精子相关抗原6(SPAG6)与TCTE3蛋白间相互作用探讨
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摘要
目的:探讨精子相关抗原6(sperm associated antigen 6,Spag6)和T复合体相关睾丸表达3(T-complex associated testis expressed 3,TCTE3)之间的相互作用。方法:PCR法观察Tcte3在雄性小鼠各组织中的表达;以小鼠睾丸c DNA为模板,扩增Tcte3序列,构建Tcte3/p GADT7、Tcte3/p EGFPN2重组质粒;以Tcte3/p EGFPN2转染CHO细胞和COS-1细胞,免疫荧光染色及Western blot检测TCTE3在细胞中的定位和表达;以Spag6/p GBKT7和Tcte3/p GADT7进行酵母双杂交实验,提取酵母蛋白后采用Western blot检测TCTE3与SPAG6之间是否存在直接的相互作用。结果:Tcte3在睾丸、脑、肝、肾、肺、脾、肌肉中均有表达;酶切和测序鉴定证实Tcte3/p GADT7和Tcte3/p EGFPN2重组质粒均构建成功;体外实验发现TCTE3主要在CHO和COS-1细胞质中表达,当与SPAG6质粒共转染后,TCTE3被募集至细胞微管中表达;酵母双杂交实验显示TCTE3/SPAG6组合后,在缺少SD/-Leu/-Trp/-His的培养板上有菌落生成,Western blot显示菌落中TCTE3、SPAG6蛋白表达显著,分子量正确。结论:精子相关抗原SPAG6与TCTE3之间存在直接的相互作用,SPAG6可募集TCTE3至细胞微管中表达。
Objective:To test the interactions between SPAG6 and Tcte3 proteins. Methods:The expression of Tcte3 was observed in different tissues of mice by PCR. The testis c DNA was used as the template to amplify Tcte3 coding sequence by PCR,following with the Tcte3/p GADT7 and Tcte3/p EGFPN2 recombinant plasmids' constructing. Immunofluorescent staining and Western blot were used to detect the location and protein express after transfecting Tcte3/p EGFPN2 into CHO cells and COS-1 cells. The interaction between SPAG6 and TCTE3 was detected by Western blot after yeast two hybrid for Tcte3/p GADT7 and SPAG6/PGBKT7. Results:Tcte3mainly expressed in testis,brain,liver,kidney,lung,spleen and muscle. The results of restriction enzyme digestion and sequencing showed the Tcte3/p GADT7 and Tcte3/p EGFPN2 recombinant plasmids were constructed successfully. TCTE3 mainly located in the whole cytoplasm in vitro,whereas it was recruited to microtubule after co-transfecting with SPAG6. In yeast two hybrid assay,the mating group of TCTE3/SPAG6 grow promoted colonies to grow in SD/-Leu/-Trp/-His plates,and Western blot assay had identified TCTE3 and SPAG6 proteins could express in these colonies. Conclusion:The directly interaction between SPAG6 and TCTE3 is confirmed,and SPAG6 could recruite TCTE3 to celluar microtubule.
Objective:To test the interactions between SPAG6 and Tcte3 proteins. Methods:The expression of Tcte3 was observed in different tissues of mice by PCR. The testis c DNA was used as the template to amplify Tcte3 coding sequence by PCR,following with the Tcte3/p GADT7 and Tcte3/p EGFPN2 recombinant plasmids' constructing. Immunofluorescent staining and Western blot were used to detect the location and protein express after transfecting Tcte3/p EGFPN2 into CHO cells and COS-1 cells. The interaction between SPAG6 and TCTE3 was detected by Western blot after yeast two hybrid for Tcte3/p GADT7 and SPAG6/PGBKT7. Results:Tcte3mainly expressed in testis,brain,liver,kidney,lung,spleen and muscle. The results of restriction enzyme digestion and sequencing showed the Tcte3/p GADT7 and Tcte3/p EGFPN2 recombinant plasmids were constructed successfully. TCTE3 mainly located in the whole cytoplasm in vitro,whereas it was recruited to microtubule after co-transfecting with SPAG6. In yeast two hybrid assay,the mating group of TCTE3/SPAG6 grow promoted colonies to grow in SD/-Leu/-Trp/-His plates,and Western blot assay had identified TCTE3 and SPAG6 proteins could express in these colonies. Conclusion:The directly interaction between SPAG6 and TCTE3 is confirmed,and SPAG6 could recruite TCTE3 to celluar microtubule.
引文
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[16]施琼,翁亚光.酵母双杂交系统筛选Mps1相互作用蛋白[J].重庆医科大学学报,2006,31(6):854-856,888.
