间日疟原虫和恶性疟原虫乳酸脱氢酶（LDH）基因核苷酸序列及LDH表位的比较分析

Jiang Li, Wang Zhen-yu, Ma Xiao-jiang, Zhang Xiao-ping, Cai Li

Changhai Municipal Center for Disease Control and Prevention, Shanghai 200336, China.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Apr;28(2):103-7.

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 beta-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 by the specific epitopes account for a small proportion in the antibody repertoire.

目的

分析间日疟原虫和恶性疟原虫乳酸脱氢酶（LDH）基因的核苷酸序列差异以及LDH表位。

方法

设计特异性引物扩增间日疟原虫和恶性疟原虫的LDH基因全长序列（GenBank登录号分别为：DQ198262和DQ060151）。对PCR产物进行测序并比较。用SYFPEITHI软件预测目的LDH抗原的表位。将间日疟原虫LDH（Pv-LDH）和恶性疟原虫LDH（Pf-LDH）基因克隆到原核表达质粒pET28a中，然后在大肠杆菌BL21(DE3)中用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。通过蛋白质印迹法和中和ELISA法分析重组Pv-LDH和Pf-LDH的免疫原性。

结果

Pf-LDH基因与参考序列（DQ198262）一致，而Pv-LDH基因与参考序列（DQ060151）在666位存在单个核苷酸差异。两个基因的编码区均含951 bp，编码316个氨基酸残基。与Pf-LDH相比，Pv-LDH的核苷酸序列同一性为75.1%，氨基酸序列同一性为90.2%。T细胞表位预测表明，有28种人类白细胞抗原（HLA）类型可识别pLDH抗原表位。共同或相似表位约占预测的180个表位的75%。Pv-LDH和Pf-LDH蛋白的特异性表位数量分别为38个和45个。蛋白质印迹分析显示，Pv-LDH重组抗原与疟疾患者血清发生反应，但其反应性远低于免疫兔血清。中和ELISA显示，Pv-LDH多克隆抗体约70.3%的反应性可被Pv-LDH抑制，而被Pf-LDH抑制的仅为30.5%。