Fu Jun, Miao Shiying, Song Wei
State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 May;29(5):507-10.
To express and purify the recombinant human sperm actin-like protein 7a (ACTL7a) N terminal (1-70) in E.coli, and prepare the corresponding rabbit anti-ACTL7a polyclonal antibodies.
We constructed the recombinant expression plasmid pGEX-6p-1-ACTL7a (1-70), transformed the plasmid into E.coli BL21 (DE3), and selected positive recombinant strain. The target protein was induced to express by IPTG and purified by nickel ions-chelating resin and glutathione sepharose 4B resin, and finally obtained by molecular sieve chromatography. The purified ACTL7a (1-70) protein were used to immunize New Zealand rabbits to prepare anti-ACTL7a polyclonal antibodies. The antibody titers were detected by ELISA, the antibody specificity by Western blotting, and the localization of ACTL7a in the spermatogenic cells of seminiferous tubule epithelium by immunofluorescence histochemistry.
The target protein ACTL7a (1-70) was successfully expressed by IPTG in E.coli, and the corresponding antiserum was obtained in the rabbits immunized with it. The titer of the antiserum reached 1:160 000 and it was proved with a good specificity.
ACTL7a was expressed in the constructed prokaryotic expression vector. After purification and immunization, high titer and specific anti-ACTL7a antibody was prepared in rabbits.
在大肠杆菌中表达并纯化重组人精子肌动蛋白样蛋白7a（ACTL7a）N端（1 - 70）片段，并制备相应的兔抗ACTL7a多克隆抗体。
构建重组表达质粒pGEX - 6p - 1 - ACTL7a（1 - 70），将其转化至大肠杆菌BL21（DE3）中，筛选阳性重组菌株。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导目标蛋白表达，先后经镍离子螯合树脂和谷胱甘肽琼脂糖4B树脂纯化，最后通过分子筛层析获得。用纯化的ACTL7a（1 - 70）蛋白免疫新西兰兔制备抗ACTL7a多克隆抗体。采用酶联免疫吸附测定（ELISA）检测抗体效价，蛋白质免疫印迹法检测抗体特异性，免疫荧光组织化学法检测ACTL7a在生精小管上皮生精细胞中的定位。
IPTG成功诱导大肠杆菌表达目标蛋白ACTL7a（1 - 70），用其免疫兔后获得相应抗血清。抗血清效价达1:160 000，且具有良好的特异性。
ACTL7a在构建的原核表达载体中得以表达。经纯化和免疫后，在兔体内制备出了高效价且特异性强的抗ACTL7a抗体。
