排卵初探[J]. 实验动物与比较医学, 2004, 24(1): 41-42.
[6]	戴丽军，叶炳飞，黄月玲. 小鼠超数排卵的比较试验[J]. 实验动物与比较医学, 2003, 23(2): 108-108.
[7]	时彦胜，周吉盛. 近交系小鼠超数排卵的研究[J]. 实验动物与比较医学, 1993, 13(4): 200-202.