CAJ | 학술논문

目的制备兔抗人PIWIL4的多克隆抗体,鉴定其特异性,并应用该抗体检测内源性PIWIL4在人各细胞系中的表达差异及细胞定位.方法构建原核表达质粒pGEX-5X-1-PIWIL4,转化大肠杆菌BL21,PIWIL4蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.融合蛋白通过切胶纯化后免疫家兔制备抗体血清.以间接酶联免疫吸附试验(ELISA)检测抗体效价,Western blot鉴定抗体特异性及检测PIWILA在各细胞系中的表达差异,免疫荧光染色观察PIWIL4的细胞定位.结果成功构建原核表达质粒,表达并纯化PIWIL4蛋白.免疫大白兔后得到PIWIL4多克隆抗体,ELISA检测抗体效价为1:20 000,Western blot和免疫组化确定抗体具有高度特异性,并成功地应用该抗体检测到PIWIL4在人多种细胞系中的表达差异及细胞定位.结论 PIWIL4蛋白及其多克隆抗体的成功制备,为进一步研究PIWILA的生物学功能奠定了基础.
목적 제비토항인PIWIL4적다극륭항체,감정기특이성,병응용해항체검측내원성PIWIL4재인각세포계중적표체차이급세포정위.방법 구건원핵표체질립pGEX-5X-1-PIWIL4,전화대장간균BL21,PIWIL4단백경이병기-β-D-류대반유당감(IPTG)유도표체.융합단백통과절효순화후면역가토제비항체혈청.이간접매련면역흡부시험(ELISA)검측항체효개,Western blot감정항체특이성급검측PIWILA재각세포계중적표체차이,면역형광염색관찰PIWIL4적세포정위.결과 성공구건원핵표체질립,표체병순화PIWIL4단백.면역대백토후득도PIWIL4다극륭항체,ELISA검측항체효개위1:20 000,Western blot화면역조화학정항체구유고도특이성,병성공지응용해항체검측도PIWIL4재인다충세포계중적표체차이급세포정위.결론 PIWIL4단백급기다극륭항체적성공제비,위진일보연구PIWILA적생물학공능전정료기출.
Objective To generate rabbit polyclonal antibody against human PIWIL4 protein, to identify its functional characterization, measure differential expression and determine the cellular localization of PIWIL4 protein in various cell lines. Methods Prokaryotic expressed plasmid pGEX-5X-1-PIWIL4 was constructed and transformed to E. coli BL21 to induce expression by IPTG. The fusion protein was injected into rabbits subcutaneously to produce polyclonal antibodies after purification by gel regaining. Enzyme linked im-munosorbent assay (ELISA) was operated to detect the titer of the antibodies, western blotting was used to identify the specificity and sensitivity of the antibodies and detect the differential expression of PIWIL4 protein in various cell lines. Meanwhile, immunofluorescence experiments were used to show cellular localization of PIWIL4 protein. Results The prokaryotic expressed plasmid was constructed correctly. PIWIL4 protein was expressed and purified, and then rabbit polyclonal antibodies against PIWIL4 were generated after immunization with the fusion protein. The titer detected by ELISA was 1:20 000. The evidence of western blotting demonstrated the specificity of the antibodies. Finally, we successfully observed the differential expression and cellular localization of PIWIL4 protein in various cell lines. Conclusion The polyclonal antibody against PIWIL4 protein has been achieved successfully. It will be propitious for the intensive functional study of PIWIL4.