【摘要】： 哺乳動物的受精過程是一系列的復雜事件。首先精子必須有正常的運動能力,這種能力保證受精過程中精卵相遇,也參與精子對卵的機械穿透作用。此外,哺乳動物精子必須在雌性生殖道內經歷成熟過程發(fā)生生理及形態(tài)學的變化才能獲得受精能力,這一現象稱為獲能。精子獲能是精子頂體反應和受精的一個重要生理前提。 與精子獲能相關的蛋白較多,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)是本單位李彥鋒博士于2007年發(fā)現的定位于鼠精子尾部纖維鞘的一種蛋白,可與CABYR蛋白相結合,因此得名。目前研究認為FSCB蛋白可能是調節(jié)精子運動和獲能的一種重要蛋白。結合對鼠精子的初步研究結果,我們對其人同源基因進行了生物信息學分析,結果顯示該人同源基因序列位于14號染色體,全長序列已公布于GenBank數據庫。 到目前為止,尚沒有任何對人FSCB蛋白進行研究的報告。鑒于同源鼠精子FSCB可能在精子運動和獲能中的重要作用,對人FSCB蛋白的生物學特性和相關作用進行深入研究是非常有意義的。 本研究通過PCR擴增人FSCB基因編碼序列,通過基因重組技術將其插入質粒pET32a(+)和pET28b(+)中,構建重組表達載體和重組菌株,分離純化蛋白后免疫動物成功制備了多克隆抗體,進行該蛋白生物學特性的初步研究。本研究的主要結果和結論如下: 1.人FSCB基因的克隆和測序:根據該FSCB蛋白的理論基因序列,設計帶適當限制性內切酶位點的引物,以人睪丸marathon-ready cDNA為模板進行PCR擴增,獲得全長序列FSCB和部分截短(truncated)序列FSCBt。測序結果與GenBank公布序列一致。 2.重組人FSCB蛋白原核表達載體的構建及原核表達:FSCB及FSCBt基因分別與線性化pMD18-T載體相連接,在大腸桿菌DH5α中擴增后,獲得NcoI/HindⅢ雙酶切片段,將其插入到表達載體pET32a(+)和pET28b(+)中,應用大腸桿菌BL21菌株在1mmol/L IPTG,30℃條件下成功進行誘導表達,并通過抗His抗體檢測,重組蛋白FSCB和FSCBt的表觀分子量分別約為230 KDa和30 KDa,均大于其理論分子量。 3.重組人FSCBt蛋白的分離純化及抗人FSCB多克隆抗體的制備:因表達載體均帶His標簽,通過Ni2+柱進行初純化,再通過分子篩層析進行第二步純化,獲得95%純度重組FSCBt蛋白。根據標準的重組蛋白免疫動物制備抗體的方案,以純化后重組FSCBt加弗氏佐劑免疫雌性家兔,間隔2周加強免疫一次,共4次,2月后獲得效價達1:105多克隆抗體。經Western blot檢測可特異性識別重組蛋白FSCB和FSCBt。 4.人FSCB蛋白表達的組織特異性分析:以所獲得的多克隆抗體通過Western blot檢測人體正常心、肺、胰、脾、肌肉和睪丸組織蛋白表達情況,確定該蛋白僅在睪丸組織內有表達。 5.免疫組化結果顯示:抗FSCB多克隆抗體可使睪丸組織內精子和部分精細胞著色,說明精子和精細胞內有FSCB表達。 6.人FSCB蛋白在精子上定位的初步研究:取正常精液標本通過間接免疫熒光法檢測,觀察到該蛋白定位于成熟精子尾部的主段。 綜上所述,本研究通過基因工程方法,原核表達了重組人FSCB蛋白,成功制備特異性的抗FSCB多克隆抗體,檢測到人FSCB在精子尾部的表達,具有睪丸特異性。這為進一步研究FSCB蛋白在精子運動和獲能中的生物學作用奠定了基礎,有助于深入理解精子獲能復雜的分子機制。
[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，

本文編號：2344054