小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定

doi:10.3969/j.issn.1004-1524.20230565

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (5): 1047-1054.DOI: 10.3969/j.issn.1004-1524.20230565

小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定

李菊^a^,^b(), 毕冬琳^a^,^b^,^c, 杨晓莉^a^,^b^,^c, 杨东亮^a^,^b^,^c, 张潇文^a^,^b^,^c, 刘方程^a^,^b^,^c, 李琼毅^a^,^b^,^c, 柏家林^a^,^b^,^c^,^*()

西北民族大学 a.细胞基质疫苗关键技术与产业化教育部工程研究中心; b.生物医学研究中心生物工程与技术国家民委重点实验室; c.生命科学与工程学院,甘肃兰州 730030

收稿日期:2023-05-04 出版日期:2024-05-25 发布日期:2024-05-29
作者简介:李菊(1995—),女,贵州毕节人,硕士,兽医师,研究方向为预防兽医学。E-mail:1226270738@qq.com
通讯作者: * 柏家林,E-mail:jlbai@xbmu.edu.cn
基金资助:
国家自然科学基金(31860696);西北民族大学中央高校基本科研业务费项目(31920190003);兰州市人才创业创新项目(2020-RC-85)

Preparation and identification of monoclonal antibodies against non-structural protein C of Peste des petits ruminants virus

LI Ju^a^,^b(), BI Donglin^a^,^b^,^c, YANG Xiaoli^a^,^b^,^c, YANG Dongliang^a^,^b^,^c, ZHANG Xiaowen^a^,^b^,^c, LIU Fangcheng^a^,^b^,^c, LI Qiongyi^a^,^b^,^c, BAI Jialin^a^,^b^,^c^,^*()

a. Engineering Research Center for Key Technology and Industrialization of Cell-Based Vaccine,Ministry of Education; b. Key Laboratory of Bioengineering and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center; c. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China

Received:2023-05-04 Online:2024-05-25 Published:2024-05-29

摘要/Abstract

摘要：

为制备小反刍兽疫病毒(PPRV)非结构蛋白C单克隆抗体,根据PPRV C基因编码多肽链氨基酸序列抗原性分析,设计合成一条含20个氨基酸的抗原肽(CRSGKPRGETPGPLLPEIMQ)和一条含21个氨基酸的筛选抗原多肽(PLRAGERGLAPQAVQHRTLIK),将它们分别与钥孔血蓝蛋白(keyhole limpet hemocyanin, KLH)和生物素羧基蛋白(biotin carboxyl carrier protein, Biotin)交联,制备获得免疫原和筛选抗原。用免疫原肌肉注射5只8~12周龄、体重约20 g的无特定病原体(specific pathogen free, SPF)级BALB/c雌性小鼠,第1次免疫后分别间隔7 d进行第2次免疫和第3次免疫,三免后21 d进行冲击免疫,冲击免疫后第3天采集血液分离血清,采用间接酶联免疫吸附法(ELISA)测得1只免疫小鼠血清抗体效价为1:312 500,2只为1:62 500。取3只小鼠脾细胞与骨髓瘤细胞SP2/0经聚乙二醇(PEG)融合制备杂交瘤细胞,通过间接ELISA筛选出26株阳性淋巴杂交瘤细胞,进一步经克隆化培养筛选出46株单克隆细胞株。通过Western blot(WB)筛选获得2株能稳定分泌特异性抗PPRV C蛋白单克隆抗体的杂交瘤细胞株, WB、间接免疫荧光(indirect immunofluorescence assay, IFA)鉴定显示,制备的单克隆抗体具有较好的灵敏性和病毒反应性。研究结果为进一步阐明C蛋白在PPRV生命周期中的作用奠定了基础,也为小反刍兽疫(PPR)诊断提供了有效的诊断试剂。

关键词: 小反刍兽疫病毒, 非结构蛋白C, 杂交瘤细胞技术, 单克隆抗体

Abstract:

To prepare monoclonal antibody against non-structural protein C of Peste des petits ruminants virus (PPRV), a 20-amino acids antigen peptide of CRSGKPRGETPGPLLPEIMQ and a 21-amino acid screening antigen peptide of PLRAGERGLAPQAVQHRTLIK were designed and synthesized based on the antigenicity analysis of the amino acid sequence of the polypeptide chain encoded by PPRV C gene. The immunogen and screening antigen were prepared by crosslinking with keyhole limpet hemocyanin (KLH) and biotin carboxyl carrier protein (Biotin), respectively. Five female specific pathogen free (SPF) grade BALB/c mice aged 8 to 12 weeks and weighing about 20 g were given intramuscular injection of immunogen for the first immunization, while the second immunization and the third immunization were given at intervals of 7 d after the first immunization, the rush immunization was given at 21 d after the third immunization, and the blood was collected and serum was separated on the third day after the rush immunization. The titer of serum antibody of one immunized mouse was 1:312 500 and the two mice was 1:62 500 by indirect enzyme linked immunosorbent assay (ELISA) detection. Three mouse spleen cells were fused with myeloma cells SP2/0 by polyethylene glycol (PEG) to produce hybridoma cells. 26 positive lymphohybridoma cells were screened by indirect ELISA, and 46 monoclonal cell lines were screened by clonal culture. Western blot (WB) screened two hybridoma cell lines that could steadily secrete monoclonal antibodies against PPRV C protein. WB and indirect immunofluorescence assay (IFA) showed that the monoclonal antibodies were of good sensitivity and viral reactivity. These results laid a foundation for further elucidating the role of C protein in the life cycle of PPRV, and also provided an effective diagnostic reagent for the detection of peste des petits ruminants (PPR).

