Objective: To analyze the polymorphism of Plasmodium lactate dehydrogenase (pLDH) gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites.
Methods: The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The pLDH genes of four human Plasmodium species were amplified using nested PCR assay and sequenced. The polymorphisms of pLDH genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB).
Results: The sequences of P. vivax LDH (PvLDH), P. falciparum LDH (PfLDH), P. ovale LDH (PoLDH) and P. malariae LDH (PmLDH) genes were obtained from 153, 29, 17 and 11 blood samples from patients with P. vivax, P. falciparum, P. ovale and P. malariae malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the PoLDH gene (π = 0.012), and the π values were all < 0.001 for PvLDH, PfLDH and PmLDH genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the "86-PGKSDKEWNRD-96" short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the "266-GQYGHS (T)-271" short-peptide was present in PvLDH and PoLDH peptide chains, while "212-EEVEGIFDR-220" was only found in the PvLDH peptide chain, and "208-LISDAE-213" was only seen in the PfLDH peptide chain.
Conclusions: The PoLDH gene polymorphism may be derived from the weak negative purification selection, while PvLDH, PfLDH and PmLDH genes may maintain a relatively conservative state. There may be two B-cell epitopes "212-EEVEGIFDR-220" and "208-LISDAE-213" in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between P. vivax and P. falciparum infections.
［摘要］ 目的 分析 4 种人体疟原虫乳酸脱氢酶 (Plasmodium lactate dehydrogenase, pLDH) 基因多态性并预测 pLDH 肽 链 B 细胞抗原表位。方法 收集传染病报告信息管理系统中登记的云南省疟疾病例血样和流行病学等信息。采用巢式 PCR 技术扩增 4 种人体疟原虫 pLDH 基因并测序, 应用 MEGA 7.0.26 和 DnaSP 5.10 软件分析 4 种人体疟原虫 pLDH 基因 DNA 序列多态性, 并采用免疫表位数据库 (IEDB) 预测 pLDH 肽链 B 细胞抗原表位。结果 分别从 153 份间日疟、29 份恶 性疟、17 份卵形疟和 11 份三日疟患者血样中获得间日疟原虫 LDH (PvLDH)、恶性疟原虫 LDH (PfLDH)、卵形疟原虫LDH (PoLDH)、三日疟原虫 LDH (PmLDH) 基因测序序列, 分别定义 15、2、4、2 种单倍型, 核苷酸多样性指数 (π) 为 0.104。PoLDH 基因种内分化程度较高, π为 0.012; PvLDH、PfLDH 和PmLDH 基因π值均 < 0.001。4 种人体疟原虫 pLDH 肽链可预测 到 4~5 个/链的 B 细胞抗原活性区, 活性得分约 0.430; 其中活性区短肽 “86-PGKSDKEWNRD-96” 为 4 种人体疟原虫共有 的B细胞抗原表位, “266-GQYGHS(T)-271” 仅出现在PvLDH和 PoLDH 肽链, PvLDH、PfLDH肽链特有的B细胞抗原表位 分别是 “212-EEVEGIFDR-220” 和 “208-LISDAE-213”。结论 PoLDH 基因多态性可能来自微弱的负向纯化选择, PvLDH、PfLDH、PmLDH 基因则可能维持了相对保守状态。pLDH 肽链近 C 端可能存在可区分间日疟、恶性疟原虫感染的B细胞 抗原表位 “212-EEVEGIFDR-220” 和 “208-LISDAE-213”。.
Keywords:
B-cell epitope; Epitope prediction; Gene polymorphism; Human