大鼠ERp57基因的原核表达及在精卵中的鉴定和定位  

The Prokaryotic expression of ERp57 gene and its localization on rat spermatozoa and oocytes

作　　者：朱晔敏[1] 刘悦[1] 郭强苏[1] 匡昉哲[1] 丁之德[1] 

机构地区：[1]上海交通大学医学院组织胚胎学教研室上海市生殖医学重点实验室,上海200025

出　　处：《中国男科学杂志》2010年第10期12-15,24,F0004,共6页Chinese Journal of Andrology

摘　　要：目的克隆大鼠ERp57基因、原核表达蛋白并进行纯化,最终在大鼠精子和卵细胞中进行鉴定及定位。方法运用RT-PCR从SD大鼠睾丸组织的总RNA中克隆大鼠ERp57 cDNA,构建ERp57原核表达质粒[pET-28a(+)/ERp57]并转化E.coli的BL21菌株,IPTG诱导蛋白表达,经Ni-NTA树脂亲和层析得到纯化的ERp57蛋白。随后,通过SDS-PAGE、Western blot和酶联免疫吸附等实验对重组蛋白进行鉴定,并利用获得的蛋白免疫家兔制备多抗。最后,利用Western blot和间接免疫荧光定位的方法研究ERp57在精子和卵细胞中的定性和定位。结果成功获得纯化后重组的大鼠ERp57蛋白,并证实大鼠成熟精子和卵细胞都存在ERp57蛋白。ERp57主要定位于附睾尾部精子头的内侧,顶体反应后定位于精子头的赤道区;卵母细胞则定位在细胞表面。结论通过对大鼠ERp57基因的原核表达和抗体的制备可为后续进一步研究该蛋白在受精过程中作用奠定实验基础,而ERp57在精卵细胞中鉴定和定位的结果表明它可能与精卵融合过程相关。Objective To clone rat ERp57 gene and express the recombinant protein; then, to generate anti-rERp57 antibodies and localize its distribution on rat spermatozoa and oocytes. Methods Rat ERp57 cDNA was amplified from total RNA of SD rat testis by RT-PCR and PCR product was subcloned into prokaryotic expression vector pET-28a（＋）. Then, this recombinant plasmid was transformed into E. coli. BL21 and recombinant ERp57 was expressed in host cells by IPTG induction, and purified by Ni-NTA resin under denature condition. Finally, rabbit anti-rat recombinant ERp57 polyclonal antibodies were generated, and the levels of ERp57 both in rat spermatozoa and oocytes were detected by Western blot and its distribution was localized by indirect immunofluorescence analysis. Results Rat ERp57 gene, protein and anti-rat rERp57 were successfully cloned, expressed and generated respectively. ERp57 existed in both rat mature spermatozoa and oocytes. Its distribution was mainly localized on the inner part of sperm head and appeared to be translocated to equatorial region after acrosome reaction. For oocytes, however, it was almost localized on the whole cell surface. Conclusion These results suggested that the distribution and expression of ERp57 in both rat spermatozoa and oocytes might be associated with the sperm-oocyte fusion.

关 键 词：ERp57 原核表达 蛋白定位 精子 卵子 

分 类 号：R392-33[医药卫生—免疫学]