Key words: Peste des petits ruminants virus, non-structural protein C, hybridoma cell fusion technique, monoclonal antibody

中图分类号:

S855.3

李菊, 毕冬琳, 杨晓莉, 杨东亮, 张潇文, 刘方程, 李琼毅, 柏家林. 小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定[J]. 浙江农业学报, 2024, 36(5): 1047-1054.

LI Ju, BI Donglin, YANG Xiaoli, YANG Dongliang, ZHANG Xiaowen, LIU Fangcheng, LI Qiongyi, BAI Jialin. Preparation and identification of monoclonal antibodies against non-structural protein C of Peste des petits ruminants virus[J]. Acta Agriculturae Zhejiangensis, 2024, 36(5): 1047-1054.

图/表 6

参考文献 24

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免疫次数 Immunization times	免疫时间 Immune time/d	每只免疫剂量 Each immune dose/μg	每只免疫佐剂 Each immune adjuvant/μg	免疫方法 Immune method
一次免疫First immunization	0	30	FCA 15	肌肉注射Intramuscular injection
二次免疫Second immunization	14	30	FIA 15	肌肉注射Intramuscular injection
三次免疫Third immunization	28	30	FIA 15	肌肉注射Intramuscular injection
冲击免疫Rush immunization	59	200	FIA 100	尾静脉注射Tail vein injection

免疫次数 Immunization times	免疫时间 Immune time/d	每只免疫剂量 Each immune dose/μg	每只免疫佐剂 Each immune adjuvant/μg	免疫方法 Immune method
一次免疫First immunization	0	30	FCA 15	肌肉注射Intramuscular injection
二次免疫Second immunization	14	30	FIA 15	肌肉注射Intramuscular injection
三次免疫Third immunization	28	30	FIA 15	肌肉注射Intramuscular injection
冲击免疫Rush immunization	59	200	FIA 100	尾静脉注射Tail vein injection

小鼠编号 Mouse No.	稀释度Dilution rate						免疫前血清 Non-immune serum (1:500)
小鼠编号 Mouse No.	1:500	1:2 500	1:12 500	1:62 500	1:312 500	1:1562 500	免疫前血清 Non-immune serum (1:500)
PPC-01	1.634	1.183	0.771	0.387	0.114	0.148	0.038
	1.807	1.030	0.531	0.381	0.127	0.058	0.039
PPC-02	1.892	0.791	0.651	0.298	0.135	0.053	0.039
	1.876	0.950	0.984	0.382	0.169	0.082	0.038
PPC-03	3.092	2.940	1.791	1.355	0.887	0.123	0.051
	3.114	2.715	2.779	1.468	0.561	0.272	0.039
PPC-04	1.531	0.518	0.548	0.175	0.091	0.055	0.039
	1.382	0.507	0.166	0.258	0.069	0.042	0.040
PPC-05	1.877	1.208	0.685	0.274	0.146	0.079	0.039
	2.094	1.221	0.737	0.195	0.138	0.061	0.038

小鼠编号 Mouse No.	稀释度Dilution rate						免疫前血清 Non-immune serum (1:500)
小鼠编号 Mouse No.	1:500	1:2 500	1:12 500	1:62 500	1:312 500	1:1562 500	免疫前血清 Non-immune serum (1:500)
PPC-01	1.634	1.183	0.771	0.387	0.114	0.148	0.038
	1.807	1.030	0.531	0.381	0.127	0.058	0.039
PPC-02	1.892	0.791	0.651	0.298	0.135	0.053	0.039
	1.876	0.950	0.984	0.382	0.169	0.082	0.038
PPC-03	3.092	2.940	1.791	1.355	0.887	0.123	0.051
	3.114	2.715	2.779	1.468	0.561	0.272	0.039
PPC-04	1.531	0.518	0.548	0.175	0.091	0.055	0.039
	1.382	0.507	0.166	0.258	0.069	0.042	0.040
PPC-05	1.877	1.208	0.685	0.274	0.146	0.079	0.039
	2.094	1.221	0.737	0.195	0.138	0.061	0.038

初筛阳性孔 Preliminary screening positive hole	亚克隆阳性孔 Subclonal positive hole
PPC-01-1D6	1A9、1E2、1E3
PPC-01-1F10	1A9、1B11、1C3
PPC-01-2F9	1C12、1D12、1E4
PPC-01-2D3	1A5、1D11
PPC-01-4A3-1E6	1B5、1C8、1E3
PPC-01-4B8-1A9	1A7、1B5、1E1
PPC-01-4E2-1A6	1A8、1C4、1E2
PPC-01-4F10	1C12、1E4、1F4
PPC-01-5H12	1A6、1B3、1B8
PPC-02-1G4-1A7	1C6、1F3、1F4
PPC-02-2C5	1A6、1E5、1F9
PPC-03-2E9	1A4、1A12、1H3
PPC-03-2H10-1H3	1B6、1D5、1E11
PPC-03-4B12-1D9	1A6、1A7、1B6
PPC-03-6B12-1H3	1C6、1G6、1H2
PPC-03-6E1	1G2、1G6

小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against non-structural protein C of Peste des petits ruminants virus

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