酿酒酵母钙通道膜蛋白单克隆抗体制备及鉴定

doi:10.16085/j.issn.1000-6613.2020-2100

摘要/Abstract

摘要：

以酿酒酵母电压门控钙通道膜蛋白（Cch1p）、牵张敏感性钙通道膜蛋白（Mid1p）和瞬时受体电位钙通道膜蛋白（Yvc1p）为研究材料，制备其单克隆抗体。采用生物信息学方法确定3种膜蛋白抗原表位，根据分析结果克隆抗原基因，并进行原核表达和表达产物分析鉴定，通过Ni²⁺-NTA树脂亲和层析技术获得重组抗原蛋白，免疫小鼠后细胞融合技术制备单克隆抗体，酶联免疫吸附测定（ELISA）检测抗体效价，免疫印迹技术检测单克隆抗体对重组纯化抗原和天然酿酒酵母钙通道膜蛋白的反应性和特异性。生物信息学分析结果表明，Cch1p、Mid1p和Yvc1p抗原表位可能分别位于1~300位氨基酸残基、359~548位氨基酸残基、1~236位氨基酸残基；克隆目的基因条带大小分别为926bp、570bp和708bp，与预期结果一致；原核表达抗原蛋白分子量分别为60000、25000和30000，Western blot检测条带正确；重组纯化抗原免疫BALB/c小鼠，细胞融合技术制备单克隆抗体，ELISA检测显示单克隆抗体效价分别高达1∶256000、1∶128000和1∶64000，Western blot检测到3种重组纯化抗原和天然酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p。这些结果说明本文制备的单克隆抗体可以成功用于检测酿酒酵母钙通道膜蛋白Cch1p、Mid1p和Yvc1p表达的相关研究。

关键词: 酿酒酵母, 钙通道膜蛋白, 分子生物学, 单克隆抗体, 生化工程

Abstract:

The monoclonal antibodies against three calcium channel membrane proteins were successfully prepared by using voltage-gated calcium channel membrane protein (Cch1p), stretch-activated calcium channel membrane protein (Mid1p), and transient receptor potential calcium channel membrane protein (Yvc1p) in Saccharomyces cerevisiae as the study materials. The epitopes of three membrane proteins were analyzed by bioinformatics methods. Based on the analysis results, the genes of three epitopes were cloned, and recombinant proteins were expressed and identified. The BALB/c mice were immunized with three recombinant antigens which were purified by Ni²⁺-NTA resin affinity chromatography, respectively. The monoclonal antibodies were generated by the cell fusion method and their titers were detected by ELISA. The reactivity and specificity of three monoclonal antibodies were tested by Western blot with three purified recombinant antigens and natural Cch1p, Mid1p and Yvc1p. The results showed that three antigen epitopes region may be located in 1—300 amino-acid residues, 359—548 amino-acid residues, and 1—236 amino-acid residues. The purpose band sizes by gene cloning were 926bp, 570bp and 708bp, which were consistent with the expected results. Furthermore, the molecular weight of three antigen proteins from prokaryotic expression systems were 60000, 25000, 30000, respectively and three bands were proved to be correct by Western blot. The ELISA results showed that three monoclonal antibodies prepared by cell fusion method presented high titers of 1∶256000, 1∶128000, and 1∶64000. These antibodies could detect the purified recombinant antigens and natural proteins of Cch1, Mid1 and Yvc1 in Western blot. These results indicated that the prepared monoclonal antibodies against Cch1p, Mid1p and Yvc1p could be successfully used for the related research about the detection of the expression of calcium channel membrane proteins in S. cerevisiae.

Key words: Saccharomyces cerevisiae, membrane protein of calcium channel, molecular biology, monoclonal antibody, biochemical engineering

中图分类号:

R392

董晓宇. 酿酒酵母钙通道膜蛋白单克隆抗体制备及鉴定[J]. 化工进展, 2021, 40(S1): 334-343.

DONG Xiaoyu. Preparation and identification of monoclonal antibodies of calcium channel membrane proteins in Saccharomyces cerevisiae[J]. Chemical Industry and Engineering Progress, 2021, 40(S1): 334-343.