[17]Rashid S,Grzmil P,Drenckhahn JD,et al.Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia[J].Reproduction,2010,139(1):99-111.
[18]Kiselak EA,Shen X,Song J,et al.Transcriptional regulation of an axonemal central apparatus gene,sperm-associated antigen 6,by a SRY-related high mobility group transcription factor,S-SOX5[J].J Biol Chem,2010,285(40):30496-30505.
[19]Silina K,Zayakin P,Kalnina Z,et al.Sperm-associated antigens as targets for cancer immunotherapy:expression pattern and humoral immune response in cancer patients[J].J Immunother,2011,34(1):28-44.
[20]Parveen Z,Bibi Z,Bibi N,et al.Disruption of murine Tcte3-3 induces tissue specific apoptosis via co-expression of Anxa5 and Pebp1[J].Comput Biol Chem,2014,53PB:214-225.
[2]汪瑶瑶.精子DNA损伤在男性不育症中的研究进展[J].现代泌尿外科杂志,2014,19(6):419-422.
[3]苏春霞,王凌,王若竹,等.不育男性精液白细胞增高对精子质量影响临床研究[J].中国实验诊断学,2015,19(4):665-666.
[4]Wang J,Li X,Zhang Z,et al.Expression of prestin in OHCs is reduced in Spag6 gene knockout mice[J].Neurosci Lett,2015,592:42-47.
[5]Teves ME,Sears PR,Li W,et al.Sperm-associated antigen 6(SPAG6)deficiency and defects in ciliogenesis and cilia function:polarity,density,and beat[J].PLo S One,2014,9(10):e107271.
[6]Sapiro R,Tarantino LM,Velazquez F,et al.Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus[J].Biol Reprod,2000,62(3):511-518.
[7]Zhang Z,Sapiro R,Kapfhamer D,et al.A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6,the mammalian orthologue of Chlamydomonas PF16[J].Mol Cell Biol,2002,22(22):7993-8004.
[8]Yan R,Hu X,Zhang Q,et al.Spag6 negatively regulates neuronal migration during mouse brain development[J].J Mol Neurosci,2015,57(4):463-469.
[9]Cooley LF,El Shikh ME,Li W,et al.Impaired immunological synapse in sperm associated antigen 6(SPAG6)deficient mice[J].Sci Rep,2016,6:25840.
[10]Noushmehr H,Weisenberger DJ,Diefes K,et al.Identification of a cpg island methylator phenotype that defines a distinct subgroup of glioma[J].Cancer cell,2010,17(5):510-522.
[11]Han YB,Feng HL,Cheung CK,et al.Expression of a novel T-complex testis expressed 5(Tctex5)in mouse testis,epididymis,and spermatozoa[J].Mol Reprod Dev,2007,74(9):1132-1140.
[12]Teshiba R,Masumoto K,Esumi G,et al.Identification of TCTE3as a gene responsible for congenital diaphragmatic hernia using a highresolution single-nucleotide polymorphism array[J].Pediatr Surg Int,2011,27(2):193-198.
[13]Li W,Mukherjee A,Wu J,et al.Sperm associated antigen 6(SPAG6)regulates fibroblast cell growth,morphology,migration and ciliogenesis[J].Sci Rep,2015,5:16506.
[14]Teves ME,Zhang Z,Costanzo RM,et al.Sperm-associated antigen-17 gene is essential for motile cilia function and neonatal survival[J].Am J Respir Cell Mol Biol,2013,48(6):765-772.
[15]王琪,张静,田杰,等.酵母双杂交RRS筛选系统对已知蛋白间相互作用的验证[J].重庆医科大学学报,2008,33(2):133-135,193.
[16]施琼,翁亚光.酵母双杂交系统筛选Mps1相互作用蛋白[J].重庆医科大学学报,2006,31(6):854-856,888.
[17]Rashid S,Grzmil P,Drenckhahn JD,et al.Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia[J].Reproduction,2010,139(1):99-111.
[18]Kiselak EA,Shen X,Song J,et al.Transcriptional regulation of an axonemal central apparatus gene,sperm-associated antigen 6,by a SRY-related high mobility group transcription factor,S-SOX5[J].J Biol Chem,2010,285(40):30496-30505.
[19]Silina K,Zayakin P,Kalnina Z,et al.Sperm-associated antigens as targets for cancer immunotherapy:expression pattern and humoral immune response in cancer patients[J].J Immunother,2011,34(1):28-44.
[20]Parveen Z,Bibi Z,Bibi N,et al.Disruption of murine Tcte3-3 induces tissue specific apoptosis via co-expression of Anxa5 and Pebp1[J].Comput Biol Chem,2014,53PB:214-225.