粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备

doi:10.3969/j.issn.1004-1524.2021.01.05

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 34-42.DOI: 10.3969/j.issn.1004-1524.2021.01.05

粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备

刘金玉¹(), 黄鹰²^,^*()

1.长江大学生命科学学院,湖北荆州434025
2.南京师范大学生命科学学院,江苏南京210023

收稿日期:2020-08-01 出版日期:2021-01-25 发布日期:2021-01-25
作者简介:*黄鹰,E-mail:hy46457690@163.com
刘金玉(1987—),女,湖北鄂州人,博士,讲师,研究方向为生物化学与分子生物学。E-mail:545782358@qq.com
通讯作者: 黄鹰
基金资助:
湖北省教育厅科学技术研究计划指导性项目(B2019036);长江大学科研发展基金(801100010142)

Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

LIU Jinyu¹(), HUANG Ying²^,^*()

1. College of Life Science, Yangtze University, Jingzhou 434025, China
2. College of Life Sciences, Nanjing Normal University, Nanjing 210023, China

Received:2020-08-01 Online:2021-01-25 Published:2021-01-25
Contact: HUANG Ying

摘要/Abstract

摘要：

采用生物信息学方法分析粟酒裂殖酵母SpTrz2蛋白的生物学特性,利用PCR方法扩增粟酒裂殖酵母yAS56菌株SpTrz2的全长和N-末端一半的编码基因,通过NdeⅠ和XhoⅠ酶切位点将SpTrz2的全长和N-末端一半编码基因定向插入pET-28a(+)原核表达载体中,并转化大肠埃希菌BL21(DE3);经过IPTG诱导表达后采用SDS-PAGE电泳切胶纯化法获得重组蛋白,以此为抗原免疫新西兰大白兔获得多克隆抗体,通过Western blotting检测抗体特异性。结果表明,SpTrz2是一个跨膜蛋白,含有3个跨膜结构域,具有多个B细胞抗原表位。SDS-PAGE电泳分析发现,粟酒裂殖酵母SpTrz2的全长和N-末端一半(SpTrz2和SpTrz2N)都以包涵体形式高效表达,其理论相对分子质量分别约为75.99 ku和44.77 ku。Western blotting检测显示,制备的兔抗SpTrz2和SpTrz2N多克隆抗体可以特异性识别天然的SpTrz2蛋白。本研究成功诱导表达、纯化重组蛋白SpTrz2和SpTrz2N,并制备了多克隆抗体,为SpTrz2蛋白的生物学功能相关研究奠定了基础。

关键词: 粟酒裂殖酵母, SpTrz2蛋白, 原核表达, 多克隆抗体

Abstract:

To bioinformatically analyse the biological characteristics of Schizosaccharomyces pombe SpTrz2 protein, both the full-length and the N-terminal half of S. pombe sptrz2 gene were amplified by PCR and inserted into pET-28a(+) expression vector through NdeⅠ and XhoⅠ restriction enzyme sites. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and the transformants were induced by IPTG. Then recombinant proteins were purified by the way of SDS-PAGE gel slices. Polyclonal antibodies were prepared by immunizing New Zealand white rabbit using the purified recombinant proteins. The specificity of antibodies was tested by Western blotting. The results showed that SpTrz2 was a transmembrane protein with three transmembrane domains and contained multiple B cell antigen epitopes. SDS-PAGE analysis suggested that the full-length and the N-terminal half of SpTrz2 protein (SpTrz2 and SpTrz2N) with a relative molecular mass of about 75.99 ku and 44.77 ku, respectively, mainly existed in a form of inclusion body. The Western blot analysis showed that polyclonal antibodies of SpTrz2 and SpTrz2N could specifically recognize natural SpTrz2 protein. In this study, recombinant SpTrz2 and SpTrz2N protein were successfully expressed and purified, and their polyclonal antibodies were prepared. All above results could provide experimental evidence for the study of biological function of SpTrz2 protein.