图/表 12

表1 引物信息

引物名称	引物序列
RT427-CCH1-16F	AGGACGCTTACGGAACCATT
RT427-CCH1-941R	GATTCCAGTTTTCTTTGAAGGGTT
RT427-MID1-1F	AGAAGTACCGATGTCTGCTCTTTG
RT427-MID1-570R	CGTATCGTCCAATGGATGAATTAC
RT427-YVC1-1F	ATGGTATCAGCCAACGGCG
RT427-YVC1-708R	TTGGTATACAGGCGCCTTTAATC

表2 引物信息

引物名称	引物序列
XA4965F	GACACGACACCATATGAGGACGCTTACGGA
XA4965R	GACACCTCGAGTTCCAGTTTTCTTTGAAGGG
XA1209FXOF	GCCTGGTGCCGCGCGGCAGCCATATGAGAAGTACCGATGT
XA1209FXOR	CAGTGGTGGTGGTGGTGGTGCTCGAGCGTATCGTCCAATG
XA1628F	GACACGACACCATATGGTATCAGCCAACGGCGACTTG
XA1628R	GACACCTCGAGTTGGTATACAGGCGCCTTTAAT

图1 酿酒酵母RNA反转录PCR扩增目的基因1—CCH1抗原基因，926bp；2—MID1抗原基因，570bp；3—YVC1抗原基因，708bp；M—RealBand DNA 分子量标准Marker (100~10000bp)

图2 菌落PCR扩增目的基因1-1~3—CCH1抗原基因，926bp；2-1~3—MID1抗原基因，570bp；3-1~3—YVC1抗原基因，708bp；M—RealBand DNA 分子量标准Marker (100~10000bp)

图3 3种重组表达膜蛋白抗原基因序列比对分析

图4 Cch1抗原蛋白、Mid1抗原蛋白和Yvc1抗原蛋白的诱导表达M—蛋白质标准品 (14400~11600)；1—诱导前总蛋白；2—20℃上清；3—20℃细胞裂解液；4—37℃上清；5—37℃细胞裂解液

表3 单克隆抗体浓度和效价

单克隆抗体	蛋白浓度/mg·mL^-1	不同抗体稀释率下的吸光值									空白对照	阴性对照
单克隆抗体	蛋白浓度/mg·mL^-1	1000^①	2000	4000	8000	16000	32000	64000	128000	256000	空白对照	阴性对照
抗Cch1p 抗体	2.6	0.934	0.903	0.849	0.809	0.661	0.494	0.319	0.227	0.159	0.051	0.042
抗Mid1p 抗体	1.0	0.994	0.985	0.871	0.738	0.551	0.368	0.260	0.196	0.129	0.071	0.076
抗Yvc1p 抗体	5.5	1.944	1.860	1.677	0.910	0.790	0.393	0.268	0.160	0.121	0.076	0.069

图5 纯化重组Cch1p、Mid1和Yvc1抗原蛋白SDS-PAGEM—蛋白质标准品 (14400~116000)；1—Cch1抗原蛋白，蛋白分子量约60000；2—Mid1抗原蛋白，蛋白分子量25000；3—Yvc1p抗原蛋白，蛋白分子量30000

图6 纯化重组Cch1抗原蛋白、Mid1抗原蛋白和Yvc1抗原蛋白的Western blot分析M—蛋白质标准品(10000~180000)；1—Cch1抗原蛋白，蛋白分子量约60000；2—Mid1抗原蛋白，蛋白分子量25000；3—Yvc1抗原蛋白，蛋白分子量30000

图7 纯化重组Cch1抗原蛋白的质谱检测结果

图8 纯化重组抗原蛋白与单克隆抗体的Western bolt分析M—蛋白质标准品(分子量25000~130000，25000~100000)；1—纯化重组Cch1抗原蛋白，蛋白分子量约60000；2—纯化重组Mid1抗原蛋白，蛋白分子量25000；3—纯化重组Yvc1抗原蛋白，蛋白分子量30000

图9 酿酒酵母天然钙通道膜蛋白与单克隆抗体的Western blot分析M—蛋白质标准品(36000~250000；38000~117000)；1-1~3—天然Cch1蛋白，蛋白分子量234600；2-1~3—天然Mid1蛋白，蛋白分子量61500；3-1~3—天然Yvc1蛋白，蛋白分子量78000

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