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

doi:10.3969/j.issn.1004-1524.20230565

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (5): 1047-1054.DOI: 10.3969/j.issn.1004-1524.20230565

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S855.3

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.

Figures/Tables 6

References 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

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

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