精子发生相关基因ZNF230基因打靶载体的构建及该基因在肿瘤组织中的表达研究
发布时间：2018-08-25 11:25
【摘要】： 精子发生是一个多阶段，多步骤持续的细胞分裂和分化过程，它受到性染色体和常染色体上一系列基因的调控。研究表明：锌指蛋白基因家族作为哺乳动物最大的基因家族之一，参与了精子发生和成熟的各个阶段的基因表达调控。精子发生相关基因ZNF230属于锌指蛋白家族，位于11p15，是我室克隆的一个与人类精子发生过程相关的基因。 本研究围绕精子发生相关基因ZNF230的功能研究，分为两个部分。 第一部分：构建小鼠锌指蛋白基因Znf230条件性打靶载体，为产生Znf230基因条件性敲除的小鼠模型做好准备工作。设计和合成特异性引物，经PCR从小鼠的基因组中扩增出5′同源臂、3′同源臂及锚定序列，长度分别为4，4，1kb的片段，反向插入pBS载体的Neo基因的两侧，从而构建小鼠Znf230条件性打靶载体Znf230-pBS(5)。经过限制性内切酶及DNA测序鉴定，证实该条件性基因打靶载体含有的同源序列与Genebank公布的基因序列一致，表明载体构建成功。PCR技术和定向克隆技术是构建条件基因打靶载体简单而可靠的方法。运用该技术获得了小鼠锌指蛋白基因Znf230的条件性打靶载体。 第二部分：癌-睾丸抗原(Cancer-testis antigen，CTA)是目前发展肿瘤免疫治疗的理想靶分子，因其在多种肿瘤组织中均有表达，但只在正常人体的睾丸组织表达，其他组织无表达。在这部分工作中我们从蛋白和mRNA水平检测了ZNF230在不同组织类型的肿瘤中的表达情况，我们发现该基因在肺癌、肝癌、肾癌、食管癌、胃癌等肿瘤中有不同程度的表达，提示该基因为CT抗原家族的新成员。
[Abstract]:Spermatogenesis is a multi-stage, multi-step process of cell division and differentiation, which is regulated by a series of genes on sex chromosome and autosomal. As one of the largest gene families in mammals, zinc finger protein gene family is involved in the regulation of gene expression in all stages of spermatogenesis and maturation. Spermatogenesis related gene ZNF230 belongs to the zinc finger protein family located at 11p15 and is a gene related to human spermatogenesis cloned in our laboratory. This study focuses on the function of spermatogenesis related gene ZNF230, which is divided into two parts. Part one: construct the Znf230 conditional targeting vector of mouse zinc finger protein gene, and prepare the mouse model for producing Znf230 gene conditional knockout. A specific primer was designed and synthesized. The 5'homologous arm 3'homologous arm and its anchoring sequence were amplified from mouse genome by PCR. The fragments with the length of 41kb were inserted into the two sides of the Neo gene of the pBS vector. The conditional targeting vector Znf230-pBS (5) of mouse Znf230 was constructed. By restriction endonuclease and DNA sequencing, it was confirmed that the homologous sequence contained in the conditional gene targeting vector was the same as that published by Genebank. The results showed that the successful construction of vector. PCR and directional cloning were simple and reliable methods to construct conditional gene targeting vector. The conditional targeting vector of mouse zinc finger protein gene Znf230 was obtained by this technique. The second part: carcinomatous testis antigen (Cancer-testis antigen,CTA) is an ideal target molecule for the development of tumor immunotherapy, because it is expressed in many kinds of tumor tissues, but only in normal human testis, but not in other tissues. In this part of our work, we detected the expression of ZNF230 in different tissue types of tumors at the protein and mRNA levels, and we found that the gene was expressed to different degrees in lung cancer, liver cancer, renal cancer, esophageal cancer, gastric cancer, and so on. It is suggested that this gene is a new member of the CT antigen family.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346;R73-3
本文编号：2202730
[Abstract]:Spermatogenesis is a multi-stage, multi-step process of cell division and differentiation, which is regulated by a series of genes on sex chromosome and autosomal. As one of the largest gene families in mammals, zinc finger protein gene family is involved in the regulation of gene expression in all stages of spermatogenesis and maturation. Spermatogenesis related gene ZNF230 belongs to the zinc finger protein family located at 11p15 and is a gene related to human spermatogenesis cloned in our laboratory. This study focuses on the function of spermatogenesis related gene ZNF230, which is divided into two parts. Part one: construct the Znf230 conditional targeting vector of mouse zinc finger protein gene, and prepare the mouse model for producing Znf230 gene conditional knockout. A specific primer was designed and synthesized. The 5'homologous arm 3'homologous arm and its anchoring sequence were amplified from mouse genome by PCR. The fragments with the length of 41kb were inserted into the two sides of the Neo gene of the pBS vector. The conditional targeting vector Znf230-pBS (5) of mouse Znf230 was constructed. By restriction endonuclease and DNA sequencing, it was confirmed that the homologous sequence contained in the conditional gene targeting vector was the same as that published by Genebank. The results showed that the successful construction of vector. PCR and directional cloning were simple and reliable methods to construct conditional gene targeting vector. The conditional targeting vector of mouse zinc finger protein gene Znf230 was obtained by this technique. The second part: carcinomatous testis antigen (Cancer-testis antigen,CTA) is an ideal target molecule for the development of tumor immunotherapy, because it is expressed in many kinds of tumor tissues, but only in normal human testis, but not in other tissues. In this part of our work, we detected the expression of ZNF230 in different tissue types of tumors at the protein and mRNA levels, and we found that the gene was expressed to different degrees in lung cancer, liver cancer, renal cancer, esophageal cancer, gastric cancer, and so on. It is suggested that this gene is a new member of the CT antigen family.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346;R73-3
【参考文献】
相关期刊论文 前1条
1 杨晓,黄培堂,黄翠芬;在小鼠进行基因打靶的研究进展[J];科学通报;2000年15期
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