【摘要】：1997年,klinefelter GR報(bào)道硝基咪唑類及其衍生物影響大鼠的生育力。進(jìn)一步分析發(fā)現(xiàn)這類化學(xué)物質(zhì)使得大鼠精子表面的一種蛋白脫落,導(dǎo)致精子活力不足。klinefelter GR將之命名為精子蛋白SP22(sperm protein 22)。SP22是廣泛表達(dá)的蛋白,在各組織中均有表達(dá)。在不同物種中均存在同源蛋白,且同源性極高。與人的致癌基因DJ1,轉(zhuǎn)錄因子RS同源性均在90%以上。與大鼠精子基因CAP1為同一基因。SP22的廣譜表達(dá),及在各物種中的極高同源性說明SP22是由一持家基因編碼,對生命活動至關(guān)重要,所以在長期進(jìn)化中其序列得以高度保守。 SP22蛋白具有多種功能。SP22抗體在大鼠體內(nèi)外均能在卵子透明帶水平上阻斷精子與卵子的結(jié)合。推測SP22直接參與頂體反應(yīng)。蛋白結(jié)構(gòu)研究顯示SP22是一種酶,它能酶解卵子透明帶幫助精子穿入卵子。SP22與致癌因子DJ1的極高同源性也說明SP22是種酶。存在于腦中的DJ1參與抗氧化機(jī)制。DJ1可與腦中具有氧化性的代謝產(chǎn)物結(jié)合并分解。DJ1的突變使得腦中的氧化物過多積累便導(dǎo)致疾病的發(fā)生。DJ1的一個氨基酸突變可直接引起早年型帕金森的發(fā)生。SP22與轉(zhuǎn)錄因子RS極其相似,存在于核內(nèi)外。在有絲分裂時部分SP22轉(zhuǎn)入核內(nèi)。但它不具有核內(nèi)定位序列,由其它核內(nèi)蛋白帶入。在核內(nèi)間接結(jié)合于核酸核蛋白RBS上。SP22可能是一個調(diào)節(jié)因子而并不是轉(zhuǎn)錄因子,參與細(xì)胞增殖過程。它與ras因子結(jié)合時具有強(qiáng)致癌活性,能導(dǎo)致大鼠NIH3T3細(xì)胞癌變。 近年來對SP22研究漸深入,但僅限于以動物實(shí)驗(yàn)階段,針對人睪丸中SP22及在男性生殖系統(tǒng)中的分布定位分析國內(nèi)外未見相關(guān)報(bào)道。因此本實(shí)驗(yàn)研究以人睪丸組織中的SP22基因?yàn)檠芯繉ο?通過RT—PCR進(jìn)行cDNA克隆,導(dǎo)入克隆T載體進(jìn)行DNA測序。所測得序列與大鼠及相關(guān)同源性基因進(jìn)行比較分析。所得基因正是人源SP22基因,進(jìn)一步證明SP22在進(jìn)化過程中具有高度保守性。將大量擴(kuò)增所獲得的人睪丸SP22基因亞克隆到表達(dá)載體pET-28(a)、pHIL-S1中,導(dǎo)入基因工程菌大腸桿菌BL21、畢赤氏酵母GS115進(jìn)行體外表達(dá)。分別在重組大腸桿菌包涵體沉淀,重組酵母菌培養(yǎng)液上清中檢測出所表達(dá)的外源蛋白。將表達(dá)出的蛋白進(jìn)行純化。原核與真核表達(dá)的重組蛋白N端都帶有6個組氨酸的His-tag(+),通過鎳離子親和層析純化。再對所得蛋白進(jìn)行鑒定分析。比較分析
[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
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陸金春,張錫然,黃宇烽;人睪丸前列腺素D合成酶的原核表達(dá)[J];醫(yī)學(xué)研究生學(xué)報(bào);2001年05期

2 高云,黃宇烽,夏欣一,馬百坤;人睪丸前列腺素D合成酶在畢赤氏酵母中的表達(dá)、純化及鑒定[J];中國生物化學(xué)與分子生物學(xué)報(bào);2003年06期

，

本文編號：2158969