Preparation and Multi-purpose Validation of Sperm-specific Protein Cabs1 Polyclonal Antibody

doi:10.13560/j.cnki.biotech.bull.1985.2020-0724

Abstract

Abstract:

This study aims to prepare anti-mouse Cabs1（calcium-binding and spermatid-specific protein 1）polyclonal antibody contributing to further analyze the Cabs1 functions in the regulation of spermiogenesis and sperm function in mice. The mouse Cabs1 gene coding sequence was optimized based on the codon degeneracy and usage bias. Next,the recombinant expression vector ET-30a（+）/Cabs1 was constructed to express recombinant protein. The recombinant protein was subcutaneously immunized New Zealand white rabbit to produce serum,and the Cabs1 polyclonal antibody was obtained by affinity and purification. Western blot and immunofluorescence experiments verified the application value of antibodies. As results,polyclonal antibodies with high titer and specificity for Cabs1 were obtained,and the results by Western blot and immunofluorescence experiments revealed that Cabs1 protein of the mouse began to be expressed in elongating spermatid and mainly located in the acrosome and the flagellar principal piece. This antibody also could be used to immunological recognition for human Cabs1. In addition,our Cabs1 antibody also recognized naturally un-denatured Cabs1 protein,suggesting that this antibody could be employed to identify and study the interaction complexes of Cabs1. In conclusion,the prepared antibody solves the problem that the commercial Cabs1 antibodies cannot recognize the mouse Cabs1 protein.

Key words: spermiogenesis, sperm function, Cabs1, prokaryotic expression, polyclonal antibody

PENG Li-zhong, ZHANG Peng, ZHOU Wen-wen, ZENG Xu-hui, ZHANG Xiao-ning. Preparation and Multi-purpose Validation of Sperm-specific Protein Cabs1 Polyclonal Antibody[J]. Biotechnology Bulletin, 2021, 37(3): 261-270.

Figures/Tables 7

References 14

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Peptide	Sequence	RT	Observed Mono m/z	Theoretical Mono m/z	Error（PPM）
T25-26	DKEDLSTTNSGLFK	13.32	518.9307	518.9264	8.4
T3-4	DNSKTPTEADIFFGADNTIPK	19.06	761.0448	761.0376	9.4
T14	EDIFLTTIDTGEK	18.79	741.3769	741.3721	6.4
T26	EDLSTTNSGLFK	14.39	656.3287	656.3250	5.7
T11	EITTPTETPNSKPK	7.98	514.9434	514.9385	9.5
T23	ETASEAESHAVLLTAVESR	15.95	667.3413	667.3359	8.2
T16	EVTTIPDITNIAEENVTEIK	21.28	743.7271	743.7214	7.7
T20	FITVFELTNSAEK	21.16	749.8970	749.8930	5.3
T20	FITVFELTNSAEK	21.16	500.2678	500.2644	6.7
T12	GSITENFIPVK	17.06	602.8360	602.8322	6.2
T13	IGNTSSPVGTVSLIDFSSNTAK	19.78	732.3832	732.3779	7.3
T13-14	IGNTSSPVGTVSLIDFSSNTAKEDIFLTTIDTGEK	22.56	915.2239	915.2150	9.7
T2	IYSRPPR	2.33	444.7582	444.7561	4.7
T9	LEDDIEGCLK	13.25	596.2835	596.2817	3.0
T17	LIVSEDRPK	8.32	352.8777	352.8731	13.1
T8-9	LKLEDDIEGCLK	15.74	478.2537	478.2499	7.9
T19	LSDSEEEK	1.37	468.7124	468.7115	2.0
T19-20	LSDSEEEKFITVFELTNSAEK	21.31	806.0700	806.0637	7.8
T6	LTPTK

Peptide	Sequence	RT	Observed Mono m/z	Theoretical Mono m/z	Error（PPM）
T25-26	DKEDLSTTNSGLFK	13.32	518.9307	518.9264	8.4
T3-4	DNSKTPTEADIFFGADNTIPK	19.06	761.0448	761.0376	9.4
T14	EDIFLTTIDTGEK	18.79	741.3769	741.3721	6.4
T26	EDLSTTNSGLFK	14.39	656.3287	656.3250	5.7
T11	EITTPTETPNSKPK	7.98	514.9434	514.9385	9.5
T23	ETASEAESHAVLLTAVESR	15.95	667.3413	667.3359	8.2
T16	EVTTIPDITNIAEENVTEIK	21.28	743.7271	743.7214	7.7
T20	FITVFELTNSAEK	21.16	749.8970	749.8930	5.3
T20	FITVFELTNSAEK	21.16	500.2678	500.2644	6.7
T12	GSITENFIPVK	17.06	602.8360	602.8322	6.2
T13	IGNTSSPVGTVSLIDFSSNTAK	19.78	732.3832	732.3779	7.3
T13-14	IGNTSSPVGTVSLIDFSSNTAKEDIFLTTIDTGEK	22.56	915.2239	915.2150	9.7
T2	IYSRPPR	2.33	444.7582	444.7561	4.7
T9	LEDDIEGCLK	13.25	596.2835	596.2817	3.0
T17	LIVSEDRPK	8.32	352.8777	352.8731	13.1
T8-9	LKLEDDIEGCLK	15.74	478.2537	478.2499	7.9
T19	LSDSEEEK	1.37	468.7124	468.7115	2.0
T19-20	LSDSEEEKFITVFELTNSAEK	21.31	806.0700	806.0637	7.8
T6	LTPTK

	Diluted from 1 mg/mL	Antibody Concen-tration（ng/mL）	Rabbit #4279_OD450nm	Rabbit #4286_OD450nm
NC	1：1 000	N/A	0.068	0.077
1	1：1 000	1 000.00	2.938	3.470
2	1：2 000	500.00	2.856	2.863
3	1：4 000	250.00	2.723	2.734
4	1：8 000	125.00	2.620	2.584
5	1：16 000	62.50	2.540	2.538
6	1：32 000	31.25	2.349	2.211
7	1：64 000	15.62	2.033	2.062
8	1：128 000	7.81	1.583	1.550
9	1：256 000	3.90	1.191	1.159
10	1：512 000	1.95	0.743	0.709
11	Blank	Blank	0.061	0.076
12	Blank	Blank	0.061	0.076
	Titer	Titer	>1：512 000	>1：512 000

	Diluted from 1 mg/mL	Antibody Concen-tration（ng/mL）	Rabbit #4279_OD450nm	Rabbit #4286_OD450nm
NC	1：1 000	N/A	0.068	0.077
1	1：1 000	1 000.00	2.938	3.470
2	1：2 000	500.00	2.856	2.863
3	1：4 000	250.00	2.723	2.734
4	1：8 000	125.00	2.620	2.584
5	1：16 000	62.50	2.540	2.538
6	1：32 000	31.25	2.349	2.211
7	1：64 000	15.62	2.033	2.062
8	1：128 000	7.81	1.583	1.550
9	1：256 000	3.90	1.191	1.159
10	1：512 000	1.95	0.743	0.709
11	Blank	Blank	0.061	0.076
12	Blank	Blank	0.061	0.076
	Titer	Titer	>1：512 000	>1：512 000

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