绵羊TMEM190、IZUM01和PDIA3之间相互作用的鉴定
发布时间：2018-12-11 19:58
【摘要】：哺乳动物的受精作用需要经历精子的获能、精子与透明带的结合、精子发生顶体反应和精卵细胞膜的融合四个过程。精卵细胞膜的融合是整个受精作用中最为关键的一步,此过程中精卵膜上都有大量的膜蛋白参与。目前已知精卵膜融合过程中最重要的蛋白相互作用是精子表面的精卵融合蛋白IZUMO1与卵子表面的精卵融合蛋白JUNO的结合。但仅仅IZUMO1-JUNO结合还不足以引起精卵膜融合,我们猜想应该还需要精子或卵子表面的其他蛋白之间的相互作用。本实验拟用免疫共沉淀和酵母双杂交技术探明绵羊精子表面的IZUMO1、PDIA3和TMEM190三个蛋白之间是否存在相互作用。以绵羊睾丸cDNA为模板首次克隆到779 bp的绵羊TMEM190 cDNA,包括543bp的开放阅读框,并提交NCBI GeneBank获得登录号:KY914491。同时克隆了绵羊Izumo1和PDIA3的cDNA序列。将克隆得到的三个基因的cDNA分别与pGEX-4T-1连接构建了原核表达载体,转入大肠杆菌表达TMEM190-GST(43.9kDa)、IZUMO1-GST(60.2kDa)和 PDIA3-GST(80.6kDa)融合蛋白。以纯化的IZUMO1-GST蛋白为抗原免疫家兔得到兔抗羊IZUMO1抗血清;以纯化的PDIA3-GST蛋白为抗原免疫小鼠得到鼠抗羊PDIA3抗血清。Western Blot检验证明得到的两种抗血清均能识别GST和GST融合蛋白。为了进行免疫共沉淀实验,我们用pCMV-HA-C、pCMV-Flag-C构建了真核表达载体:pCMV-IZUMO1-HA、pCMV-IZUMO1-Flag、pCMV-PDIA3-HA、pCMV-PDIA3-Flag、pCMV-TMEM190-HA 和 pCMV-TMEM190-Flag。将 pCMV-IZUMO1-HA 与 pCMV-PDIA3-Flag、pCMV-TMEM190-HA 与 pCMV-PDIA3-Flag、pCMV-TMEM190-HA与pCMV-IZUMO1-Flag分别共转染293T细胞,用兔抗HA多克隆抗体为IP抗体,用自制的鼠抗羊PDIA3抗血清检测IZUMO1-HA与PDIA3-Flag和TMEM190-HA与PDIA3-Flag的相互作用,出现预期共沉淀蛋白条带。用自制的兔抗羊IZUMO1抗血清检测TMEM190-HA与IZUMO1-Flag的相互作用,没有出现预期共沉淀蛋白条带。为了进行酵母双杂交实验,我们用pGAD-T7、pGBK-T7构建了酵母双杂交载体:pGAD-IZUMO1、pGBK-IZUMO1、pGAD-PDIA3、pGBK-PDIA3、pGAD-TMEM190 和 pGBK-TMEM190,并将连 pGAD 的载体转化 Y2HGold,连 pGBK的载体转化 Y187,将 IZUMO1(Y187)与 PDIA3(Y2HGold)、IZUMO1(Y2H Gold)与 TMEM190(Y187)和 PDIA3(Y2HGold)与 TMEM190(Y187)杂交。出现了三叶草结构的杂交体后,涂SD/-Trp-Leu二缺平板,IZUMO1(Y2H Gold)与TMEM190(Y187)杂交平板生长一个蓝色菌落,另外两组大部分为蓝色菌落。继续涂SD/-Ade-His-Leu-Trp四缺平板后,IZUMO1(Y2H Gold)与TMEM190(Y187)杂交平板没有菌落生长,另外两组有大量蓝色菌落生长,表明IZUMO1与TMEM190不能发生相互作用,而IZUMO1与PDIA3、PDIA3与TMEM190存在相互作用。最后免疫共沉淀实验和酵母双杂交实验得到了一致的结论,即在酵母和293T细胞中,IZUMO1和PDIA3、PDIA3和TMEM190存在相互作用,而IZUMO1和TMEM190不存在相互作用。
[Abstract]:Fertilization in mammals involves four processes: capacitation of sperm, combination of sperm and pellucida, acrosome reaction of spermatozoa and fusion of spermatozoa cell membrane. The fusion of spermatozoa membrane is the most important step in the whole fertilization process, in which a large number of membrane proteins are involved. The most important protein interaction in spermatozoa fusion is the binding of spermatozoa fusion protein (IZUMO1) on sperm surface to egg fusion protein (JUNO) on egg surface. However, IZUMO1-JUNO binding alone is not enough to induce spermatozoa fusion, and we suspect that interaction between other proteins on the surface of sperm or egg should be needed. The aim of this study was to investigate the interaction between IZUMO1,PDIA3 and TMEM190 on the sperm surface of sheep by immunoprecipitation and yeast two-hybrid technique. Sheep TMEM190 cDNA, first cloned into 779 bp using sheep testis cDNA as template, includes the open reading box of 543bp, and submits NCBI GeneBank to obtain login number: KY914491. At the same time, the cDNA sequences of sheep Izumo1 and PDIA3 were cloned. The cDNA of the three genes were ligated with pGEX-4T-1 respectively to construct the prokaryotic expression vector, and then transferred into E. coli to express TMEM190-GST (43.9kDa), IZUMO1-GST (60.2kDa) and PDIA3-GST (80.6kDa) fusion proteins. Rabbit anti-sheep IZUMO1 antiserum was obtained by immunizing rabbits with purified IZUMO1-GST