Key words: Schizosaccharomyces pombe, SpTrz2 protein, prokaryotic expression, polyclonal antibody

中图分类号:

Q939.9

刘金玉, 黄鹰. 粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 34-42.

LIU Jinyu, HUANG Ying. Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 34-42.

图/表 9

表1 用于扩增目的片段的引物序列

Table 1 PCR primers for the amplification of target gene fragments

引物名称 Primer name	引物序列(5'→3') Sequences(5'→3')	限制性内切酶 Restriction enzyme
trz2-fo	GGGAATTCCATATGAAAGCTTCTCTTCTGGTTCCA	NedⅠ
trz2-re	CCGCTCGAGTATCGTTTCTCGCTGCTTGC	XhoⅠ
trz2N-dw	CCGCTCGAGTCCTTCATTTTCAAAGGGTAAAGGA	XhoⅠ

图1 长型tRNase ZL N-末端一半的氨基酸序列比对 ScTrz1-N,芽殖酵母长型tRNase ZL蛋白的N-末端一半序列;SpTrz2-N,粟酒裂殖酵母SpTrz2蛋白的N-末端一半序列;DmTrz1-N,果蝇tRNase ZL蛋白的N-末端一半序列;ELAC2-N,人的ELAC2蛋白的N-末端一半序列;黑线,假模体II、可变臂、GP模体和类Walker A模体;黑色标记表示相同氨基酸残基的保守性≥80%;灰色标记表示相似氨基酸残基;括号内的数字为氨基酸序列的位置。

Fig.1 Sequence alignment of the N-terminal halves of tRNase ZLs ScTrz1-N, the N-terminal halve of tRNase ZL protein from S. cerevisiae; SpTrz2-N, the N-terminal halve of SpTrz2 protein from S. pombe; DmTrz1-N, the N-terminal halve of tRNase ZL protein from D. melanogaster; ELAC2-N, the N-terminal halve of ELAC2 protein from Homo sapiens; Black lines indicated ψ motif II, flexible arm, GP and Walker A-like motifs; Identical residues that were conserved in at least 80% of the aligned sequences were shaded in black; Similar residues were shaded in grey; Numbers in parentheses were the positions of the amino acid sequences.

图2 SpTrz2蛋白的跨膜结构域

Fig.2 Transmembrane domain of SpTrz2 protein

图3 SpTrz2蛋白的B细胞表位 A,SpTrz2蛋白的N-末端一半的B细胞表位;B,SpTrz2蛋白C-末端一半的B细胞表位。

Fig.3 B cell epitopes in SpTrz2 protein A,The B cell epitopes in the N-terminal halve of SpTrz2 protein; B, The B cell epitopes in the C-terminal halve of SpTrz2 protein.

表2 SpTrz2 蛋白N端B细胞表位的氨基酸序列

Table 2 Amino acid sequence of B cell epitope in the N-terminal halve of SpTrz2 protein

编号 No.	开始 Start	结束 End	肽段序列 Peptide sequence	氨基酸长度 Length/aa
1	22	24	YSW	3
2	39	43	KSKRN	5
3	55	57	LNP	3
4	90	97	QASNYGGK	8
5	128	130	GIQ	3
6	134	138	GLHAP	5
7	164	173	FSSEDNADAT	10
8	202	208	KEAAGVF	7
9	219	228	PFGPSNGKLC	10
10	233	242	VLSKDGTTWI	10
11	246	254	QVVGPPRKR	9
12	325	333	PVFQRNKGR	9
13	349	360	PSTLDTQTQLPE	12
14	378	389	CKISESPSYSPV	12

表3 SpTrz2蛋白 C端B细胞表位的氨基酸序列

Table 3 Amino acid sequence of B cell epitope in the C-terminal halve of SpTrz2 protein

