精子特异性蛋白Cabs1多克隆抗体的制备及多用途验证

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

摘要/Abstract

摘要：

制备抗小鼠Cabs1（Calcium-binding and spermatid-specific protein 1）多克隆抗体,以便进一步利用小鼠模型解析Cabs1蛋白在精子变形与精子功能调控中的功能。以密码子的简并性和偏好性为原则,优化小鼠Cabs1基因编码序列,构建重组表达载体ET-30a（+）/Cabs1,原核表达系统表达重组蛋白,免疫新西兰大白兔获得血清,通过亲和纯化得到Cabs1多克隆抗体。Western Blot、免疫荧光实验验证抗体的应用价值。结果显示,获得了高效价、特异性识别Cabs1的多克隆抗体。Western Blot、免疫荧光实验发现小鼠Cabs1蛋白开始表达于长型精子阶段,在成熟精子中主要定位于顶体和主段。同时进一步发现该抗体还可以识别人Cabs1蛋白。此外,Cabs1抗体也可以识别天然未变性的Cabs1蛋白,用于鉴定和研究Cabs1蛋白的互作复合物。制备的抗体解决了目前商业化的Cabs1抗体均不能识别小鼠来源的Cabs1蛋白的问题。

关键词: 精子变形, 精子功能, Cabs1, 原核表达, 多克隆抗体

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

彭利忠, 张鹏, 周雯雯, 曾旭辉, 张小宁. 精子特异性蛋白Cabs1多克隆抗体的制备及多用途验证[J]. 生物技术通报, 2021, 37(3): 261-270.

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.

图/表 7

参考文献 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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