protein. The purified PDIA3-GST protein was used as antigen to immunize mice with mouse anti-sheep PDIA3 antiserum. Western Blot test showed that the two antisera could recognize both GST and GST fusion proteins. In order to carry out immunoprecipitation experiment, we constructed eukaryotic expression vectors: pCMV-IZUMO1-HA,pCMV-IZUMO1-Flag,pCMV-PDIA3-HA,pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-TMEM190-Flag. using pCMV-HA-C,pCMV-Flag-C. PCMV-IZUMO1-HA and pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-IZUMO1-Flag were co-transfected into 293T cells respectively. Rabbit anti-HA polyclonal antibody was used as IP antibody. The interaction between IZUMO1-HA and PDIA3-Flag and between TMEM190-HA and PDIA3-Flag was detected by using the self-made mouse anti-sheep PDIA3 antiserum and the expected coprecipitation protein bands appeared. The interaction between TMEM190-HA and IZUMO1-Flag was detected by rabbit anti-sheep IZUMO1 antiserum and there were no expected coprecipitation protein bands. In order to carry out yeast two-hybrid experiment, we constructed yeast two-hybrid vector by pGAD-T7,pGBK-T7: pGAD-IZUMO1,pGBK-IZUMO1,pGAD-PDIA3,pGBK-PDIA3,pGAD-TMEM190 and pGBK-TMEM190, and transformed the vector of linked pGAD into Y2HGold. and the vector of pGBK into Y187. IZUMO1 (Y187) and PDIA3 (Y2HGold), IZUMO1 (Y2H Gold) and TMEM190 (Y187) and PDIA3 (Y2HGold) were hybridized with TMEM190 (Y187). After the hybrid of clover structure appeared, the SD/-Trp-Leu plate was coated, the IZUMO1 (Y2H Gold) and TMEM190 (Y187) hybrid plate grew a blue colony, and the other two groups were mostly blue colony. After the SD/-Ade-His-Leu-Trp plate was continued to be coated, the IZUMO1 (Y2H Gold) and TMEM190 (Y187) hybrid plates had no colony growth, and the other two groups had a large number of blue colonies, indicating that IZUMO1 and TMEM190 could not interact, while IZUMO1 and PDIA3, could not interact with each other. PDIA3 interacts with TMEM190. Finally, the results of immunoprecipitation test and yeast two-hybrid experiment showed that there was interaction between IZUMO1 and PDIA3,PDIA3 and TMEM190 in yeast and 293T cells, but there was no interaction between IZUMO1 and TMEM190 in yeast and 293T cells.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;S826
本文编号：2373135
[Abstract]:Fertilization in mammals involves four processes: capacitation of sperm, combination of sperm and pellucida, acrosome reaction of spermatozoa and fusion of spermatozoa cell membrane. The fusion of spermatozoa membrane is the most important step in the whole fertilization process, in which a large number of membrane proteins are involved. The most important protein interaction in spermatozoa fusion is the binding of spermatozoa fusion protein (IZUMO1) on sperm surface to egg fusion protein (JUNO) on egg surface. However, IZUMO1-JUNO binding alone is not enough to induce spermatozoa fusion, and we suspect that interaction between other proteins on the surface of sperm or egg should be needed. The aim of this study was to investigate the interaction between IZUMO1,PDIA3 and TMEM190 on the sperm