CAJ | 학술논문

构建美利奴绵羊磷脂酶 C zeta（PLCζ）融合蛋白表达载体，并在原核细胞内表达及纯化，为其特异性抗体的制备及生物学功能研究奠定基础。用 PCR 技术扩增出 PLCζ基因片段，亚克隆到原核表达载体pCzn1中，导入 Arctic Express TM （DE3）感受态细胞，IPTG 诱导表达，收样后进行 SDS-PAGE 电泳或Western blot 检验 PLCζ的表达情况。用镍离子亲和层析的方法大量纯化融合蛋白。结果显示，重组质粒经 PCR、酶切和测序鉴定证明载体构建正确。pCzn1表达载体在 Arctic Express TM （DE3）表达菌中能很好地表达融合蛋白。表达纯化后可获得了分子量约74 ku 的融合蛋白，符合预期大小。成功构建 pCzn1-PLCζ原核表达载体，表达并纯化了其融合蛋白，Western blot 试验表明其蛋白分子的完整性良好，这将有利于对 PLCζ蛋白进行深入的研究。
구건미리노면양린지매 C zeta（PLCζ）융합단백표체재체，병재원핵세포내표체급순화，위기특이성항체적제비급생물학공능연구전정기출。용 PCR 기술확증출 PLCζ기인편단，아극륭도원핵표체재체pCzn1중，도입 Arctic Express TM （DE3）감수태세포，IPTG 유도표체，수양후진행 SDS-PAGE 전영혹Western blot 검험 PLCζ적표체정황。용얼리자친화층석적방법대량순화융합단백。결과현시，중조질립경 PCR、매절화측서감정증명재체구건정학。pCzn1표체재체재 Arctic Express TM （DE3）표체균중능흔호지표체융합단백。표체순화후가획득료분자량약74 ku 적융합단백，부합예기대소。성공구건 pCzn1-PLCζ원핵표체재체，표체병순화료기융합단백，Western blot 시험표명기단백분자적완정성량호，저장유리우대 PLCζ단백진행심입적연구。
The construction of PLCζfusion protein expression vector in Merino sheep,was made to expresse fusion protein in prokaryotic cells.The protein was purified for the preparation of specific antibody and the study of biological function.PLCζgene fragment amplified by PCR was subcloned into the prokaryotic ex-pression vector pCzn1 and then introducted into the Arctic Express TM (DE3)competent cells.IPTG was used to induce the expression.The situation of PLCζ expression was tested by SDS-PAGE and Western blott after receiving the samples.The fusion protein was purified in quantity by the approach to Nickelion affinity chromatography.The recombinant plasmid was identified by PCR,restriction enzyme digestion and sequencing proved that the vector construction was correct.pCzn1 expression vector can well expressed the fusion protein in the Arctic Express TM (DE3)bacteria.After the expression and purification,the 74 ku fusion protein was obtained,which is consistents with expected size.PCzn1-PLCζprokaryotic expression vector was successfully constructed.Western blot tests was used to show the good integrity of its protein molecules.This result is helpful for deep study on PLCζprotein.