疫苗候选抗原Pvs48/45在中缅边境的遗传多样性特点

Genetic diversity and natural selection of Pvs48/45 in the China-Myanmar border

目的了解间日疟原虫传播阻断疫苗候选抗原Pvs48/45在中缅边境地区的基因多态性和自然选择特点。方法提取39例来自中缅边境主要流行区(Laiza)的间日疟原虫基因组DNA,采用巢式PCR(nest-PCR)扩增Pvs48/45基因开放阅读区(Open reading frame,ORF);通过MEGA 4.0、DnaSP v5.10和NETWORK 4.6对Pvs48/45基因多态性和自然选择特点进行分析。结果研究发现了6个突变位点,均为非同义突变(E35K,H211N,K250N,D335Y,A376T,K418R),导致了5种单倍型;Pvs48/45基因的核苷酸多态性水平极低(π=0.00064),CRDⅢ区最高(π=0.00163);多种中性检验均表明Pvs48/45基因处于平衡选择;种群进化分析和单倍型网络图显示Pvs48/45具有较强的地理分化,且H2型为中缅边境地区频率最高的单倍型。结论Pvs48/45在中缅边境地区遗传多样性很低,且在进化过程中经历平衡选择,提示其可作为传播阻断疫苗候选靶点;Pvs48/45在全球具有较强的地理分化,应根据地区遗传特点开发更加有效的传播阻断疫苗。

In order to understand the genetic diversity of Pvs48/45, a protein located on the surface of female gametocytes and regarded as a promising transmission-blocking vaccines(TBVs) candidate antigen of Plasmodium vivax(P. vivax), 39 finger-prick blood samples of P. vivax cases were collected from the China-Myanmar border epidemic area(Laiza) and P. vivax infections were diagnosed by light microscopy and confirmed by PCR targeting the 18 S rRNA. Pvs48/45 gene was amplified by nest-PCR with specific primers and the PCR-products were directly sequenced. Polymorphism analyses were carried out through the MEGA 4.0, DnaSP v5.10 and NETWORK 4.6 software. When aligned with the Sal-1 reference sequence(PlasmoDB: PVX083235), the ultimately obtained Pvs48/45 sequences showed only six non-synonymous mutations resulting in six amino acid substitutions and five haplotypes(Hd=0.633). Nucleotide diversity of Pvs48/45 open reading frame(ORF) was 0.00064, while in three cysteine-rich domains(CRDs) of the Pvs48/45 were 0.00053, 0 and 0.00163, respectively. Multiple neutral tests indexes targeting the ORF and three CRDs deviated from zero positively, indicating that Pvs48/45 was undergoing balancing selection in the China-Myanmar border. High degrees of genetic differentiation existed among populations in the China-Myanmar border, Vanuatu and Colombia than very low between China-Myanmar border and Thailand;this may be caused by a geographical barrier. Haplotype network showed that H2 was the predominant haplotype in the China-Myanmar border and it was sharing with Thailand other than Vanuatu and Colombia. Overall Pvs48/45 displayed limited genetic diversity and experienced balancing selection, indicating that it was potential candidate target for TBVs. The Pvs48/45 had strong population differentiation, suggesting the necessity to develop effective vaccines based on regional genetic characteristics.