綿羊TMEM190、IZUM01和PDIA3之間相互作用的鑒定
發(fā)布時(shí)間：2018-12-11 19:58
【摘要】：哺乳動(dòng)物的受精作用需要經(jīng)歷精子的獲能、精子與透明帶的結(jié)合、精子發(fā)生頂體反應(yīng)和精卵細(xì)胞膜的融合四個(gè)過程。精卵細(xì)胞膜的融合是整個(gè)受精作用中最為關(guān)鍵的一步,此過程中精卵膜上都有大量的膜蛋白參與。目前已知精卵膜融合過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與卵子表面的精卵融合蛋白JUNO的結(jié)合。但僅僅IZUMO1-JUNO結(jié)合還不足以引起精卵膜融合,我們猜想應(yīng)該還需要精子或卵子表面的其他蛋白之間的相互作用。本實(shí)驗(yàn)擬用免疫共沉淀和酵母雙雜交技術(shù)探明綿羊精子表面的IZUMO1、PDIA3和TMEM190三個(gè)蛋白之間是否存在相互作用。以綿羊睪丸cDNA為模板首次克隆到779 bp的綿羊TMEM190 cDNA,包括543bp的開放閱讀框,并提交NCBI GeneBank獲得登錄號(hào):KY914491。同時(shí)克隆了綿羊Izumo1和PDIA3的cDNA序列。將克隆得到的三個(gè)基因的cDNA分別與pGEX-4T-1連接構(gòu)建了原核表達(dá)載體,轉(zhuǎn)入大腸桿菌表達(dá)TMEM190-GST(43.9kDa)、IZUMO1-GST(60.2kDa)和 PDIA3-GST(80.6kDa)融合蛋白。以純化的IZUMO1-GST蛋白為抗原免疫家兔得到兔抗羊IZUMO1抗血清;以純化的PDIA3-GST蛋白為抗原免疫小鼠得到鼠抗羊PDIA3抗血清。Western Blot檢驗(yàn)證明得到的兩種抗血清均能識(shí)別GST和GST融合蛋白。為了進(jìn)行免疫共沉淀實(shí)驗(yàn),我們用pCMV-HA-C、pCMV-Flag-C構(gòu)建了真核表達(dá)載體:pCMV-IZUMO1-HA、pCMV-IZUMO1-Flag、pCMV-PDIA3-HA、pCMV-PDIA3-Flag、pCMV-TMEM190-HA 和 pCMV-TMEM190-Flag。將 pCMV-IZUMO1-HA 與 pCMV-PDIA3-Flag、pCMV-TMEM190-HA 與 pCMV-PDIA3-Flag、pCMV-TMEM190-HA與pCMV-IZUMO1-Flag分別共轉(zhuǎn)染293T細(xì)胞,用兔抗HA多克隆抗體為IP抗體,用自制的鼠抗羊PDIA3抗血清檢測IZUMO1-HA與PDIA3-Flag和TMEM190-HA與PDIA3-Flag的相互作用,出現(xiàn)預(yù)期共沉淀蛋白條帶。用自制的兔抗羊IZUMO1抗血清檢測TMEM190-HA與IZUMO1-Flag的相互作用,沒有出現(xiàn)預(yù)期共沉淀蛋白條帶。為了進(jìn)行酵母雙雜交實(shí)驗(yàn),我們用pGAD-T7、pGBK-T7構(gòu)建了酵母雙雜交載體:pGAD-IZUMO1、pGBK-IZUMO1、pGAD-PDIA3、pGBK-PDIA3、pGAD-TMEM190 和 pGBK-TMEM190,并將連 pGAD 的載體轉(zhuǎn)化 Y2HGold,連 pGBK的載體轉(zhuǎn)化 Y187,將 IZUMO1(Y187)與 PDIA3(Y2HGold)、IZUMO1(Y2H Gold)與 TMEM190(Y187)和 PDIA3(Y2HGold)與 TMEM190(Y187)雜交。出現(xiàn)了三葉草結(jié)構(gòu)的雜交體后,涂SD/-Trp-Leu二缺平板,IZUMO1(Y2H Gold)與TMEM190(Y187)雜交平板生長一個(gè)藍(lán)色菌落,另外兩組大部分為藍(lán)色菌落。繼續(xù)涂SD/-Ade-His-Leu-Trp四缺平板后,IZUMO1(Y2H Gold)與TMEM190(Y187)雜交平板沒有菌落生長,另外兩組有大量藍(lán)色菌落生長,表明IZUMO1與TMEM190不能發(fā)生相互作用,而IZUMO1與PDIA3、PDIA3與TMEM190存在相互作用。最后免疫共沉淀實(shí)驗(yàn)和酵母雙雜交實(shí)驗(yàn)得到了一致的結(jié)論,即在酵母和293T細(xì)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;S826
本文編號(hào)：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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;S826
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 王耀新;綿羊TMEM190、IZUM01和PDIA3之間相互作用的鑒定[D];內(nèi)蒙古大學(xué);2017年
，本文編號(hào)：2373135
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