大鼠RSA14-44基因功能的初步研究
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摘要
在本研究中，选择RSA14-44基因作为研究对象。RSA14-44基因位于大鼠染色体4q31，该基因是中国协和医科大学医学分子生物学国家重点实验室通过激光捕获显微切割(Laser Capture Microdissection，LCM)技术，构建大鼠初级精母细胞cDNA削减文库，并进一步用斑点杂交(Dot Blotting)分析，通过在互连网上的比对获得的。全长为1124bp，可读编码框自100bp_684bp，编码194个氨基酸，推测其编码蛋白质分子量为21.8Kda。
首先，我们利用RT-PCR检测了RSA14-44在多个组织中的表达，证明其在睾丸、附睾入脑组织中的呈高表达，这为进一步对其其功能的研究提供了新的思路。
其次，将RSA14-44 cDNA克隆到pET30a(+)载体，在大肠杆菌中进行了pET30a(+)-14融合蛋白的表达与纯化。以纯化的pET30a(+)-14融合蛋白免疫新西兰大白兔，最后获得了抗pET30a(+)-14融合蛋白的多克隆抗体。利用该抗体进行的免疫组化研究显示，RSA14-44蛋白定位于大鼠睾丸生精上皮组织中的初级精母细胞的细胞质部位和支持细胞，而在已经变形的精子细胞中则未观察到阳性信号。
最后，我们又构建了包含RSA14-44 cDNA的pAS2-1-14载体，并以其作为诱饵质粒，进行人睾丸cDNA文库的酵母双杂交筛选，获得了一条编码人20S蛋白酶体蛋白PSMB5的cDNA片段，PSMB5蛋白氨基酸序列全长为264个氨基酸，我们所获得的序列编码该3’端蛋白编码框内全部氨基酸(191个氨基酸)。PSMB5蛋白是26S蛋白酶体的一个重要组成部分，是泛素-蛋白酶体体系(Ubiquitin-proteasome system)的核心，主要对细胞内发生错误折叠、突变或非正常的短期存在的蛋白质进行修饰或降解。依据NCBI已有的PSMB5核苷酸序列设计引物，利用PCR技术成功克隆了PSMB5的cDNA编码区的部分序列。将PSMB5 cDNA克隆到表达载体pET-30a(+)和pGEX4T-1中，进行了His-tagged-PSMB5和GST-PSMB5融合蛋白的原核表达，并利用表达蛋白验证了PSMB5和RSA14-44蛋白体外结合。同时，构建了pCDNA-Flag-PSMB5真核融合蛋白克隆，用于转染Hela胞和CHO细胞，进一步在真核细胞体内验证了RSA14-44和PSMB5的结合。将RSA14-44cDNA和PSMB5克隆到pEGFP-N1载体，构建成功绿色荧光融合表达质粒pEGFP-N1-14和pEGFP-N1-PSMB5，将两种质粒载体分别转染CHO细胞后，借助碘化丙啶染色的细胞化学荧光技术和激光共聚焦显微镜观察发现，RSA14-44蛋白都定位在CHO细胞的细胞质，PSMB5在胞质和胞浆中均有表达。将RSA14-44和PSMB5 cDNA分别克隆
博士学位论文
摘要
P占外拐J蛋白存在相互作用，为此，我们研究了RSA14一44和尸5外招J相互作用后对泛素
水解酶酶活性的影响，发现二者单独作用时都可显著提高泛素水解酶酶活性，共同
转染细胞时极显著地提高了酶活性，这表明两者相互作用可能对生物学功能具有协
同作用。
研究中酵母双杂交实验及体内外验证的结果首次表明，RSA14一44和p占外拐J蛋白
在体内外存在相互作用;从尺SA14一44和只5外拐J蛋白在细胞及组织中的定位，细胞蛋
白酶体活性分析结果推测，在精子发生过程中，RSA14一44和尸5外招J相互作用，在生
殖细胞中调节细胞的增殖和分裂，在支持细胞中两者协同来调节精子发育过程中的
蛋白降解。
关键词:RsA14一4
酵母双杂交蛋白表达
205及265蛋白酶体
RSA14-44 gene was obtained from the cDNA library of rat prirnay by the method of laser capture microdissection (LCM) combined wmVsuppressive subtractive hybridization (SSH). The full-length of RSA-14-44 is 1124 bp, and it contains a 582 bp open reading frame, encoding a 194 aa protein. The sequence has been submitted to Genbank, the accession number is AY149343. The transcription patterns of RSA-14-44 in multiple tissues were analysedbvRT-PCR and the results showed that RSA-14-44 is transcribed in most organs and high eranscrib testis, epididymis and brain. RSA-14-44 was cloned into the prokaryotic expression vector pET30a(+) and the protein of interest was expressed in E.Coli BL21 with a high quantity. The fusion protein was purified by affinity chromatography. Immunohistochemical analysis was performed for the localization of RSA-14-44 protein in rat testis tissues. It was showed that RSA-14-44 protein mainly appears in primary spermatid and sertoli cell, and some signals also existed in the mature sperms For further understanding the biological function of RSA-14-44, RSA-14-44 cDNA (cloned into pAS2-l plasmid) was chosen as the "bait" to screen human testis cDNA library using yeast two-hybrid system and the results showed that the 5 subunit of human 20S proteasome (PSMB5) is capable of binding to RSA-14-44. To confirm the interaction between RSA-14-44 and PSMB5, the protein was expressed as fusion proteins. The recombinant protein was purified with affinity chromatography and the polyclonarbodies of PSMB5 were raised by immunizing the abbit. In turn, the interaction of RSA-14-44 and PSMB5 was confirmed by the in vitro binding and GST-pull down experiments. Then, their interaction in the in vivo system was verified by coimmunoprecipatation. To confirm their interaction in