人精子蛋白FSCB的基因克
发布时间：2018-11-20 06:25
【摘要】： 哺乳动物的受精过程是一系列的复杂事件。首先精子必须有正常的运动能力,这种能力保证受精过程中精卵相遇,也参与精子对卵的机械穿透作用。此外,哺乳动物精子必须在雌性生殖道内经历成熟过程发生生理及形态学的变化才能获得受精能力,这一现象称为获能。精子获能是精子顶体反应和受精的一个重要生理前提。 与精子获能相关的蛋白较多,包括SP-10,FA-1,YLP-12,CABYR等。本文所研究的目的蛋白FSCB(Fibrous Sheath Cabyr-Binding,FSCB)是本单位李彦锋博士于2007年发现的定位于鼠精子尾部纤维鞘的一种蛋白,可与CABYR蛋白相结合,因此得名。目前研究认为FSCB蛋白可能是调节精子运动和获能的一种重要蛋白。结合对鼠精子的初步研究结果,我们对其人同源基因进行了生物信息学分析,结果显示该人同源基因序列位于14号染色体,全长序列已公布于GenBank数据库。 到目前为止,尚没有任何对人FSCB蛋白进行研究的报告。鉴于同源鼠精子FSCB可能在精子运动和获能中的重要作用,对人FSCB蛋白的生物学特性和相关作用进行深入研究是非常有意义的。 本研究通过PCR扩增人FSCB基因编码序列,通过基因重组技术将其插入质粒pET32a(+)和pET28b(+)中,构建重组表达载体和重组菌株,分离纯化蛋白后免疫动物成功制备了多克隆抗体,进行该蛋白生物学特性的初步研究。本研究的主要结果和结论如下: 1.人FSCB基因的克隆和测序:根据该FSCB蛋白的理论基因序列,设计带适当限制性内切酶位点的引物,以人睾丸marathon-ready cDNA为模板进行PCR扩增,获得全长序列FSCB和部分截短(truncated)序列FSCBt。测序结果与GenBank公布序列一致。 2.重组人FSCB蛋白原核表达载体的构建及原核表达:FSCB及FSCBt基因分别与线性化pMD18-T载体相连接,在大肠杆菌DH5α中扩增后,获得NcoI/HindⅢ双酶切片段,将其插入到表达载体pET32a(+)和pET28b(+)中,应用大肠杆菌BL21菌株在1mmol/L IPTG,30℃条件下成功进行诱导表达,并通过抗His抗体检测,重组蛋白FSCB和FSCBt的表观分子量分别约为230 KDa和30 KDa,均大于其理论分子量。 3.重组人FSCBt蛋白的分离纯化及抗人FSCB多克隆抗体的制备:因表达载体均带His标签,通过Ni2+柱进行初纯化,再通过分子筛层析进行第二步纯化,获得95%纯度重组FSCBt蛋白。根据标准的重组蛋白免疫动物制备抗体的方案,以纯化后重组FSCBt加弗氏佐剂免疫雌性家兔,间隔2周加强免疫一次,共4次,2月后获得效价达1:105多克隆抗体。经Western blot检测可特异性识别重组蛋白FSCB和FSCBt。 4.人FSCB蛋白表达的组织特异性分析:以所获得的多克隆抗体通过Western blot检测人体正常心、肺、胰、脾、肌肉和睾丸组织蛋白表达情况,确定该蛋白仅在睾丸组织内有表达。 5.免疫组化结果显示:抗FSCB多克隆抗体可使睾丸组织内精子和部分精细胞着色,说明精子和精细胞内有FSCB表达。 6.人FSCB蛋白在精子上定位的初步研究:取正常精液标本通过间接免疫荧光法检测,观察到该蛋白定位于成熟精子尾部的主段。 综上所述,本研究通过基因工程方法,原核表达了重组人FSCB蛋白,成功制备特异性的抗FSCB多克隆抗体,检测到人FSCB在精子尾部的表达,具有睾丸特异性。这为进一步研究FSCB蛋白在精子运动和获能中的生物学作用奠定了基础,有助于深入理解精子获能复杂的分子机制。
[Abstract]:The process of fertilization in a mammal is a series of complex events. First, the sperm must have a normal ability to exercise, which ensures that the sperm eggs meet during the fertilization process and also in the mechanical penetration of the sperm to the egg. In addition, mammalian sperm must undergo a physiological and morphological change in the female reproductive tract in order to obtain the ability to fertilise, which is known as an acquisition. Sperm capture can be an important physiological prerequisite for sperm acrosomal reaction and fertilization. More protein-related proteins, including SP-10, FA-1, YLP-12, CA BYR et al. The target protein FSCB (Fibreus Sheath Cabyr-Binding, FSCB) studied in this paper is a protein which is found in the tail fiber of the mouse sperm in 2007, and can be combined with the CABYR protein. The name is therefore named. The current study found that the FSCB protein may be one of the types of sperm motility and energy In combination with the results of the preliminary study on the mouse sperm, we have carried on the bioinformatics analysis of the human homologous gene. The results show that the homologous gene sequence of the human is located on chromosome 14, and the full-length sequence has been published in GenBan. k database. So far, there is no one-to-one FSCB In view of the important role of the mouse sperm FSCB in the sperm movement and the capture, the biological characteristics and related effects of the human FSCB protein are in-depth. The gene coding sequence of human FSCB was amplified by PCR and inserted into