CAJ | 학술논문

目的通过pGEX-2T原核表达载体表达蜂毒肽.方法将人工合成整合了肠激酶作用位点的蜂毒肽基因,插入载体pGEX-2T,构建蜂毒肽与谷胱甘肽S-转移酶(GST)融合表达栽体pGEX-MEL.重组质粒转化E.coli BL21(DE3)诱导袁达,超声破碎菌体,SDS-PAGE检测蛋白表达情况,取超声上清,通过GST亲和色谱系统纯化目的融合蛋白,酶切通过溶血实验检测其溶血活性.结果 SDS-PAGE结果显示,表达了相对分子质量约为30 000的融合蛋白,目的融合蛋白量约占菌体总蛋白量的29.5%.经Western blot鉴定,表达的GST融合蛋白与GST抗体有强烈的交叉反应,说明成功表达了目的融合蛋白.取超声上清,亲和色谱纯化融合蛋白,纯度为95%.肠激酶切割得到了人工天然蜂毒肤,测定蜂毒肽回收率为80%.结论通过原核表达系统成功表达蜂毒肽,具有和天然蜂毒相同的溶血活性.
목적 통과pGEX-2T원핵표체재체표체봉독태.방법 장인공합성정합료장격매작용위점적봉독태기인,삽입재체pGEX-2T,구건봉독태여곡광감태S-전이매(GST)융합표체재체pGEX-MEL.중조질립전화E.coli BL21(DE3)유도원체,초성파쇄균체,SDS-PAGE검측단백표체정황,취초성상청,통과GST친화색보계통순화목적융합단백,매절통과용혈실험검측기용혈활성.결과 SDS-PAGE결과현시,표체료상대분자질량약위30 000적융합단백,목적융합단백량약점균체총단백량적29.5%.경Western blot감정,표체적GST융합단백여GST항체유강렬적교차반응,설명성공표체료목적융합단백.취초성상청,친화색보순화융합단백,순도위95%.장격매절할득도료인공천연봉독부,측정봉독태회수솔위80%.결론 통과원핵표체계통성공표체봉독태,구유화천연봉독상동적용혈활성.
Purpose Melittin was expressed in prokaryotic vector pGEX-2T for production. Methods Melittin gene synthesized with enterokinase digested sequence,the gene was cloned into vector pGEX-2T,and constructed a recombinant plasmid of pGEX-MEL. Then the recombinant vector was introduced into E. coli BL21 (DE3)for expression. Fusion protein was purified by affinity chromatography. Hemolytic activity of Melittin was detected. Results Analysis result showed that the expression products accumulate in the cells to about 29.5 % of total cell protein. Detection of western blot using ant-GST as the first antibody showed that a special blot was revealed among the expression products. It certified that we have succeeded in expressing the fusion protein. SDS-PAGE showed that most part of the products is resoluble. The purity of obtained protein is 95% , by through GST affinity chromatography system. Melittin is harvested with a recovery of 80% by EK digestion. Test results showed melittin has good hemolytic activity. Conclusion We have expressed Melittin successfully by prokaryotic expression system.