the cell, RSA-14-44 and PSMB5 cDNAs were correspondingly cloned into pEGFP-Nl and pDsRedl-Nl vectors. The colocalization for RSA-14-44 and PSMB5 in CHO cells was also observed. The results demonstrated that RSA-14-44 and PSMB5 were overlay perfectly, suggesting that RSA-14-44 and PSMB5 can interact with each other to function in eukaryotic cells.Previous studies have shown that PSMB5 is related with the enzyme activity of 20S proteasome, so we investlgated the effect of RSA 14-44 and PSMB5 on the activity of 20S and 26S proteasome. It is interesting that the activityof both 20S and 26S proteasome were highly improved in the hela cell trasfected with RSA 14-44 and PSMB5. In addition, the activityof both 20S and 26S proteasome were significatly improved when the cells were cotransfected with RSA 14-44 and PSMB5. It is Wuggested that the overexpression of both RSA 14-44 and PSMB5 can increase the activityof 20S and 26S
proteasome, moreover the activityof both 20S and 26S proteasome were more significantly improved when their cooperation.To find the with their signi ficance of the interaction between RSA14-44 and PSMB5, immunohistpchemical analyst was performed for PSMB5 protein. It was shown that PSMB5 existed in the spermatogonia and Sertoli cells. This is consistent with the results of RS A-14-44. In conclusion, it seems that the interaction betwe there two proteins may play a role in spermatogenesis. It is speculated that the interaction between them may promote the proliferation and division in germ cells, and regulate protein degomeration in sretoli cells. Future experiments need be made to confirm the hypothesis.
首先，我们利用RT-PCR检测了RSA14-44在多个组织中的表达，证明其在睾丸、附睾入脑组织中的呈高表达，这为进一步对其其功能的研究提供了新的思路。
其次，将RSA14-44 cDNA克隆到pET30a(+)载体，在大肠杆菌中进行了pET30a(+)-14融合蛋白的表达与纯化。以纯化的pET30a(+)-14融合蛋白免疫新西兰大白兔，最后获得了抗pET30a(+)-14融合蛋白的多克隆抗体。利用该抗体进行的免疫组化研究显示，RSA14-44蛋白定位于大鼠睾丸生精上皮组织中的初级精母细胞的细胞质部位和支持细胞，而在已经变形的精子细胞中则未观察到阳性信号。
最后，我们又构建了包含RSA14-44 cDNA的pAS2-1-14载体，并以其作为诱饵质粒，进行人睾丸cDNA文库的酵母双杂交筛选，获得了一条编码人20S蛋白酶体蛋白PSMB5的cDNA片段，PSMB5蛋白氨基酸序列全长为264个氨基酸，我们所获得的序列编码该3’端蛋白编码框内全部氨基酸(191个氨基酸)。PSMB5蛋白是26S蛋白酶体的一个重要组成部分，是泛素-蛋白酶体体系(Ubiquitin-proteasome system)的核心，主要对细胞内发生错误折叠、突变或非正常的短期存在的蛋白质进行修饰或降解。依据NCBI已有的PSMB5核苷酸序列设计引物，利用PCR技术成功克隆了PSMB5的cDNA编码区的部分序列。将PSMB5 cDNA克隆到表达载体pET-30a(+)和pGEX4T-1中，进行了His-tagged-PSMB5和GST-PSMB5融合蛋白的原核表达，并利用表达蛋白验证了PSMB5和RSA14-44蛋白体外结合。同时，构建了pCDNA-Flag-PSMB5真核融合蛋白克隆，用于转染Hela胞和CHO细胞，进一步在真核细胞体内验证了RSA14-44和PSMB5的结合。将RSA14-44cDNA和PSMB5克隆到pEGFP-N1载体，构建成功绿色荧光融合表达质粒pEGFP-N1-14和pEGFP-N1-PSMB5，将两种质粒载体分别转染CHO细胞后，借助碘化丙啶染色的细胞化学荧光技术和激光共聚焦显微镜观察发现，RSA14-44蛋白都定位在CHO细胞的细胞质，PSMB5在胞质和胞浆中均有表达。将RSA14-44和PSMB5 cDNA分别克隆
博士学位论文
摘要
P占外拐J蛋白存在相互作用，为此，我们研究了RSA14一44和尸5外招J相互作用后对泛素
水解酶酶活性的影响，发现二者单独作用时都可显著提高泛素水解酶酶活性，共同
转染细胞时极显著地提高了酶活性，这表明两者相互作用可能对生物学功能具有协
同作用。
研究中酵母双杂交实验及体内外验证的结果首次表明，RSA14一44和p占外拐J蛋白
在体内外存在相互作用;从尺SA14一44和只5外拐J蛋白在细胞及组织中的定位，细胞蛋
白酶体活性分析结果推测，在精子发生过程中，RSA14一44和尸5外招J相互作用，在生
殖细胞中调节细胞的增殖和分裂，在支持细胞中两者协同来调节精子发育过程中的
蛋白降解。
关键词:RsA14一4
酵母双杂交蛋白表达
205及265蛋白酶体
