CAJ | 학술논문

目的获得原核表达GST-Syntenin1融合蛋白,制备高效价的抗Syntenin1多克隆抗体.方法 RT-PCR扩增Syntenin1 cDNA开放阅读框(CDS)序列,亚克隆到编码GST的pGEX-4T-2原核表达载体上,将编码Syntenin1的pGEX-4T-2-Syntenin1重组质粒化学转化BL21(DE3)感受态细胞,IPTG诱导GST-Syntenin1融合蛋白表达,对融合蛋白亲和纯化.SDS-PAGE鉴定后,将纯化的融合蛋白辅以弗氏佐剂,免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体效价及特异性.结果成功制备GST-Syntenin1融合蛋白及多克隆抗体.Western blot结果表明Syntenin1兔多克隆抗体效价可达到1∶20 000,且与Syntenin1蛋白特异结合.结论成功制备Syntenin1多克隆抗体,为进一步研究Syntenin1基因的功能奠定了基础.
목적 획득원핵표체GST-Syntenin1융합단백,제비고효개적항Syntenin1다극륭항체.방법 RT-PCR확증Syntenin1 cDNA개방열독광(CDS)서렬,아극륭도편마GST적pGEX-4T-2원핵표체재체상,장편마Syntenin1적pGEX-4T-2-Syntenin1중조질립화학전화BL21(DE3)감수태세포,IPTG유도GST-Syntenin1융합단백표체,대융합단백친화순화.SDS-PAGE감정후,장순화적융합단백보이불씨좌제,면역신서란대백토제비다극륭항체,병용Western blot검측항체효개급특이성.결과 성공제비GST-Syntenin1융합단백급다극륭항체.Western blot결과표명Syntenin1토다극륭항체효개가체도1∶20 000,차여Syntenin1단백특이결합.결론 성공제비Syntenin1다극륭항체,위진일보연구Syntenin1기인적공능전정료기출.
Objective To express the GST fusion protein, GST-Synteninl in E. colt, and to prepare the polyclonal antibody of Synteninl. Methods CDS fragment of Synteninl was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Synteninl recombinant. The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG. The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE. The purified fusion protein was mixed with the Freund's adjuvant, and then injected into New Zealand white rabbits by hypodermic injection. The polyclonal antibody titer and specification were identified by Western blot. Results Synteninl polyclonal antibody-bind Syteninl protein specifically and the antiserum tiger reached to 1:20 000. Conclusion The Synteninl polyclonal antibody with high titer and high specificity was prepared successfully. This will be very helpful for the further study on Synteninl function and molecule mechanism of cancer metastasis.