本文選題：LYC4 + LYC3��； 參考：《復(fù)旦大學(xué)》2005年博士論文

【摘要】：隨著社會的發(fā)展和人類的進(jìn)步,人們越來越關(guān)心健康的話題。近年來由于性傳播疾病發(fā)病率的增加,生殖健康成為公眾關(guān)注的焦點之一。同時,很多臨床調(diào)查報告也顯示男女生殖道的繼發(fā)性急、慢性感染是引起不育的重要原因。伴隨著人類基因組計劃的初步完成及后基因組計劃的展開,鑒定的與生殖相關(guān)的分子越來越多,一些人體基因開始在生殖系統(tǒng)找到了功能點。從幾個主要的生殖科學(xué)期刊可以感受到這些分子生物學(xué)研究對生殖生物學(xué)的巨大推動作用以及今后在應(yīng)用方面的潛力。 本論文第一部分的工作是關(guān)于一個人體附睪特異表達(dá)的免疫活性蛋白基因的克隆和功能初步研究。該基因被命名為LYC4,并在GenBank中注冊(accession number:AF 326749)。此基因編碼148個氨基酸,其N端的18個氨基酸為一強的信號肽序列。同源分析顯示LYC4成熟蛋白和人體溶菌酶有38%的同源度,和其他溶菌酶也有一定同源度。其8個形成二硫鍵的半胱氨酸的保守結(jié)構(gòu)等特征符合c型溶菌酶,屬c型溶菌酶/α-乳清蛋白超基因家族。LYC4與我實驗室已克隆的人體LYC1、LYC2、LYC3及由人體基因組序列預(yù)測的LYC5,以及c型溶菌酶/α-乳清蛋白超基因家族的其他成員的ClustalW分析顯示LYC4與LYC1、LYC2、LYC3、LYC5單獨聚為一枝,提示他們組成人體類溶菌酶家族。 Northern blot分析LYC4在人體16種組織中的表達(dá)譜,發(fā)現(xiàn)LYC4在睪丸組織中特異表達(dá)。進(jìn)一步的人體睪丸、附睪組織的原位雜交(In SituHybridization,ISH)顯示LYC4高豐度表達(dá)于人體附睪頭部的上皮細(xì)胞,在附睪體和附睪尾的上皮細(xì)胞也有表達(dá)。從附睪的近端至遠(yuǎn)端基本呈表達(dá)下降的趨勢。而在睪丸的曲線精管中沒有表達(dá)信號。 用自己制備的LYC4抗體作睪丸、附睪的免疫組化分析發(fā)現(xiàn),LYC4蛋白在附睪上皮內(nèi)的表達(dá)豐度從附睪頭到附睪尾逐漸減弱,對應(yīng)了ISH的研究。在附睪管腔內(nèi)也有陽性染色。用Western blot的方法在人體精液及小鼠的附睪頭、附睪體和附睪尾,均檢測到約14kDa的預(yù)期分子量的蛋白條帶。 用his-tag融合表達(dá)的方法成功表達(dá)了人LYC4蛋白。純化后的蛋白顯示出對革蘭氏陽性菌M. Luteus和B.subtilis的殺菌活性,而對革蘭氏陰性菌E. coli無明顯殺菌活性。LYC4顯示出對婦科門診患者的陰道混菌樣本具有一定的殺滅活性,同時LYC4表現(xiàn)出對這些混菌樣本的特定細(xì)菌的偏愛性殺滅。用表達(dá)載體pcDNA3.1C所作的細(xì)胞轉(zhuǎn)染瞬時表達(dá)實驗也支持LYC4
[Abstract]:With the development of society and the progress of human beings, people are more and more concerned about the topic of health. In recent years, reproductive health has become one of the public concerns due to the increasing incidence of sexually transmitted diseases. At the same time, many clinical reports also show that male and female genital tract secondary acute, chronic infection is an important cause of infertility. With the initial completion of the human genome project and the development of the post-genome project, more and more molecules related to reproduction have been identified, and some human genes have begun to find functional points in the reproductive system. From several major journals of reproductive science, we can see that these molecular biology studies have great impetus to reproductive biology and their potential application in the future. The first part of this thesis is about the cloning and functional study of an immunoreactive protein gene specifically expressed in human epididymis. The gene was named LYC4 and was registered in GenBank with session number:AF 326749. This gene encodes 148 amino acids, and 18 amino acids at the N-terminal are a strong signal peptide sequence. Homology analysis showed that LYC4 mature protein had 38% homology with human lysozyme and had some homology with other lysozyme. The conserved structure of cysteine, which forms disulfide bond, is consistent with c type lysozyme. ClustalW analysis of c type lysozyme / 偽 -whey protein supergene family. LYC4, human LYC1, LYC2, LYC3 and LYC5 predicted by human genome sequence, as well as other members of the c type lysozyme / 偽 -whey protein supergene family, showed that LYC1, LYC2LYC3 and other members of the C-type lysozyme / 偽 -whey protein supergene family were identified by ClustalW analysis. LYC4 and LYC2 LYC3 LYC5 were clustered into a single branch. It suggests that they form a family of human lysozyme. Northern blot was used to analyze the expression profiles of LYC4 in 16 kinds of human tissues. It was found that LYC4 was specifically expressed in testis. In situ hybridization of human testis and epididymis showed that LYC4 was highly expressed in the epithelial cells of the head of the human epididymis, and also in the epididymal body and tail of the epididymis. From the proximal end to distal end of epididymis, the expression was decreased. There is no signal in the testicular curved seminiferous duct. Using the LYC4 antibody prepared by ourselves as testis, the immunohistochemical analysis of epididymis showed that the abundance of LYC4 protein in epididymal epithelium decreased gradually from the head of epididymis to the tail of epididymis, corresponding to the study of ISH. Positive staining was also found in the epididymal lumen. The expected molecular weight protein bands of 14kDa were detected in human semen, epididymal head, epididymal body and cauda epididymidis by Western blot. Human LYC4 protein was successfully expressed by his-tag fusion expression. The purified protein showed bactericidal activity against gram-positive bacteria M. Luteus and B.subtilis, but not against Gram-negative bacteria E. coli. LYC4 showed certain bactericidal activity against vaginal mixed bacteria samples of gynecological outpatients. At the same time, LYC4 showed preference for specific bacteria in these mixed bacteria samples. The transient expression experiment of cell transfection with expression vector pcDNA3.1C also supports LYC4.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：Q78

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前2條

1 王繼文;中國主要家鵝品種分子系統(tǒng)進(jìn)化研究[D];四川農(nóng)業(yè)大學(xué);2003年

2 張莉;人α-LA與LacZ基因在牛乳腺上皮細(xì)胞中的共同表達(dá)研究[D];東北農(nóng)業(yè)大學(xué);2008年

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本文編號：1974057