發(fā)布時間：2018-02-10 08:56

  本文關(guān)鍵詞： 綿羊 IZUMO2 基因克隆 免疫組織化學(xué) 酵母雙雜交　出處：《內(nèi)蒙古大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：受精作用是將上一代的基因組傳遞給下一代,并開始一個新生命體生長的細胞機制的總和。在哺乳動物中,精卵融合是通過精子和卵子表面多種分子的相互作用完成的,但目前對這些分子的相互作用機制還不清楚。到目前為止鑒定出若干參與精卵融合的蛋白質(zhì),其中精子蛋白有IZUMO蛋白家族、CRISPs家族蛋白、SLLP1、PDIA3、TSSK6、SPESP1和EQUATORIN等,卵子上有JUNO等蛋白�，F(xiàn)在的研究多集中在這些蛋白質(zhì)之間的相互作用模式上。本研究克隆了綿羊Izumo2 cDNA序列,原核表達并純化GST-IZUMO2融合蛋白,用純化后的融合蛋白免疫小鼠,制備小鼠抗綿羊IZUMO2腹水多克隆抗體,研究IZUMO2蛋白在睪丸和在精子上的定位。本研究還使用酵母雙雜交技術(shù)初步探究了IZUMO2 與 IZUMO1、IZUMO3、IZUMO4、PDIA3、TSSK6、SPESP1、SLLP1、 CRISP1、MN9之間的相互作用。以綿羊睪丸組織總cDNA為模板,根據(jù)GenBank序列(XM 004015399.1)設(shè)計引物克隆綿羊Izumo2 cDNA,全長為813 bp,其中編碼區(qū)為666 bp, BLAST結(jié)果顯示其與GenBank數(shù)據(jù)庫中的綿羊預(yù)測序列相似度為98.6%�？寺〉玫降木d羊Izumo2 cDNA序列編碼222個氨基酸,其中從N端開始前15個氨基酸為信號肽,第186-206個氨基酸構(gòu)成跨膜結(jié)構(gòu)域。構(gòu)建原核表達質(zhì)粒pGEX-Izumo2,轉(zhuǎn)化E. coli BL21(DE3)感受態(tài)菌,用IPTG誘導(dǎo)表達了分子量約為50 kDa的GST-IZUMO2融合蛋白,并采用SDS-PAGE切膠純化法純化GST-IZUM02融合蛋白。用純化后的融合蛋白免疫小鼠,制備并純化小鼠抗綿羊IZUM02腹水多克隆抗體。用純化后的抗體進行免疫組織化學(xué)研究IZUM02蛋白在睪丸和精子上的定位,結(jié)果顯示IZUMO2蛋白在綿羊睪丸組織的各期細胞中均會表達；在精子發(fā)生頂體反應(yīng)前IZUMO2蛋白位于精子的頭部,當(dāng)精子發(fā)生頂體反應(yīng)后IZUM02蛋白消失。為了探尋IZUM02蛋白與精子上的其他精卵融合相關(guān)蛋白的關(guān)系,本研究利用酵母雙雜交技術(shù)初步探究了IZUMO2與IZUMO1、IZUMO3、IZUMO4、 PDIA3、TSSK6、SPESP1、SLLP1、CRISP1、MN9之間的相互作用,結(jié)果未發(fā)現(xiàn)相互作用。
[Abstract]:Fertilization is the sum of the cellular mechanisms that transmit the genomes of the previous generation to the next generation and begin the growth of a new organism. In mammals, sperm and egg fusion is accomplished by the interaction of multiple molecules on the surface of the sperm and egg. However, the interaction mechanism of these molecules is not clear. Up to now, a number of proteins involved in spermatozoa fusion have been identified. Among them, sperm proteins include IZUMO protein family, CRISPs family protein, SLLP1PDIA3, TSSK6, SPESP1 and EQUATORIN, etc. There are JUNO and other proteins on the egg. The current research focuses on the interaction pattern of these proteins. We cloned the sheep Izumo2 cDNA sequence, expressed and purified the GST-IZUMO2 fusion protein in prokaryotic, and immunized mice with the purified fusion protein. Mouse anti-sheep IZUMO2 ascites polyclonal antibody was prepared to study the localization of IZUMO2 protein in testis and sperm. The interaction between IZUMO2 and IZUMO1, IZUMO3, IZUMO3, TSSK6, SPESP1, SLLP1, CRISP1MN9 was studied using yeast two-hybrid technique. According to the GenBank sequence XM004015399.1), a primer was designed to clone sheep Izumo2 cDNA. the length of Izumo2 was 813 BP, and the coding region was 666bp.The BLAST result showed that the similarity between Izumo2 cDNA and the predicted sheep sequence in GenBank database was 98.6.The cloned sheep Izumo2 cDNA sequence encoded 222 amino acids. The first 15 amino acids were signal peptides from the N-terminal, and the 186-206 amino acids formed transmembrane domain. The prokaryotic expression plasmid pGEX-Izumo2 was constructed and transformed into E. coli BL21DE3) -receptive bacteria. The GST-IZUMO2 fusion protein with molecular weight of about 50 kDa was induced and expressed by IPTG. GST-IZUM02 fusion protein was purified by SDS-PAGE gel purification. Mice were immunized with the purified fusion protein. Mouse anti-sheep IZUM02 ascites polyclonal antibody was prepared and purified. The localization of IZUM02 protein on testis and spermatozoa was studied by immunohistochemistry with purified antibody. The results showed that IZUMO2 protein was expressed in all stages of sheep testicular cells. IZUMO2 protein is located in the head of sperm before acrosome reaction, and IZUM02 protein disappears after acrosome reaction. In order to explore the relationship between IZUM02 protein and other spermatozoa fusion related proteins, The interaction between IZUMO2 and IZUMO3 IZUMO4, PDIA3TSSK6 SPESP1 and SLLP1CRISP1MN9 was preliminarily investigated by yeast two-hybrid technique. No interaction was found.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826;Q78

【參考文獻】

相關(guān)碩士學(xué)位論文 前1條

1 鄂爾渾塔娜;絨山羊Izumo3 cDNA的克隆及在睪丸和精子上的定位觀察[D];內(nèi)蒙古大學(xué);2012年

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本文編號：1500143