surface of sheep by immunoprecipitation and yeast two-hybrid technique. Sheep TMEM190 cDNA, first cloned into 779 bp using sheep testis cDNA as template, includes the open reading box of 543bp, and submits NCBI GeneBank to obtain login number: KY914491. At the same time, the cDNA sequences of sheep Izumo1 and PDIA3 were cloned. The cDNA of the three genes were ligated with pGEX-4T-1 respectively to construct the prokaryotic expression vector, and then transferred into E. coli to express TMEM190-GST (43.9kDa), IZUMO1-GST (60.2kDa) and PDIA3-GST (80.6kDa) fusion proteins. Rabbit anti-sheep IZUMO1 antiserum was obtained by immunizing rabbits with purified IZUMO1-GST protein. The purified PDIA3-GST protein was used as antigen to immunize mice with mouse anti-sheep PDIA3 antiserum. Western Blot test showed that the two antisera could recognize both GST and GST fusion proteins. In order to carry out immunoprecipitation experiment, we constructed eukaryotic expression vectors: pCMV-IZUMO1-HA,pCMV-IZUMO1-Flag,pCMV-PDIA3-HA,pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-TMEM190-Flag. using pCMV-HA-C,pCMV-Flag-C. PCMV-IZUMO1-HA and pCMV-PDIA3-Flag,pCMV-TMEM190-HA and pCMV-IZUMO1-Flag were co-transfected into 293T cells respectively. Rabbit anti-HA polyclonal antibody was used as IP antibody. The interaction between IZUMO1-HA and PDIA3-Flag and between TMEM190-HA and PDIA3-Flag was detected by using the self-made mouse anti-sheep PDIA3 antiserum and the expected coprecipitation protein bands appeared. The interaction between TMEM190-HA and IZUMO1-Flag was detected by rabbit anti-sheep IZUMO1 antiserum and there were no expected coprecipitation protein bands. In order to carry out yeast two-hybrid experiment, we constructed yeast two-hybrid vector by pGAD-T7,pGBK-T7: pGAD-IZUMO1,pGBK-IZUMO1,pGAD-PDIA3,pGBK-PDIA3,pGAD-TMEM190 and pGBK-TMEM190, and transformed the vector of linked pGAD into Y2HGold. and the vector of pGBK into Y187. IZUMO1 (Y187) and PDIA3 (Y2HGold), IZUMO1 (Y2H Gold) and TMEM190 (Y187) and PDIA3 (Y2HGold) were hybridized with TMEM190 (Y187). After the hybrid of clover structure appeared, the SD/-Trp-Leu plate was coated, the IZUMO1 (Y2H Gold) and TMEM190 (Y187) hybrid plate grew a blue colony, and the other two groups were mostly blue colony. After the SD/-Ade-His-Leu-Trp plate was continued to be coated, the IZUMO1 (Y2H Gold) and TMEM190 (Y187) hybrid plates had no colony growth, and the other two groups had a large number of blue colonies, indicating that IZUMO1 and TMEM190 could not interact, while IZUMO1 and PDIA3, could not interact with each other. PDIA3 interacts with TMEM190. Finally, the results of immunoprecipitation test and yeast two-hybrid experiment showed that there was interaction between IZUMO1 and PDIA3,PDIA3 and TMEM190 in yeast and 293T cells, but there was no interaction between IZUMO1 and TMEM190 in yeast and 293T cells.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;S826
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1 王耀新;绵羊TMEM190、IZUM01和PDIA3之间相互作用的鉴定[D];内蒙古大学;2017年
，本文编号：2373135
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