学术
摘 要:
目的：原核表达小鼠睾丸生精新基因SRG4, 并对重组蛋白进行纯化, 为研究SRG4的生物学功能奠定基础.方法：应用RT-PCR从小鼠睾丸中扩增SRG4 C端包含一个Sad1_UNC like domain的587 bp片段, 将PCR产物克隆至pUCm-T载体.随后, cDNA片段亚克隆至带有6个组氨酸标签的原核表达载体PQE-30中, 测序鉴定.将重组质粒PQE-30-SRG4转化大肠杆菌M15, IPTG诱导, 表达产物以Western blot进行鉴定, 并进一步通过Ni-NTA Agarose亲合层析纯化.结果：成功地构建了原核表达质粒PQE-30-SRG4.IPTG诱导4 ～5 h, SRG4融合蛋白表达量达最高.Western blot分析证实, IPTG诱导表达的蛋白是SRG4融合蛋白.Ni-NTA Agarose纯化后得到较纯的SRG4融合蛋白.结论：重组质粒PQE-30-SRG4能在大肠杆菌M15中表达, 包含Sad1_UNC like domain的纯化SRG4融合蛋白可用于研究SRG4在精子发生中的生物学功能.[著者文摘]
文章出处:
《细胞与分子免疫学杂志》-2007年23卷8期 -701-703页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1007-8738（2007）08-0701-03
Prokaryotic expression and purification of SRG4, a novel mouse spermatogenesis gene
XING Xiao-wei, YUAN Hong, WANG Wei（ Cell Transplantation ＆ Gene Therapy Center, the Third Xiangy Hospital of Central South University, Changsha 410013, China ）
Abstract:
AIM： To express SRG4, a novel mouse spermatogenesis gene in E. coli and purify its fusion protein. METHODS： RT-PCR was used to amplify the 586 bp fragment that was located＇ in SRG4 C-end and included a Sadl_ UNC like domain. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into PQE-30, a prokaryotic expression vector with 6 x His tag. PQE-30-SRG4 was sequenced and transformed into E. co/i M15. The expression of histidine-tagged fusion protein was induced by IPTG. The histidine-tagged fusion protein was identified by Western blot and purified by Ni-NTA Agarose. RESULTS： The recombinant plasmid PQE30-SRG4 was constructed successfully and was expressed in E. coli M15. The expression of the fusion protein reached the top at 4 -5 h after it was induced by IPTG. The fusion protein SRG4 with 6 x His tag was confirmed by Western blot and was purified by Ni-NTA Agarose. CONCLUSION： The recombinant plasmid PQE-30-SRG4 can be expressed in E. coil M15. The purified fusion protein including a Sadl_UNC like domain can be used for studying the biological function of SRG4 in spermatogenesis.[著者文摘]
Key words:
SRG4; clone; prokaryotic expression; fusionprotein; spermatogenesis
基金资助:
国家自然科学基金资助项目（30600681）;湖南省自然科学基金资助项目（05JJ40044）
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