摘要
目的对间日疟原虫和恶性疟原虫乳酸脱氢酶(LDH)基因的序列及重组抗原的表位进行比较分析。方法根据GenBank中已知的pLDH基因序列(登录号为DQ198262和DQ060151)设计特异性引物,体外扩增间日疟原虫和恶性疟原虫LDH的全长基因,对两序列进行比对分析,用SYFPEITHI软件进行表位预测分析。将Pv-LDH和Pf-LDH基因克隆入pET28a表达载体,转化至大肠埃希菌BL21(DE3)株,加入异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,Westernblotting和中和ELISA法鉴定重组蛋白。结果Pv-LDH和Pf-LDH基因的编码区全长均为951bp,编码316个氨基酸,Pf-LDH与参考序列DQ198262完全一致,而Pv-LDH与参考序列DQ060151仅在第666位有一个核苷酸的变异;两者的核苷酸和氨基酸序列的一致性分别为75.1%和90.2%。T细胞表位预测分析结果显示,能识别pLDH抗原表位的人类白细胞抗原(HLA)分子类型共28种,预测的表位数约为180个,相同或相似的表位约占总表位数的75%,Pv-LDH和Pf-LDH特异性表位分别为38个和45个。Westernblotting分析显示,Pv-LDH重组抗原可被疟疾患者血清识别,但反应强度明显低于Pv-LDH重组抗原免疫兔血清。中和ELISA试验结果显示,Pv-LDH抗原对多克隆抗体最高抑制率可达70.3%,而Pf-LDH抗原的最高抑制率仅为30.5%。结论Pv-LDH和Pf-LDH基因的核苷酸序列及其重组抗原均有差异,特异性表位诱导的抗体在整个抗体谱中相对较少。
Objective To analyze the difference of nucleotide sequences of lactate dehydrogenase （LDH） gene and LDH epitopes of Plasmodium vivax and P. falciparum. Methods Specific primers were designed to amplify the full-length LDH gene sequence of P. vivax and P. falciparum （GenBank accession number： DQ198262 and DQ060151 respectively）. The PCR products were sequenced and compared. The epitopes of objective LDH antigens were predicted by SYFPEITHI software. Pv-LDH and Pf-LDH genes were cloned into prokaryotic plasmid pET28a, then expressed in E. coli BL21（DE3） with isopropyl β-D-1-thiogalactopyranoside （IPTG） induction. The immunogenicity of the recombinants Pv-LDH and Pf-LDH was analyzed by Western blotting and neutralization ELISA assays. Results Pf-LDH gene was same to reference sequences（DQ198262）, while there is a single nucleotide difference at the position 666 between Pv-LDH gene and reference sequences （DQ060151）. The coding region of the two genes contained 951 bp encoding a 316-amino-acid residue. Compared with Pf-LDH, Pv-LDH showed a nucleotide sequence identity of 75.1%, and an amino acid sequence identity of 90.2%. T cell epitope prediction indicated that there were 28 human leukocyte antigen （HLA） types which could recognize pLDH antigen epitopes. The common or similar epitopes accounted for about 75% of the predicted 180 epitopes. The number of specific epitopes of Pv-LDH and Pf-LDH proteins was 38 and 45, respectively. Western blotting analysis showed that the Pv-LDH recombinant antigen reacted with the sera of malaria patients, and the reactivity was much lower than that of sera of immunized rabbit. Neutralization ELISA showed that about 70.3% reactivity of Pv-LDH polyclonal antibodies could be suppressed by Pv-LDH, while only 30.5% by Pf-LDH. Conclusion There are differences in DNA sequences of LDH gene and LDH epitopes between P. vivax and P. falciparum. The antibodies induced bythe specific epitopes account for a small proportion in the antibody repertoire.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2010年第2期103-107,共5页
Chinese Journal of Parasitology and Parasitic Diseases
关键词
间日疟原虫
恶性疟原虫
乳酸脱氢酶
抗原表位
Plasmodium vivax
Plasmodium falciparum
Lactate dehydrogenase
Antigen epitope