RSA14-44 gene was obtained from the cDNA library of rat prirnay by the method of laser capture microdissection (LCM) combined wmVsuppressive subtractive hybridization (SSH). The full-length of RSA-14-44 is 1124 bp, and it contains a 582 bp open reading frame, encoding a 194 aa protein. The sequence has been submitted to Genbank, the accession number is AY149343. The transcription patterns of RSA-14-44 in multiple tissues were analysedbvRT-PCR and the results showed that RSA-14-44 is transcribed in most organs and high eranscrib testis, epididymis and brain. RSA-14-44 was cloned into the prokaryotic expression vector pET30a(+) and the protein of interest was expressed in E.Coli BL21 with a high quantity. The fusion protein was purified by affinity chromatography. Immunohistochemical analysis was performed for the localization of RSA-14-44 protein in rat testis tissues. It was showed that RSA-14-44 protein mainly appears in primary spermatid and sertoli cell, and some signals also existed in the mature sperms For further understanding the biological function of RSA-14-44, RSA-14-44 cDNA (cloned into pAS2-l plasmid) was chosen as the "bait" to screen human testis cDNA library using yeast two-hybrid system and the results showed that the 5 subunit of human 20S proteasome (PSMB5) is capable of binding to RSA-14-44. To confirm the interaction between RSA-14-44 and PSMB5, the protein was expressed as fusion proteins. The recombinant protein was purified with affinity chromatography and the polyclonarbodies of PSMB5 were raised by immunizing the abbit. In turn, the interaction of RSA-14-44 and PSMB5 was confirmed by the in vitro binding and GST-pull down experiments. Then, their interaction in the in vivo system was verified by coimmunoprecipatation. To confirm their interaction in the cell, RSA-14-44 and PSMB5 cDNAs were correspondingly cloned into pEGFP-Nl and pDsRedl-Nl vectors. The colocalization for RSA-14-44 and PSMB5 in CHO cells was also observed. The results demonstrated that RSA-14-44 and PSMB5 were overlay perfectly, suggesting that RSA-14-44 and PSMB5 can interact with each other to function in eukaryotic cells.Previous studies have shown that PSMB5 is related with the enzyme activity of 20S proteasome, so we investlgated the effect of RSA 14-44 and PSMB5 on the activity of 20S and 26S proteasome. It is interesting that the activityof both 20S and 26S proteasome were highly improved in the hela cell trasfected with RSA 14-44 and PSMB5. In addition, the activityof both 20S and 26S proteasome were significatly improved when the cells were cotransfected with RSA 14-44 and PSMB5. It is Wuggested that the overexpression of both RSA 14-44 and PSMB5 can increase the activityof 20S and 26S
proteasome, moreover the activityof both 20S and 26S proteasome were more significantly improved when their cooperation.To find the with their signi ficance of the interaction between RSA14-44 and PSMB5, immunohistpchemical analyst was performed for PSMB5 protein. It was shown that PSMB5 existed in the spermatogonia and Sertoli cells. This is consistent with the results of RS A-14-44. In conclusion, it seems that the interaction betwe there two proteins may play a role in spermatogenesis. It is speculated that the interaction between them may promote the proliferation and division in germ cells, and regulate protein degomeration in sretoli cells. Future experiments need be made to confirm the hypothesis.
引文
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80. 金冬雁等译。分子克隆实验指南。科学出版社，(2002)
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78.Patricio Morales, Eduardo Pizarro, Milene Kong, Marcos Jara. Extracellular localization of proteasomes in human sperm. Mol. Reprod. Dev. 68:115-124(2004)
79.C. Yan Cheng and Dolores D. Mruk. Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development。Physiol Rev 82: 825–874(2002)
80. 金冬雁等译。分子克隆实验指南。科学出版社，(2002)