人睾丸精子蛋白SP22的基因工程表达及抗体制备
发布时间：2018-08-02 09:18
【摘要】：1997年,klinefelter GR报道硝基咪唑类及其衍生物影响大鼠的生育力。进一步分析发现这类化学物质使得大鼠精子表面的一种蛋白脱落,导致精子活力不足。klinefelter GR将之命名为精子蛋白SP22(sperm protein 22)。SP22是广泛表达的蛋白,在各组织中均有表达。在不同物种中均存在同源蛋白,且同源性极高。与人的致癌基因DJ1,转录因子RS同源性均在90%以上。与大鼠精子基因CAP1为同一基因。SP22的广谱表达,及在各物种中的极高同源性说明SP22是由一持家基因编码,对生命活动至关重要,所以在长期进化中其序列得以高度保守。 SP22蛋白具有多种功能。SP22抗体在大鼠体内外均能在卵子透明带水平上阻断精子与卵子的结合。推测SP22直接参与顶体反应。蛋白结构研究显示SP22是一种酶,它能酶解卵子透明带帮助精子穿入卵子。SP22与致癌因子DJ1的极高同源性也说明SP22是种酶。存在于脑中的DJ1参与抗氧化机制。DJ1可与脑中具有氧化性的代谢产物结合并分解。DJ1的突变使得脑中的氧化物过多积累便导致疾病的发生。DJ1的一个氨基酸突变可直接引起早年型帕金森的发生。SP22与转录因子RS极其相似,存在于核内外。在有丝分裂时部分SP22转入核内。但它不具有核内定位序列,由其它核内蛋白带入。在核内间接结合于核酸核蛋白RBS上。SP22可能是一个调节因子而并不是转录因子,参与细胞增殖过程。它与ras因子结合时具有强致癌活性,能导致大鼠NIH3T3细胞癌变。 近年来对SP22研究渐深入,但仅限于以动物实验阶段,针对人睾丸中SP22及在男性生殖系统中的分布定位分析国内外未见相关报道。因此本实验研究以人睾丸组织中的SP22基因为研究对象,通过RT—PCR进行cDNA克隆,导入克隆T载体进行DNA测序。所测得序列与大鼠及相关同源性基因进行比较分析。所得基因正是人源SP22基因,进一步证明SP22在进化过程中具有高度保守性。将大量扩增所获得的人睾丸SP22基因亚克隆到表达载体pET-28(a)、pHIL-S1中,导入基因工程菌大肠杆菌BL21、毕赤氏酵母GS115进行体外表达。分别在重组大肠杆菌包涵体沉淀,重组酵母菌培养液上清中检测出所表达的外源蛋白。将表达出的蛋白进行纯化。原核与真核表达的重组蛋白N端都带有6个组氨酸的His-tag(+),通过镍离子亲和层析纯化。再对所得蛋白进行鉴定分析。比较分析
[Abstract]:In 1997, Klinefelter gr reported the effects of nitroimidazole and its derivatives on the fertility of rats. Further analysis showed that this kind of chemical compounds caused a protein on the sperm surface of rats to fall off, resulting in sperm motility. Klinefelter gr named sperm protein SP22 (sperm protein 22) .SP22 is a widely expressed protein, which is expressed in all tissues. Homologous proteins exist in different species and have high homology. The homology with human oncogene DJ 1 and transcription factor RS is more than 90%. The wide spectrum expression of the same gene. SP22 as the rat spermatozoa gene CAP1 and its high homology in various species indicate that SP22 is encoded by a housekeeping gene and is essential for life activity. Therefore, its sequence has been highly conserved in the long term evolution. SP22 protein has many functions. SP22 antibody in vivo and in vitro can be at the level of egg pellucida in rats. Block the combination of sperm and eggs. It is speculated that SP22 is directly involved in acrosome reaction. Protein structure studies show that SP22 is an enzyme that can hydrolyze the pellucida of the egg to help sperm penetrate into the egg. SP22 is highly homologous to the oncogenic factor DJ1. It also indicates that SP22 is an enzyme. DJ1 in the brain participates in antioxidant mechanism. DJ1 binds to oxidizing metabolites in the brain and decomposes the mutation of. DJ1 so that the excessive accumulation of oxides in the brain leads to the occurrence of disease. A amino acid mutation of DJ1 can directly lead to the occurrence of disease. The genesis of early type Parkinson. SP22 is very similar to that of transcription factor RS. It exists inside and outside the nucleus. Part of the SP22 is transferred into the nucleus during mitosis. However, it does not have a nuclear localization sequence and is carried by other nuclear proteins. SP22 may be a regulatory factor and not a transcriptional factor, which is involved in cell proliferation. It has strong carcinogenic activity when combined with ras factor and can cause carcinogenesis of rat NIH3T3 cells. In recent years, the research on SP22 has been deepened, but only in the animal experiment stage. There are no reports about the distribution and localization of SP22 in human testis and in the male reproductive system at home and abroad. Therefore, the SP22 gene in human testis was cloned by RT-PCR and cloned into T vector for DNA sequencing. The sequence was compared with rat and related homologous genes. The obtained gene is the human SP22 gene, which further proves that SP22 is highly conserved in the course of evolution. The human testis SP22 gene was subcloned into the expression vector pET-28 (a) pIL-S1, and was introduced into Escherichia coli BL21 and Pichia pastoris GS115 for expression in vitro. The extraneous proteins were detected in the supernatant of recombinant Escherichia coli inclusion body and recombinant yeast culture medium respectively. The expressed protein was purified. Both prokaryotic and eukaryotic expressed recombinant protein His-tag (), containing 6 histidine was purified by nickel ion affinity chromatography. The protein was identified and analyzed. Comparative analysis