编号 No.	开始 Start	结束 End	肽段序列 Peptide sequence	氨基酸长度 Length/aa
1	9	14	SATCPT	6
2	48	53	GTNTEP	6
3	83	87	KANTN	5
4	128	130	TVT	3
5	167	177	YSGDTRPNEKL	11
6	193	198	FEDDLK	6
7	203	211	QRQHSTASE	9
8	232	248	RSYDADFLPPDWTIYPK	17
9	250	253	KTIY	4

图4 重组质粒pET28a-sptrz2和pET28a-sptrz2N的构建 M,DNA分子质量标准;1,sptrz2基因PCR扩增产物;2,sptrz2N基因PCR扩增产物;3~4,重组质粒pET28a-sptrz2的PCR鉴定;5~6,重组质粒pET28a-sptrz2N的PCR鉴定。

Fig.4 Construction of recombinant plasmids of pET28a-sptrz2 and pET28a-sptrz2N M, DNA marker; 1, PCR product of sptrz2; 2, PCR product of sptrz2N; 3 and 4, Identification of recombinant plasmid pET28a-sptrz2 by PCR; 5 and 6, Identification of recombinant plasmid pET28a-spTrz2N by PCR.

图5 重组蛋白SpTrz2和SpTrz2N诱导表达的SDS-PAGE分析 M,蛋白质分子质量标准;1,未经诱导的SpTrz2重组菌株;2,IPTG诱导的SpTrz2重组菌株;3,诱导后的上清;4,诱导后的沉淀,箭头标示重组蛋白SpTrz2;5,未经诱导的SpTrz2N重组菌株;6,IPTG诱导的SpTrz2N重组菌株;7,诱导后的上清;8,诱导后的沉淀,箭头标示重组蛋白SpTrz2N。

Fig.5 SDS-PAGE analysis of induced expression of recombinant protein SpTrz2 and SpTrz2N M, Protein molecular weight marker; 1, SpTrz2 recombinant strain non-induced; 2, SpTrz2 recombinant strain induced with IPTG; 3, The supernatant of SpTrz2 recombinant strain induced with IPTG; 4, The pellet of SpTrz2 recombinant strain induced with IPTG, the arrow indicated recombinant protein SpTrz2; 5, SpTrz2N recombinant strain non-induced; 6, SpTrz2N recombinant strain induced with IPTG; 7, The supernatant of SpTrz2N recombinant strain induced with IPTG; 8, The pellet of SpTrz2N recombinant strain induced with IPTG, the arrow indicated recombinant protein SpTrz2N.

图6 SpTrz2和SpTrz2N多克隆抗体的Western blotting检测 T,全细胞蛋白;PMS,去除线粒体的上清;Mt,线粒体。

Fig.6 Western blotting of polyclonal antibodies of SpTrz2 and SpTrz2N protein T, Total cell extracts; PMS, Postmitochondrial supernatants; Mt, Mitochondria.

