Big-BALB/c小鼠亚系选育前后的抗体制备效率比较研究

doi:10.12300/j.issn.1674-5817.2023.035

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (6): 612-618.DOI: 10.12300/j.issn.1674-5817.2023.035

Big-BALB/c小鼠亚系选育前后的抗体制备效率比较研究

王丹¹(), 张晓璐², 王妍³, 傅博², 王文栋², 刘京³, 张甦寅³, 武怡荷³, 吴德国¹, 杜小燕², 战大伟³, 章秀林⁴()(), 李长龙²()()

^1.中国科学院遗传学与发育生物学研究所, 北京 100101
^2.首都医科大学基础医学院, 北京 100069
^3.斯贝福(北京)生物技术有限公司, 北京 102100
^4.中国医学科学院阜外医院, 北京 100037

收稿日期:2023-03-17 修回日期:2023-06-12 出版日期:2023-12-25 发布日期:2024-01-06
通讯作者: 章秀林（1995—），女，博士，研究实习员，主要从事医学遗传学研究。E-mail： xiulinzh@163.com。ORCID： 0000-0003-2964-4769
李长龙（1976—），男，博士，副教授/副研究员，主要从事实验动物资源开发及遗传与发育研究。E-mail: li-changlong@126.com。ORCID: 0000-0001-6832-785X
作者简介:王丹（1980—），女，本科，工程师，主要从事单克隆抗体制备及水生动物饲养工作。E-mail： danwang@genetics.ac.cn。ORCID：0009-0007-2203-7890
基金资助:
国家重点研发计划项目课题“基因编辑、SPF级和无菌级实验动物质量评价技术标准研究”(2022YFF0711005);国家重点研发计划青年科学家项目“基于SNP技术建立我国常用实验动物遗传质量评价技术体系”(2021YFF0703200);国家自然科学基金“长爪沙鼠长期感染的幽门螺杆菌遗传变异及对胃内微生物种群和病理变化的影响”(32070537);北京市自然科学基金-北京市教育委员会重点项目“幽门螺杆菌调节RXR诱发长爪沙鼠胃癌的机制研究”(KZ20210025037)

Study on the Antibody Production Efficiency in Modified Big-BALB/c Mice

Dan WANG¹(), Xiaolu ZHANG², Yan WANG³, Bo FU², Wendong WANG², Jing LIU³, Suyin ZHANG³, Yihe WU³, Deguo WU¹, Xiaoyan DU², Dawei ZHAN³, Xiulin ZHANG⁴()(), Changlong LI²()()

^1.The Institute of Genetics and Developmental Biology in Chinese Academy of Science, Beijing 100101, China
^2.School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China
^3.SPF (Beijing) Biotechnology Co. , Ltd. , Beijing 102100, China
^4.Fuwai Hospital of Chinese Academy of Medical Sciences. , Beijing 100037, China

Received:2023-03-17 Revised:2023-06-12 Published:2023-12-25 Online:2024-01-06
Contact: ZHANG Xiulin (ORCID: 0000-0003-2964-4769), E-mail: xiulinzh@163.com;
LI Changlong (ORCID: 0000-0001-6832-785X), E-mail: li-changlong@126.com.

