中缅边境恶性疟原虫裂殖子表面蛋白1,2基因多态性研究

Genotyping and polymorphism analysis of merozoite surface protein 1 and 2 of Plasmodium falciparum isolates from China-Myanmar border areas

目的 对云南省及边境地区恶性疟原虫的裂殖子表面蛋白（merozoite surface protein，MSP）1，2基因进行分型研究，确定其等位基因的类型和分布特征，结合当地的恶性疟原虫株流行病学信息，为该地区疟疾的防治提供科学依据。 方法 从缅甸拉咱、那威和我国云南省西双版纳勐腊、德宏瑞丽、保山腾冲采集恶性疟患者血样。用巢式PCR（nest-PCR）扩增18S rRNA基因，确定感染疟原虫的种类。对检测为恶性疟原虫以及恶性疟原虫/间日疟原虫混合感染的样本，进行恶性疟原虫MSP-1、MSP-2基因的扩增并作测序、验证与序列分析。 结果 共采集89份恶性疟样本，经18S rRNA基因检测，确定间日疟9例，恶性疟78例和混合感染2例。在检测为恶性疟和混合感染的80份样本中，69例扩增出MSP-1基因片段，77例扩增出MSP-2基因片段。在MSP-1等位基因中，以MAD20型68.75%为主，RO33型23.75%和K1型20.00%次之。来源于勐腊的样本均未检出RO33型和K1型；MSP-2等位基因FC27型和3D7型的感染率均为91.25%，无明显的优势虫株；MSP-1和MSP-2基因多克隆样本所占百分比与多重性感染（multiplicity of infection，MOI）分别为22.50%、1.81和86.25%、3.51。MSP-1 和MSP-2等位基因目的片段多样性与其原虫密度之间存在相关性（Spearman’s r=0.496，P＜0.05；Spearman’s r=0.240，P＜0.05）。MSP-1和MSP-2等位基因测序结果表明，在FC27型基因序列3＇端发现1个新的APK序列，在3D7基因型序列中检测到1个新的PAT重复序列和其它19个新的序列。 结论 云南省及边境地区恶性疟原虫分离株MSP-1等位基因存在MAD20型、K1型和RO33型3种类型，以MAD20型为优势虫株，勐腊样本未发现K1型和RO33型；MSP-2等位基因存在FC27型和3D7型2种类型，其优势均不明显。

Objective To determine the genotypas of merozoite surface protein-1（MSP-1） and merozoite surface protein-2 （MSP-2） of P.falciparum isolates from China-Myanmar border areas, and provide the important information for prevention and treatment of malaria in local areas. Methods The extracted DNA of all isolates underwent the nested PCR using specific primers of 185 rRNA gene to identify their malaria species. Then, for the samples identified as P. falciparum or P. falciparum and P. vivax, based on the different allele type sequences of MSP-1 and MSP-2, the specific primers were designed to amplify the MSP-1 and MSP-2 by nested PCR respectively to identify their genotypes, and the PCR products were sent to sequence for analyzing the polymorphism of MSP-1 and 2. Results Totally 89 samples from 5 counties/cities were collected and 9,78, 2 were infected with P. vivax, P. falciparum or both of them respectively by the nest-PCR. For the MSP-1 gene, from 69 out of 80 isolates of P. falciparum, gene fragments of MSP-1 were amplified with the MAD20- type allele being dominant（68.75%）, followed by RO33-type allele（23.75%） and Kl-type allele（20.00%） while there were not RO33- and K1-type allele in Mengla＇s study area of Xishuangbanna. Regarding the MSP-2 gene, from 77 out of 80 isolates of P.falciparum, gene fragments of MSP-2 were amplified with the FC27-type allele and 3D7-type allele being the same dominant （91.25%）. The percentage of muhiclonal isolates and total multiplicity of P.falciparum infection （MOI） were 22.50%, 1.81 and 86.25%, 3.51 for MSP-1 and MSP-2, respectively. The association between multiplicity of the target genes and parasite density showed a significant positive correlation （Spearman＇s r=0.496, P〈0.05） and （Spearman＇s r=0.240, P〈0.05） for MSP-1 and MSP-2, respectively calculated by Spearman＇s correlation coefficients of SPSS. Otherwise, the 3D7 clone sequence contains one PAT amino acid and nineteen different amino acids except for those reported before,and the APK motif at the 3＇ end of FC27 allele is also a new sequence. Conchtsion The three allelic types of MSP1 gene, MAD20-, RO33- and Kl-type, and two allelic types of MSP-2 gene, FC27- and 3D7-type, exist in Laza and Nawei of Myanmar, and Mengla, Ruili, Tengchong of Yunnan Province. MAD20-type was the predominant MSP1 allelic family in our study sites while there were not K1- and RO33-type alleles in Mengla city of Xishuangbanna, and the FC27- and 3D7-type of MSP-2 allelic family had the same frequency.