摘要
目的在大肠杆菌中表达恶性疟原虫谷氨酸脱氢酶(GDH)与谷胱甘肽S-转移酶融合蛋白,测定重组蛋白的免疫活性。方法采用PCR方法特异性扩增恶性疟原虫(海南株)GDH基因,双酶切后克隆入pGEX-4T-1载体中进行诱导表达,重组蛋白纯化后免疫小鼠制备特异性血清,并用琼脂双向扩散法检测效价,ELISA、Western blotting检测重组抗原的免疫活性。结果获到了重组表达的抗原蛋白,表达产物能与鼠抗恶性疟原虫血清发生特异反应,并能诱导小鼠产生特异性体液免疫应答,免疫琼脂扩散法检测抗体滴度为1∶16。结论恶性疟原虫GDH在大肠杆菌中获得高效表达且表达产物具有良好的抗原性。
Objective To express the fusion protein of glutamate dehydrogenase (GDH) with glutathione S-transferase (GST) of Plasmodium falciparum FCC1/HN in E.coli BL21 and assess the immunocompetence of the recombinant protein. Methods GDH gene of P.falciparum was specifically amplified with PCR, followed by double enzyme digestion and cloning the gene fragment into pGEX-4T-1 vector for the expression of the fusion protein GST. The recombinant plasmid was transformed into E.coli BL21. Four mice (Kunming strain) were immunized with purified recombinant protein (antigen) and the polyclonal antibodies produced in response to the treatment were collected. Enzyme-linked immunosorbent assay and Western blotting were carried out to examine the immunocompetence of the recombinant protein. Results The fusion protein was successfully expressed, which exhibited specific reaction with the sera obtained from mice immunized with P.falciparum. Specific humoral responses were elicited after introducing the fusion protein in mice and the specific antibody titer was 1:16 in agar diffusion assay. Conclusion GDH of P.falciparum may have successful expression in E.coli BL21 and the expressed protein possesses high antigenicity.
出处
《第一军医大学学报》
CSCD
北大核心
2002年第10期883-887,共5页
Journal of First Military Medical University
基金
全军医药卫生科研基金(96L034)
关键词
疟原虫
恶性
谷氨酸脱氢酶
免疫活性
Plasmodium falciparum
glutamate dehydrogenase
immunocompetence