参考文献 25

[1]	PHIZICKY E M, HOPPER A K. tRNA biology charges to the front[J]. Genes & Development, 2010,24(17):1832-1860. URL PMID
[2]	MÖRL M, MARCHFELDER A. The final cut: the importance of tRNA 3'-processing[J]. EMBO Reports, 2001,2(1):17-20. URL PMID
[3]	MARAIA R J, LAMICHHANE T N. 3' processing of eukaryotic precursor tRNAs[J]. Wiley Interdisciplinary Reviews: RNA, 2011,2(3):362-375. URL PMID
[4]	VOGEL A, SCHILLING O, SPÄTH B, et al. The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties[J]. Biological Chemistry, 2005,386(12):1253-1264. URL PMID
[5]	DAIYASU H, OSAKA K, ISHINO Y, et al. Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold[J]. FEBS Letters, 2001,503(1):1-6. DOI URL PMID
[6]	WANG Z K, ZHENG J, ZHANG X J, et al. Identification and sequence analysis of metazoan tRNA 3'-end processing enzymes tRNase Zs[J]. PLoS One, 2012,7(9):e44264. DOI URL PMID
[7]	FAN L J, WANG Z K, LIU J Y, et al. A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2[J]. BMC Evolutionary Biology, 2011,11:219.
[8]	ZHAO W, YU H Y, LI S Z, et al. Identification and analysis of candidate fungal tRNA 3'-end processing endonucleases tRNase Zs, homologs of the putative prostate cancer susceptibility protein ELAC2[J]. BMC Evolutionary Biology, 2010,10(1):1-16.
[9]	TAVTIGIAN S V, SIMARD J, TENG D H F, et al. A candidate prostate cancer susceptibility gene at chromosome 17p[J]. Nature Genetics, 2001,27(2):172-180. DOI URL PMID
[10]	MA M, LI DE LA SIERRA-GALLAY I, LAZAR N, et al. The crystal structure of Trz1, the long form RNase Z from yeast[J]. Nucleic Acids Research, 2017,45(10):6209-6216. DOI URL PMID
[11]	DUBROVSKY E B, DUBROVSKAYA V A, LEVINGER L, et al. Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo[J]. Nucleic Acids Research, 2004,32(1):255-262. DOI URL PMID
[12]	GAN X H, YANG J, LI J, et al. The fission yeast Schizosaccharomyces pombe has two distinct tRNase Z^Ls encoded by two different genes and differentially targeted to the nucleus and mitochondria[J]. The Biochemical Journal, 2011,435(1):103-111. DOI URL PMID
[13]	ZHAO Z, SU W C, YUAN S, et al. Functional conservation of tRNase Z^L among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans[J]. The Biochemical Journal, 2009,422(3):483-492. DOI URL PMID
[14]	刘金玉, 谷陈建, 杨杰, 等. 细菌和人短型tRNase Z^S与粟酒裂殖酵母长型tRNase Z^L功能保守性研究[J]. 生命科学研究, 2013,17(5):387-393.
	LIU J Y, GU C J, YANG J, et al. Functional conservation between bacterial and human tRNase Z^S and Schizosaccharomyces pombe tRNase Z^L[J]. Life Science Research, 2013,17(5):387-393.(in Chinese with English abstract)
[15]	LARKIN M A, BLACKSHIELDS G, BROWN N P, et al. Clustal W and Clustal X version 2.0[J]. Bioinformatics (Oxford, England), 2007,23(21):2947-2948. DOI URL PMID
[16]	MORENO S, KLAR A, NURSE P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe[J]. Methods in Enzymology, 1991,194:795-823. DOI URL PMID
[17]	HAACK T B, KOPAJTICH R, FREISINGER P, et al. ELAC2 mutations cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy[J]. The American Journal of Human Genetics, 2013,93(2):211-223. DOI URL PMID
[18]	LOPEZ SANCHEZ M I G, MERCER T R, DAVIES S M K, et al. RNA processing in human mitochondria[J]. Cell Cycle, 2011,10(17):2904-2916. URL PMID
[19]	XIE X, DUBROVSKY E B. Knockout of Drosophila RNase Z^L impairs mitochondrial transcript processing, respiration and cell cycle progression[J]. Nucleic Acids Research, 2015,43(21):10364-10375. DOI URL PMID
[20]	VAFAI S B, MOOTHA V K. Mitochondrial disorders as windows into an ancient organelle[J]. Nature, 2012,491(7424):374-383. DOI URL PMID
[21]	ZHANG X J, ZHAO Q Q, HUANG Y. Partitioning of the nuclear and mitochondrial tRNA 3'-end processing activities between two different proteins in Schizosaccharomyces pombe[J]. The Journal of Biological Chemistry, 2013,288(38):27415-27422. DOI URL PMID
[22]	SHANG J J, WU L, YANG Y M, et al. Overexpression of Schizosaccharomyces pombe tRNA 3'-end processing enzyme Trz2 leads to an increased cellular iron level and apoptotic cell death[J]. Fungal Genetics and Biology, 2019,122:11-20. DOI URL PMID
[23]	BUSSINEAU C M, SHUSTER J R. Genetic stability of protein expression systems in yeast[J]. Developments in Biological Standardization, 1994,83:13-19. URL PMID
[24]	ÇELIK E, ÇALıK P. Production of recombinant proteins by yeast cells[J]. Biotechnology Advances, 2012,30(5):1108-1118. DOI URL PMID
[25]	KHOW O, SUNTRARACHUN S. Strategies for production of active eukaryotic proteins in bacterial expression system[J]. Asian Pacific Journal of Tropical Biomedicine, 2012,2(2):159-162. DOI URL PMID

粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备

Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 25

相关文章 15

编辑推荐

Metrics

本文评价

[1]	沈峥嵘, 戴远兴, 郭留明, 汪芷瑶, 张恒木. 中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用[J]. 浙江农业学报, 2024, 36(9): 2042-2050.
[2]	李天恩, 周思含, 孙洪超, 付媛, 石团员, 闫文朝. 鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达[J]. 浙江农业学报, 2024, 36(3): 503-514.
[3]	张余, 金明伟, 任丽, 章毅颖, 赵洪, 刘昆, 邓姗, 褚云霞, 李寿国, 张靖立, 黄静艳, 陈海荣. 辣椒CaERF70的表达特征和转录自激活活性分析[J]. 浙江农业学报, 2024, 36(10): 2247-2256.
[4]	宋传生, 康晓飞, 樊庆忠, 王俊刚, 石雪, 张子汝, 谭青青, 曾小娇, 刘芳, 李英赛, 侯常跃. 枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达[J]. 浙江农业学报, 2023, 35(8): 1763-1772.
[5]	刘琪, 曹影丽, 魏宁波, 杨侃侃, 梁月巧, 宋祥军, 邵颖, 涂健, 祁克宗. 鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用[J]. 浙江农业学报, 2023, 35(5): 1028-1036.
[6]	薛姣雄, 赵婷芳, 张倩, 唐青海, 高翠翠, 赵铖, 张妍, 全飞杨, 刘婷, 杨灿, 杨海, 王文秀. 犬冠状病毒S1蛋白特异性小分子抗体Fab的制备[J]. 浙江农业学报, 2023, 35(11): 2568-2583.
[7]	唐国亮, 张玉宝, 王若愚, 王亚军, 赵霞, 苏学思, 金卫杰. 魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2022, 34(11): 2471-2481.
[8]	朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.
[9]	赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞. 稻瘟病菌MGG-01005的表达纯化与生物信息学分析[J]. 浙江农业学报, 2021, 33(3): 470-478.
[10]	高娉娉, 温华婷, 赵美, 王婧. 粟酒裂殖酵母接种方式对黑比诺干红葡萄酒品质的影响[J]. 浙江农业学报, 2021, 33(12): 2397-2405.
[11]	苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 104-111.
[12]	黄小珍, 乔中全, 曾慧杰, 李永欣, 何钢, 王晓明. 紫薇花器官发育调控基因LiFUL1的分离与表达分析[J]. 浙江农业学报, 2020, 32(7): 1206-1214.
[13]	陈韫陆, 单颖, 罗浩, 徐计东, 赵灵燕, 方维焕, 李肖梁. 猪Ⅲ型干扰素原核表达及其抗病毒效果研究[J]. 浙江农业学报, 2020, 32(5): 779-788.
[14]	蒲路莎, 苏世博, 陈肖韩, 赵丽丽, 陈洪岩. 鹅源星状病毒ORF2基因原核表达及遗传进化分析[J]. 浙江农业学报, 2020, 32(5): 789-797.
[15]	王元红, 邢雪, 李传峰, 朱杰, 王勇, 刘光清. 猫传染性腹膜炎病毒AH1905株N基因的生物信息学分析及原核表达[J]. 浙江农业学报, 2020, 32(3): 406-414.