摘要/Abstract

摘要：

目的比较利用BALB/c和Big-BALB/c（简称B-BALB/c）两种亚系小鼠制备鼠痘及鼠肝炎单克隆抗体的效率，为今后通过杂交瘤进行体内诱导单克隆抗体制备时实验动物的选择提供参考。方法从常规繁育的BALB/c小鼠群体中选择4周龄体重超过正常动物5%（后期选种标准为超过10%）的个体进行选种并单独繁育，经10代选育制备B-BALB/c小鼠亚系。选择10～11周龄、雌雄各半的BALB/c小鼠和B-BALB/c小鼠各40只，分别接种用液体石蜡预处理后的鼠痘单克隆抗体杂交瘤细胞株G23或鼠肝炎单克隆抗体杂交瘤细胞株Y15，然后通过体内诱生法获取含单克隆抗体的小鼠腹水，利用间接ELISA法检验抗体效价，按照品系、性别和接种杂交瘤类型对小鼠进行分组，分析腹水产量、抗体效价和抗体产量以评估两种亚系小鼠的抗体制备效率。结果经10代选育后获得的10周龄雄性和雌性B-BALB/c小鼠体重分别比同周龄BALB/c小鼠增加了22.3%和12.8%。抗体制备过程中，B-BALB/c小鼠的耐受力比BALB/c小鼠更好，能够适应二次腹水采集；与BALB/c小鼠相比，B-BALB/c小鼠腹水产量和抗体效价均显著提高，尤以杂交瘤细胞株G23接种组更明显（均P＜0.000 1）。接种杂交瘤细胞株G23或Y15后，B-BALB/c小鼠的平均抗体产量（14.99×10⁴U和33.22×10⁴U）高于BALB/c小鼠（5.33×10⁴U和19.31×10⁴U）（均P＜0.01）。接种杂交瘤细胞株G23后，B-BALB/c小鼠的平均单位体重的抗体产量（0.55×10⁴ U/g）高于BALB/c小鼠（0.23×10⁴ U/g）（P＜0.000 1）。接种杂交瘤细胞株Y15后，雌性B-BALB/c和BALB/c小鼠的单位体重抗体产量高于同亚系的雄性B-BALB/c小鼠（均P＜0.01）。结论 B-BALB/c小鼠在单克隆抗体体内诱导制备中可作为BALB/c小鼠的替代选择，能够达到减少实验动物使用数量、降低人力成本并提高抗体制备效率的目的。

关键词: 单克隆抗体, 制备效率, 杂交瘤, BALB/c小鼠, Big-BALB/c小鼠

Abstract:

Objective To compare the preparation efficiency of mouse pox and mouse hepatitis antibodies between two substrains of BALB/c and Big-BALB/c (B-BALB/c) mice, and to provide a theoretical basis and reference for the selection of laboratory animals in the preparation of monoclonal antibodies induced in vivo through hybridoma. Methods Individuals weighing more than 5% of the weight of normal animals at 4 weeks of age (the criterion for late selection is more than 10%) were selected from a population of conventionally bred BALB/c mice and bred individually, and a subline of B-BALB/c mice was prepared after 10 generations of selection. A total of 40 BALB/c mice and 40 B-BALB/c mice aged 10 to 11 weeks, half male and half female, were selected and inoculated with the mousepox monoclonal antibody hybridoma cell line G23 or the murine hepatitis monoclonal antibody hybridoma cell line Y15 pre-treated with liquid paraffin, respectively. Mice ascites containing monoclonal antibodies were obtained by in vivo induction. The antibody titer was tested by indirect ELISA. The mice were grouped based on the sub-strains, gender and inoculation type of hybridoma to analyze the ascites production, antibody titer and antibody production, and to evaluate the antibody preparation efficiency of the two BALB/c mouse sub-strains. Results After 10 generations of breeding, the body weight of 10-week-old male and female B-BALB/c mice increased by 22.3% and 12.8%, respectively, compared with BALB/c mice of the same age. Compared with BALB/c mice, B-BALB/c mice had better tolerance and adaptation to secondary ascites collection. Compared with BALB/c mice, the ascites production and antibody titer during the preparation of antibodies in B-BALB/c mice were significantly increased, especially in the hybridoma cell line G23 vaccination group (both P＜0.000 1) . After inoculation with the hybridoma cell lines G23 or Y15, the average antibody production of B-BALB/c mice (14.99×10⁴ U and 33.22×10⁴ U) was higher than that of BALB/c mice (5.33×10⁴ U and 19.31×10⁴ U) (both P＜0.01). After inoculation with hybridoma cell line G23, the average antibody production per unit body weight of B-BALB/c mice (0.55×10⁴ U/g) was higher than that of BALB/c mice (0.23×10⁴ U/g) (P＜0.000 1). And the antibody production per unit body weight of female B-BALB/c or BALB/c mice was higher than that of male B-BALB/c or BALB/c mice (both P＜0.01). Conclusion B-BALB/c mice can be used as an alternative to BALB/c mice in the in vivo induction of monoclonal antibody preparation, which can achieve the purpose of reducing the number of experimental animals used, lowering the labor cost, and improving the efficiency of antibody preparation.

