大肠埃希菌中疟原虫MSP1-17编码基因蛋白重组及表达  

Expression and recombination of Plasmodium yoelii merozoite surface protein 1 gene in Escherichia coli

作　　者：张庆军[1] 陈国英[1] 黄希宝[1] 由井克之 

机构地区：[1]湖北省预防医学科学院,武汉430079 [2]日本长崎大学医学部感染与免疫系

出　　处：《中国媒介生物学及控制杂志》2007年第5期390-393,共4页Chinese Journal of Vector Biology and Control

基　　金：国家留学基金委员会部分资助项目(留学出19993025);湖北省卫生厅科技基金项目(HBWK2002B)

摘　　要：目的为免疫原性及疟疾特异性早期诊断研究，以及高效抗红内期疟原虫复合多价基因疫苗的研究提供基础。方法（1）以鼠约氏疟原虫MSP1编码基因为模板，片段自4855到5271共416bp，扩增出PyMSP1 DNA片段，将该片段克隆入质粒pPGEX-2T，构建含有GST编码的质粒pGEX-2T-PyMSP1-17。（2）用上述构建的质粒植入大肠埃希菌DH-5α表达重组蛋白。结果（1）构建出约氏疟原虫裂殖子表面蛋白1融合基因片段真核重组表达质粒。（2）获得MSP1-GST融合蛋白，在大肠埃希菌DH一5a收获到重组的约氏疟原虫裂殖子表面蛋白浓度为46mg／L。（3）原核表达的蛋白产物免疫小鼠后分离血清，采用间接免疫荧光法（IFA）检测疟原虫，滴度在1：400～1：1000之间均可识别IFA血片中的疟原虫。结论设计重组MSP1-17基因在大肠埃希菌中真核系统内能够得到充分表达，并具有免疫原性和抗原性。无论是作为DNA疫苗研究的候选者抑或是制备特异性抗体进行疟疾早期诊断都具有重要的意义；由于该抗原的表达量高、免疫原性强，是目前很有希望的疟疾疫苗候选抗原之一。Objective To construct recombinant plasmid pPGEX-2T-PyMSP1V30 bearing the gene coding for Merozoite surface protein 1 of Plasmodium yoelii and expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. The aim is to explore the possiblity of immunogenicity detecting and early specific diagnosis of malaria, and provide the research base in studying on higher efficiency polyvalent gene vaccine anti-erythrocytic stage parasites further. Methods Using polymerase chain reaction（PCR） technique, the Merozoite surface protein 1 was amplified from genomic DNA of Plasmodium yoelii amplifying to a 416 bp of 4855-5271 fragment, then cloned into the plasmid pPGEX-2T, and pyMSPI-17 was constructed, pyMSPI-17 expressed the fusion protein in E.coli DH-5α. Results The recombinant PGEX-2T-PyMSPI-17 contained the MSPI-17 from P. yoelii. The fusion 41 kilodalton protein of GST-MSP1 was collected and purified with the total concentration of 46 mg/L. These recombinants proteins were used to immunize C3H/He mice. The specificity and subtyping of the antibodies from the immunized C3H/He mice was carried out. Furthermore, showed reactivity with P. vivax, P. cynomolgi, P. knowlesi and P. coatneyi schizonts by immunofluorescence assays （IFA） with the titer 1：400-1：1000. Conclusion It could highly express fusion protein in E. coli that recombinant Merozoite surface protein 1 of P. yoelii and the fusion protein possessing the immunogenicity and antigenicity. It is a scientific base for malaria early diagnosis study. With in higher production of fusion protein and better immunogenicity it maybe used as an efficient candidate genetic vaccine against the asexual blood stages of plasmodium.

关 键 词：约氏疟原虫 裂殖子表面蛋白 基因重组 

分 类 号：R382.31[医药卫生—医学寄生虫学] R378.21[医药卫生—基础医学]