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R321
本文编号：2158969
[Abstract]:In 1997, Klinefelter gr reported the effects of nitroimidazole and its derivatives on the fertility of rats. Further analysis showed that this kind of chemical compounds caused a protein on the sperm surface of rats to fall off, resulting in sperm motility. Klinefelter gr named sperm protein SP22 (sperm protein 22) .SP22 is a widely expressed protein, which is expressed in all tissues. Homologous proteins exist in different species and have high homology. The homology with human oncogene DJ 1 and transcription factor RS is more than 90%. The wide spectrum expression of the same gene. SP22 as the rat spermatozoa gene CAP1 and its high homology in various species indicate that SP22 is encoded by a housekeeping gene and is essential for life activity. Therefore, its sequence has been highly conserved in the long term evolution. SP22 protein has many functions. SP22 antibody in vivo and in vitro can be at the level of egg pellucida in rats. Block the combination of sperm and eggs. It is speculated that SP22 is directly involved in acrosome reaction. Protein structure studies show that SP22 is an enzyme that can hydrolyze the pellucida of the egg to help sperm penetrate into the egg. SP22 is highly homologous to the oncogenic factor DJ1. It also indicates that SP22 is an enzyme. DJ1 in the brain participates in antioxidant mechanism. DJ1 binds to oxidizing metabolites in the brain and decomposes the mutation of. DJ1 so that the excessive accumulation of oxides in the brain leads to the occurrence of disease. A amino acid mutation of DJ1 can directly lead to the occurrence of disease. The genesis of early type Parkinson. SP22 is very similar to that of transcription factor RS. It exists inside and outside the nucleus. Part of the SP22 is transferred into the nucleus during mitosis. However, it does not have a nuclear localization sequence and is carried by other nuclear proteins. SP22 may be a regulatory factor and not a transcriptional factor, which is involved in cell proliferation. It has strong carcinogenic activity when combined with ras factor and can cause carcinogenesis of rat NIH3T3 cells. In recent years, the research on SP22 has been deepened, but only in the animal experiment stage. There are no reports about the distribution and localization of SP22 in human testis and in the male reproductive system at home and abroad. Therefore, the SP22 gene in human testis was cloned by RT-PCR and cloned into T vector for DNA sequencing. The sequence was compared with rat and related homologous genes. The obtained gene is the human SP22 gene, which further proves that SP22 is highly conserved in the course of evolution. The human testis SP22 gene was subcloned into the expression vector pET-28 (a) pIL-S1, and was introduced into Escherichia coli BL21 and Pichia pastoris GS115 for expression in vitro. The extraneous proteins were detected in the supernatant of recombinant Escherichia coli inclusion body and recombinant yeast culture medium respectively. The expressed protein was purified. Both prokaryotic and eukaryotic expressed recombinant protein His-tag (), containing 6 histidine was purified by nickel ion affinity chromatography. The protein was identified and analyzed. Comparative analysis
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R321
【参考文献】
相关期刊论文 前2条
1 陆金春,张锡然,黄宇烽;人睾丸前列腺素D合成酶的原核表达[J];医学研究生学报;2001年05期
2 高云,黄宇烽,夏欣一,马百坤;人睾丸前列腺素D合成酶在毕赤氏酵母中的表达、纯化及鉴定[J];中国生物化学与分子生物学报;2003年06期
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