Key words: Monocloning antibody, Production efficiency, Hybridomas, BALB/c mice, Big-BALB/c mice

中图分类号:

R-332

王丹, 张晓璐, 王妍, 傅博, 王文栋, 刘京, 张甦寅, 武怡荷, 吴德国, 杜小燕, 战大伟, 章秀林, 李长龙. Big-BALB/c小鼠亚系选育前后的抗体制备效率比较研究[J]. 实验动物与比较医学, 2023, 43(6): 612-618.

Dan WANG, Xiaolu ZHANG, Yan WANG, Bo FU, Wendong WANG, Jing LIU, Suyin ZHANG, Yihe WU, Deguo WU, Xiaoyan DU, Dawei ZHAN, Xiulin ZHANG, Changlong LI. Study on the Antibody Production Efficiency in Modified Big-BALB/c Mice[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 612-618.

图/表 4

表1 不同亚系小鼠接种杂交瘤细胞后平均腹水产量和平均抗体效价

Table 1 The individual average ascites production and antibody titer of mice from different sub-strains after hybridoma inoculation

杂交瘤细胞株 Cell lines	小鼠亚系 Sub-strains of mice	小鼠数量/只 Number of mice n	平均腹水产量/mL Average ascites production/mL	平均抗体效价 (×10⁴)/ (U·mL^-1) Average antibody titer (×10⁴)/(U·mL^-1)	平均抗体产量 (×10⁴)/U Average antibody production (×10⁴)/U
G23	BALB/c	20	2.24±0.52	2.38±1.09	5.33±2.62
	B-BALB/c	20	3.21±0.76^****	4.76±1.68^****	14.99±5.96^****
Y15	BALB/c	20	2.88±0.66^▲	6.65±3.23^▲▲	19.31±10.91^▲▲
	B-BALB/c	20	4.19±1.16^{**** ▲▲}	7.73±5.38 ^▲▲▲	33.22±28.13^**▲▲

图1 不同性别BALB/c和B-BALB/c小鼠接种杂交瘤细胞后的腹水产量、抗体效价及产量注：A，腹水产量；B，抗体效价；C，抗体产量。所有数据均显示为平均值±标准差，****P<0.000 1，***P<0.001，**P<0.01，*P<0.05，nsP＞0.05。B-BALB/c是由BALB/c小鼠定向选育10代后建立的新亚种，命名为Big-BALB/c小鼠。

Figure 1 The ascites production， antibody titer and production of female and male BALB/c and B-BALB/c mice after hybridoma inoculationNote：A, Ascites production; B, Antibody titer; C，Antibody production. All data are shown as means ± SD. ****P<0.000 1, ***P<0.001, **P<0.01, *P < 0.05, nsP＞0.05. B-BALB/c is a new subspecies established after 10 generations of targeted breeding of BALB/c mice, named as Big-BALB/c mice.

表2 不同亚系小鼠接种杂交瘤细胞后平均单位体重的腹水产量和抗体产量

Table 2 The average ascites production per unit body weight and antibody production of different sub-strains mice after hybridoma inoculation

杂交瘤细胞株 Cell lines	小鼠亚系 Sub-strains of mice	小鼠数量/只 Number of mice n	平均单位体重腹水产量/(mL·g^-1) The average ascites production per unit body weight/(mL·g^-1)	平均单位体重抗体产量 (×10⁴)/（U·g^-1) The average antibody production per unit body weight(×10⁴)/(U·g^-1)
G23	BALB/c	20	0.10±0.025	0.23±0.11
G23	B-BALB/c	20	0.12±0.031^***	0.55±0.22^****
Y15	BALB/c	20	0.13±0.038^▲▲	0.86±0.55^▲▲▲
Y15	B-BALB/c	20	0.16±0.055^**▲	1.27±1.19^▲▲▲

