人体LYC4基因克隆暨LYC4、LYC3、Cdv-1R与生殖关系的初步研究

发布时间：2018-06-03 20:22

本文选题：LYC4 + LYC3　；参考：《复旦大学》2005年博士论文

【摘要】：随着社会的发展和人类的进步,人们越来越关心健康的话题。近年来由于性传播疾病发病率的增加,生殖健康成为公众关注的焦点之一。同时,很多临床调查报告也显示男女生殖道的继发性急、慢性感染是引起不育的重要原因。伴随着人类基因组计划的初步完成及后基因组计划的展开,鉴定的与生殖相关的分子越来越多,一些人体基因开始在生殖系统找到了功能点。从几个主要的生殖科学期刊可以感受到这些分子生物学研究对生殖生物学的巨大推动作用以及今后在应用方面的潜力。本论文第一部分的工作是关于一个人体附睾特异表达的免疫活性蛋白基因的克隆和功能初步研究。该基因被命名为LYC4,并在GenBank中注册(accession number:AF 326749)。此基因编码148个氨基酸,其N端的18个氨基酸为一强的信号肽序列。同源分析显示LYC4成熟蛋白和人体溶菌酶有38%的同源度,和其他溶菌酶也有一定同源度。其8个形成二硫键的半胱氨酸的保守结构等特征符合c型溶菌酶,属c型溶菌酶/α-乳清蛋白超基因家族。LYC4与我实验室已克隆的人体LYC1、LYC2、LYC3及由人体基因组序列预测的LYC5,以及c型溶菌酶/α-乳清蛋白超基因家族的其他成员的ClustalW分析显示LYC4与LYC1、LYC2、LYC3、LYC5单独聚为一枝,提示他们组成人体类溶菌酶家族。 Northern blot分析LYC4在人体16种组织中的表达谱,发现LYC4在睾丸组织中特异表达。进一步的人体睾丸、附睾组织的原位杂交(In SituHybridization,ISH)显示LYC4高丰度表达于人体附睾头部的上皮细胞,在附睾体和附睾尾的上皮细胞也有表达。从附睾的近端至远端基本呈表达下降的趋势。而在睾丸的曲线精管中没有表达信号。用自己制备的LYC4抗体作睾丸、附睾的免疫组化分析发现,LYC4蛋白在附睾上皮内的表达丰度从附睾头到附睾尾逐渐减弱,对应了ISH的研究。在附睾管腔内也有阳性染色。用Western blot的方法在人体精液及小鼠的附睾头、附睾体和附睾尾,均检测到约14kDa的预期分子量的蛋白条带。用his-tag融合表达的方法成功表达了人LYC4蛋白。纯化后的蛋白显示出对革兰氏阳性菌M. Luteus和B.subtilis的杀菌活性,而对革兰氏阴性菌E. coli无明显杀菌活性。LYC4显示出对妇科门诊患者的阴道混菌样本具有一定的杀灭活性,同时LYC4表现出对这些混菌样本的特定细菌的偏爱性杀灭。用表达载体pcDNA3.1C所作的细胞转染瞬时表达实验也支持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 / 伪 -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 / 伪 -whey protein supergene family, showed that LYC1, LYC2LYC3 and other members of the C-type lysozyme / 伪 -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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q78

【共引文献】