the plasmid pET32a (+) and pET28b (+) by the gene recombination technique, and the recombinant expression vector and the recombinant strain were constructed, and the purified protein was isolated. The immune animals successfully prepared polyclonal antibodies, a preliminary study on the biological characteristics of the protein The main results and conclusions of this study are as follows: 1. Cloning and sequencing of human FSCB gene: according to the theoretical gene sequence of the FSCB protein, a primer with an appropriate restriction endonuclease site is designed, and human testis marython-and carrying out PCR amplification on the ready cDNA as a template to obtain a full-length sequence FSCB and a partial interception. 鐭，
本文编号：2344054
[Abstract]:The process of fertilization in a mammal is a series of complex events. First, the sperm must have a normal ability to exercise, which ensures that the sperm eggs meet during the fertilization process and also in the mechanical penetration of the sperm to the egg. In addition, mammalian sperm must undergo a physiological and morphological change in the female reproductive tract in order to obtain the ability to fertilise, which is known as an acquisition. Sperm capture can be an important physiological prerequisite for sperm acrosomal reaction and fertilization. More protein-related proteins, including SP-10, FA-1, YLP-12, CA BYR et al. The target protein FSCB (Fibreus Sheath Cabyr-Binding, FSCB) studied in this paper is a protein which is found in the tail fiber of the mouse sperm in 2007, and can be combined with the CABYR protein. The name is therefore named. The current study found that the FSCB protein may be one of the types of sperm motility and energy In combination with the results of the preliminary study on the mouse sperm, we have carried on the bioinformatics analysis of the human homologous gene. The results show that the homologous gene sequence of the human is located on chromosome 14, and the full-length sequence has been published in GenBan. k database. So far, there is no one-to-one FSCB In view of the important role of the mouse sperm FSCB in the sperm movement and the capture, the biological characteristics and related effects of the human FSCB protein are in-depth. The gene coding sequence of human FSCB was amplified by PCR and inserted into the plasmid pET32a (+) and pET28b (+) by the gene recombination technique, and the recombinant expression vector and the recombinant strain were constructed, and the purified protein was isolated. The immune animals successfully prepared polyclonal antibodies, a preliminary study on the biological characteristics of the protein The main results and conclusions of this study are as follows: 1. Cloning and sequencing of human FSCB gene: according to the theoretical gene sequence of the FSCB protein, a primer with an appropriate restriction endonuclease site is designed, and human testis marython-and carrying out PCR amplification on the ready cDNA as a template to obtain a full-length sequence FSCB and a partial interception. 鐭，
本文编号：2344054
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