图2 不同性别BALB/c和B-BALB/c小鼠接种杂交瘤后单位体重的腹水产量及抗体产量注：所有数据均显示为平均值±标准差，****P<0.000 1，***P<0.001，**P<0.01，*P<0.05，nsP＞0.05。B-BALB/c是由BALB/c小鼠定向选育10代后建立的新亚种，命名为Big-BALB/c小鼠。

Figure 2 Ascites production and antibody production per 1 g body weight of female and male BALB/c and B-BALB/c mice after hybridoma inoculationNote： All data are shown as means±SD. ****P<0.000 1， ***P<0.001， **P<0.01， *P<0.05， nsP＞0.05. B-BALB/c is a new subspecies established after 10 generations of targeted breeding of BALB/c mice, named as Big-BALB/c mice.

参考文献 23

1	杨松鑫, 冯建远, 郭旋, 等. 小分子化合物单克隆抗体的制备及其在动物医学领域的应用[J]. 中国畜牧兽医, 2021, 48(3):1121-1131. DOI: 10.16431/j.cnki.1671-7236.2021.03.037 .
	YANG S X, FENG J Y, GUO X, et al. Preparation of monoclonal antibodies for small molecular compounds and their applications in the field of veterinary medicine[J]. China Animal Husb Vet Med, 2021, 48(3):1121-1131. DOI: 10.16431/j.cnki.1671-7236.2021.03.037 .
2	MINISTRO J, MANUEL A M, GONCALVES J. Therapeutic antibody engineering and selection strategies[J]. Adv Biochem Eng Biotechnol, 2020, 171:55-86. DOI: 10.1007/10_2019_116 .
3	MAJID A R, JAVAD R M, MALIHE P, et al. Scale-up production and characterization of anti-human cardiac troponin I monoclonal antibody in ascitic fluid of balb/c mice[J]. J Immunoassay Immunochem, 2017, 38(4):389-399. DOI: 10.1080/15321819.2016.1274263 .
4	HILGERS J, VAN NIE R, IVÁNYI D, et al. Genetic differences in BALB/c sublines[M]//Current Topics in Microbiology and Immunology. Berlin, Heidelberg: Springer Berlin Heidelberg, 1985:19-30. DOI: 10.1007/978-3-642-70740-7_3 .
5	DAM S A, JAGER A, OOMEN C A, et al. Inhibitory control in BALB/c mice sub-strains during extinction learning[J]. Eur Neuropsychopharmacol, 2019, 29(4):509-518. DOI: 10.1016/j.euroneuro.2019.02.007 .
6	SEETHARAMAIAH G S, LAND K J. Differential susceptibility of BALB/c and BALB/cBy mice to Graves' hyperthyroidism[J]. Thyroid, 2006, 16(7):651-658. DOI:10.1089/thy.2006.16.651 .
7	FARKAS B, BOLDIZSAR F, TARJANYI O, et al. BALB/c mice genetically susceptible to proteoglycan-induced arthritis and spondylitis show colony-dependent differences in disease penetrance[J]. Arthritis Res Ther, 2009, 11(1):R21. DOI: 10.1186/ar2613 .
8	POTTER M, WAX J S, BLANKENHORN E. BALB/c subline differences in susceptibility to plasmacytoma induction[J]. Curr Top Microbiol Immunol, 1985, 122:234-241. DOI: 10.1007/978-3-642-70740-7_33 .
9	TEUSCHER C, SMITH S M, TUNG K S. Experimental allergic orchitis in mice: III. Differential susceptibility and resistance among BALB/c sublines[J]. J Reprod Immunol, 1987, 10(3):219-230. DOI: 10.1016/0165-0378(87)90088-x .
10	KANG M J, GONG J E, KIM J E, et al. Influence of three BALB/c substrain backgrounds on the skin tumor induction efficacy to DMBA and TPA cotreatment[J].Lab Animal Res, 2020, 36(1):1-12. DOI: 10.1186/s42826-020-00063-z .
