Abstract:

Objective To analyze the gene sequence polymorphisms and predict antigen epitope of Plasmodium falciparum merozoite surface protein-3 (PfMSP-3) from different infection sources of P. falciparum in Yunnan Province. Methods Blood samples on filter paper and the corresponding epidemiology data of patients with falciparum malaria reported through the Reporting System of Chinese Center for Disease Control and Prevention, were collected from August 2012 to December 2016. P. falciparum DNA was extracted, and the PfMSP-3 gene was amplified with semi-nest PCR. PCR products were sequenced, and all sequences were aligned with the reference sequences LN999944.1 of Pf3D7 isolate and U08851.1 of PfK1 isolate in GenBank. The haplotype number, expected heterozygsity (He), and genetic differentiation (Fst) of PfMSP-3 gene in different Plasmodium populations were analyzed by MEGA 5.04 and Arlequin 3.5.2.2 softwares. The haplotype media evolution tree was constructed with the Network 4.6.0 software. The mean number of non-synonymous substitutions per non-synonymous site (Ka) and synonymous mutations per synonymous site (Ks) were calculated with the DnaSP 5.10 software. T/B cell epitopes of PfMSP-3 peptides were analyzed and predicted using the IEDB online software.  Results A total of 190 blood samples were collected, of which 181 produced PfMSP-3 fragments at 1 100 bp, and 167 PCR products were sequenced successfully, including 121 from Myanmar, 37 from Africa and 9 from indigenous cases in Yunnan. Alignment results showed that there were 36 haplotypes in the 167 DNA sequences, with an He of (0.409 + 0.183) and a Ka/Ks of 0.98. The 36 haplotypes evolved along the direction from Pf3D7 type (Ⅰ group), to transitional type (Ⅱ group), then PfK1 (Ⅲ group). Among them, 7 haplotypes including H_1 and H_9 were Pf3D7 type, 6 haplotypes including H_7 and H_8 were the transitional type, and 23 haplotypes including H_2 and H_3 were the PfK1 type. The Pf3D7 type, transitional type and PfK1 type accounted for 49.7% (83/167), 12.6% (21/167) and 37.7% (63/167), respectively. The genetic differentiation of PfMSP-3 was highest between African and Myanmar P. falciparum populations (Fst = 0.049) and lowest between African and Yunnan populations (Fst = 0.032). The peptide chains of 36 haplotypes of PfMSP-3 had a high hydrophilicity than hydrophobicity, with indexes of 1.050~2.535 and -2.583~-3.544, respectively. The activity score of T cell epitope was -1.1. The average activity score of B cells was > 0.5, more specifically ranked as Pf3D7 type ＞ transitional type ＞ PfK1 type.  Conclusion The PfMSP-3 gene of P. falciparum populations from different infection sources has evolved into 3 genotypes, and the B cell epitopes are predicted to be more active than T cell epitopes in PfMSP-3 proteins of all genotypes.
  

Key words: Plasmodium falciparum, Merozoite surface protein-3, Antigen epitope, Polymorphism, Yunnan Province