恶性疟原虫青蒿素抗性相关k13基因及其C-末端功能域的克隆及表达
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摘要
目的探索在大肠埃希菌中表达恶性疟原虫青蒿素抗性相关基因k13及其C-末端propeller结构域,为后续蛋白结构分析及青蒿素抗性机制等功能研究奠定基础。方法以恶性疟原虫基因组DNA为模板、PCR扩增k13及其C-末端基因片段并分别克隆至pET-28a载体,转化大肠杆菌BL21(DE3),经异丙基-β-d-硫代半乳糖苷(isopropyl-β-dthiogalactoside,IPTG)诱导,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和Western blot检测重组蛋白的表达。再分别用恶性疟和间日疟患者血清进行重组蛋白rK13和rK13-propeller的Western blot检测。结果 PCR扩增得到全长k13基因和k13-propeller片段,并构建了重组质粒pET-28a-k13和pET-28a-k13-propeller。SDS-PAGE电泳和Western blot检测表明,两重组蛋白均表达,且重组蛋白rK13可与恶性疟患者血清反应,rK13-propeller既可与恶性疟又可与间日疟患者血清反应。结论利用原核表达系统成功表达重组蛋白rK13和rK13-propeller,两重组蛋白能够与疟疾患者血清反应,且与不同种疟疾的患者血清反应的特异性有所不同。
Objective To express artemisinin resistance related k13 gene and its C-terminal domain of Plasmodium falciparum in Escherichia coli, we establish the foundation for the further study on protein structure and the mechanism of artemisinin resistance. Methods We utilized Plasmodium falciparum genomic DNA as template, the k13 and k13-propeller fragments were amplified by PCR and then cloned into the expression vector pET-28a. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and the expression was induced by IPTG. The recombinant proteins were detected by SDS-PAGE and confirmed by Western blot. The proteins were also examined by Western blot using sera from patients infected with Plasmodium falciparum and Plasmodium vivax. Results The full-length k13 gene and k13-propeller fragments were amplified by PCR and the expression vectors were successfully constructed. Results of SDS-PAGE and Western blot using his-tag monoclonal antibody indicated that the recombinant proteins were expressed. Western blot analysis also showed,the rK13 protein could react with sera from falciparum malaria patients, and rK13-propeller protein could be recognized by sera from both falciparum and vivax patients. Conclusion The rK13 and rK13-propeller proteins can be expressed successfully in E. coli. The recombinant proteins can be recognized by sera from malaria patients and showed different binding specificities.
Objective To express artemisinin resistance related k13 gene and its C-terminal domain of Plasmodium falciparum in Escherichia coli, we establish the foundation for the further study on protein structure and the mechanism of artemisinin resistance. Methods We utilized Plasmodium falciparum genomic DNA as template, the k13 and k13-propeller fragments were amplified by PCR and then cloned into the expression vector pET-28a. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and the expression was induced by IPTG. The recombinant proteins were detected by SDS-PAGE and confirmed by Western blot. The proteins were also examined by Western blot using sera from patients infected with Plasmodium falciparum and Plasmodium vivax. Results The full-length k13 gene and k13-propeller fragments were amplified by PCR and the expression vectors were successfully constructed. Results of SDS-PAGE and Western blot using his-tag monoclonal antibody indicated that the recombinant proteins were expressed. Western blot analysis also showed,the rK13 protein could react with sera from falciparum malaria patients, and rK13-propeller protein could be recognized by sera from both falciparum and vivax patients. Conclusion The rK13 and rK13-propeller proteins can be expressed successfully in E. coli. The recombinant proteins can be recognized by sera from malaria patients and showed different binding specificities.
引文
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[21]ALASSANE M,SOUVIK B,TRUPTI P,et al.A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria[J].Nature,2015,520(7549):683-687.
[2]WHITE N J.Delaying antimalarial drug resistance with combination chemotherapy[J].Parassitologia,1999,41(1):301-308.
[3]NOEDL H,SE Y,SCHAECHER K,et al.Evidence of artemisininresistant malaria in western Cambodia[J].New England Journal of Medicine,2008,359(24):2619-2620.
[4]PHYO A P,NKHOMA S,STEPNIEWSKA K,et al.Emergence of artemisinin-resistant malaria on the western border of Thailand:a longitudinal study[J].The Lancet,2012,379(9830):1960-1966.
[5]KYAW M P,NYUNT M H,CHIT K,et al.Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar[J].PLo S One,2013,8(3):e57689.
[6]ASHLEY E A,DHORDA M,FAIRHURST R M,et al.Spread of artemisinin resistance in Plasmodium falciparum malaria[J].New England Journal of Medicine,2014,371(5):411-423.
[7]ARIEY F,WITKOWSKI B,AMARATUNGA C,et al.A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[J].Nature,2014,505(7481):50-55.
[8]STRAIMER J,GN?DIG N F,WITKOWSKI B,et al.K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[J].Science,2015,347(6220):428-431.
[9]AMARATUNGA C,WITKOWSKI B,DEK D,et al.Plasmodium falciparum founder populations in western Cambodia have reduced artemisinin sensitivity in vitro[J].Antimicrobial agents and chemotherapy,2014,58(8):4935-4937.
[10]LUN Z R,FERREIRA P E,FU L C.Artemisinin resistance in Plasmodium falciparum[J].The Lancet Infectious Diseases,2014,14(6):450-451.
[11]TALUNDZIC E,CHENET S M,GOLDMAN I F,et al.Genetic analysis and species specific amplification of the artemisinin resistance-associated Kelch propeller domain in P.falciparum and P.vivax[J].Plo S one,2015,10(8):e0136099.
[12]TUN K M,IMWONG M,LWIN K M,et al.Spread of artemisininresistant Plasmodium falciparum in Myanmar:a cross-sectional survey of the K13 molecular marker[J].The Lancet infectious diseases,2015,15(4):415-421.
[13]王梦,王娜娜,吕志跃.脑型疟疾的病因、症状和诊治进展[J].热带医学杂志,2016(11):1465-1467.
[14]AMARATUNGA C,SRENG S,SUON S,et al.Artemisinin-resistant Plasmodium falciparum in Pursat province,western Cambodia:a parasite clearance rate study[J].The Lancet infectious diseases,2012,12(11):851-858.
[15]WITKOWSKI B,AMARATUNGA C,KHIM N,et al.Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia:in-vitro and ex-vivo drug-response studies[J].The Lancet Infectious Diseases,2013,13(12):1043-1049.
[16]MéNARD D,KHIM N,BEGHAIN J,et al.A worldwide map of Plasmodium falciparum K13-propeller polymorphisms[J].New England Journal of Medicine,2016,374(25):2453-2464.
[17]黄旸木,曹俊.中国在抗寄生虫病医疗产品研发中的贡献--2015年诺贝尔生理学或医学奖的启示[J].中国血吸虫病防治杂志,2016,28(4):349-352.
[18]ADAMS J,KELSO R,COOLEY L.The kelch repeat superfamily of proteins:propellers of cell function[J].Trends in Cell Biology,2000,10(1):17-24.
[19]BHATTACHARJEE S,STAHELIN R V,SPEICHER K D,et al.Endoplasmic reticulum PI(3)P lipid binding targets malaria proteins to the host cell[J].Cell,2012,148(1):201-212.
[20]江海龙,卢丽琨,常学辉,等.复方磷酸萘酚喹片治疗非洲恶性疟疾35例[J].中国热带医学,2016,16(2):195-196.
[21]ALASSANE M,SOUVIK B,TRUPTI P,et al.A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria[J].Nature,2015,520(7549):683-687.