11	STEWART F, CALLANDER A, GARWES D J. Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice[J]. J Immunol Methods, 1989, 119(2):269-275. DOI: 10.1016/0022-1759(89)90406-7 .
12	周晓丽, 袁秀芳, 杜晓莉, 等. 重组猪干扰素-α单克隆抗体的制备和鉴定[J]. 浙江农业学报, 2015, 27(3):332-337. DOI: 10.3969/j.issn.1004-1524.2015.03.03 .
	ZHOU X L, YUAN X F, DU X L, et al. Preparation and identification of monoclonal antibodies against recombinant porcine interfer-α[J]. Acta Agric Zhejiangensis, 2015, 27(3):332-337. DOI: 10.3969/j.issn.1004-1524.2015.03.03 .
13	DE ANGELIS I, RICCERI L, VITALE A. The 3R principle: 60 years taken well. Preface[J]. Ann Ist Super Sanita, 2019, 55(4):398-399. DOI: 10.4415/ANN_19_04_15 .
14	FOLTZ I N, GUNASEKARAN K, KING C T. Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform[J]. Immunol Rev, 2016, 270(1):51-64. DOI: 10.1111/imr.12409 .
15	王卉, 刘红芹, 王爱先, 等. 重组载体免疫小鼠制备抗人CD33单克隆抗体[J]. 细胞与分子免疫学杂志, 2020, 36(2):169-174. DOI: 10.1038/sj.cr.7290338 .
	WANG H, LIU H Q, WANG A X, et al. Preparation of monoclonal antibody against human CD33 by immunizing mice with recombinant vector[J]. Chin J Cell Mol Immunol, 2020, 36(2):169-174. DOI: 10.1038/sj.cr.7290338 .
16	郑燕, 厉旭云, 高铃铃, 等. 动物实验中贯彻动物福利的探讨[J]. 基础医学教育, 2022, 24(6):431-435. DOI: 10.13754/j.issn2095-1450.2022.06.14 .
	ZHENG Y, LI X Y, GAO L L, et al. Safeguarding animal welfare in animal experiments in Chinese context[J]. Basic Med Educ, 2022, 24(6):431-435. DOI: 10.13754/j.issn2095-1450.2022.06.14 .
17	YOKOYAMA W M. Production of monoclonal antibody supernatant and ascites fluid[J]. Curr Protoc Mol Biol, 2008, 83(1): 1-10. DOI: 10.1002/0471142727.mb1110s83 .
18	EZZATIFAR F, MAJIDI J, BARADARAN B, et al. Large scale generation and characterization of anti-human IgA monoclonal antibody in ascitic fluid of BALB/c mice[J]. Adv Pharm Bull, 2015, 5(1):97-102. DOI: 10.5681/apb.2015.013 .
19	刘雨晴, 尹宝靓, 姚毅波, 等. 不完全弗氏佐剂诱导的髓样细胞抑制T细胞活性[J]. 中国免疫学杂志, 2015(3):344-346. DOI: 10.3969/j.issn.1000-484X.2015.03.012 .
	LIU Y Q, YIN B J, YAO Y B, et al. Incomplete Freund's adjuvant-induced myeloid cells inhibit T cell activity[J]. Chin J Immun, 2015(3):344-346. DOI: 10.3969/j.issn.1000-484X.2015.03.012 .
20	HOOGENRAAD N J, WRAIGHT C J. The effect of pristane on ascites tumor formation and monoclonal antibody production[J]. Methods Enzymol, 1986, 121:375-381. DOI: 10.1016/0076-6879(86)21036-8 .
21	QIAN C Y, YANG Y R, XU Q, et al. Characterization of an Escherichia coli-derived triple-type chimeric vaccine against human papillomavirus types 39, 68 and 70[J]. NPJ Vaccines, 2022, 7(1):134. DOI: 10.1038/s41541-022-00557-y .
22	LIN Y H, YAN Y, ZHANG H F, et al. Salvianolic acid A alleviates renal injury in systemic lupus erythematosus induced by pristane in BALB/c mice[J]. Acta Pharm Sin B, 2017, 7(2):159-166. DOI: 10.1016/j.apsb.2016.07.001 .
23	SHOENFELD Y, AGMON-LEVIN N. 'ASIA'-Autoimmune/inflammatory syndrome induced by adjuvants[J]. J Autoimmun, 2011, 36(1):4-8. DOI: 10.1016/j.jaut.2010.07.003 .

