人体疟原虫乳酸脱氢酶基因多态性及B细胞表位预测

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”。

Analysis of lactate dehydrogenase gene polymorphisms and prediction of B cell epitopes in four human Plasmodium species

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.