[1]	程继帅, 牟唐维, 柳蕾, 吴化叶, 李琦涵. BALB/c小鼠经不同途径感染单纯疱疹病毒Ⅱ型的病理学比较分析[J]. 实验动物与比较医学, 2020, 40(1): 15-21.
[2]	雷山, 刘强, 黄维金, 王佑春. 小鼠品系、性别和被毛对可视化假病毒感染模型的影响[J]. 实验动物与比较医学, 2019, 39(6): 423-428.
[3]	邹芦, 戴天娥, 卢晓, 艾晓杰. 替来他明/唑拉西泮联合右美托咪定对BALB/c小鼠麻醉效果观察[J]. 实验动物与比较医学, 2019, 39(4): 310-313.
[4]	李迎, 杜小燕, 崔晓霞, 马兰芝, 尚世臣, 赵志兵, 赵权, 李桂军, 王冬平, 陈振文. CMU/1和CMU/2长爪沙鼠与F344大鼠及BALB/c小鼠4项凝血指标分析[J]. 实验动物与比较医学, 2018, 38(1): 57-59.
[5]	顾剑洁, 葛蓉, 徐伟超, 刘丽均. 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.
[6]	邵宝珠, 唐其凤, 曹丽丽. BALB/c小鼠不同体质量范围对重组人促红素注射液体内活性测定的影响[J]. 实验动物与比较医学, 2016, 36(3): 216-218.
[7]	周洁, 高诚, 胡建华. 小鼠肝炎病毒双抗体夹心ELISA检测方法的建立[J]. 实验动物与比较医学, 2015, 35(1): 6-9.
[8]	侯倩女, 俞宏, 钱小丽. BALB/c小鼠口服诱导EGFP融合标签免疫耐受的观察[J]. 实验动物与比较医学, 2013, 33(1): 72-73.
[9]	马建国, 严望军, 刘铁龙, 肖建如, 周许辉, 贾连顺. 针对MAG, OMgp, NogoA基因的scFv (OMgp)-9R-siRNA 复合物的制备和鉴定[J]. 实验动物与比较医学, 2012, 32(4): 311-317.
[10]	郑海明, 赵航, 郑萍. 葡聚糖硫酸钠和偶氮氧甲烷建立小鼠溃疡性结肠炎癌变模型[J]. 实验动物与比较医学, 2012, 32(1): 47-50.
[11]	柴青，高文杰，谢炜，潘建忠，居颂光，张学光，薛智谋. 激发型鼠抗人4-1BBL单克隆抗体的研制[J]. , 2010, 30(4): 289-292.
[12]	高文杰，柴青，张学光，居颂光，薛智谋. 阻断型鼠抗人FL单克隆抗体的制备[J]. , 2010, 30(4): 293-296.
[13]	顾卫忠1，王晓东1，鲍世民2，邢正弘1，陈国强1. 玩具对小鼠生长繁育性能影响初探[J]. , 2008, 28(2): 131-132.
[14]	敖红1，许兰文1，杨萍1，金玫蕾2. 4个与营养代谢相关基因对小鼠的血清生化指标的影响[J]. , 2007, 27(3): 186-189.
[15]	姚健, 胡樱, 夏萍, 李春生. 三种处理饲料饲喂BALB/c小鼠的效价分析[J]. , 2005, 25(1): 47-48.

Big-BALB/c小鼠亚系选育前后的抗体制备效率比较研究

Study on the Antibody Production Efficiency in Modified Big-BALB/c Mice

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 4

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价