| Malaria is one of the most harmful parasitic diseases which is still popular in many developing countries. After many years'effort of prevention and treatment, malaria has been under control generally in China. On the other hand, malaria is still endemic in some provinces such as Hainan , Yunnan and Guangxi provinces. An important reason is that Plasmodium falciparum produce drug-resistence to traditional antimalarial such as chloroquine. At the same time, research and development of malaria vaccine and new drug make little advancement, which leads to great difficulties to control malaria, especially falciparum malaria. Therefore study of malaria vaccine candidate antigens and drug-resistant mechanism of falciparum has become a hot spot at present.This study includes two main parts: one was gene typing of merozoite surface protein 1 and 2 of falciparum isolates from Hainan province. The other was polymorphism analysis of chloroquine resistant genes of P. falciparum.P. falciparum isolates with different gene-types probably have different pathogenicity, antigenicity and drug-sensitivity. The genotypes of merozoite surface protein 1 (PfMSP1) and surface protein 2 (PfMSP2) of falciparum isolates from Hainan province were determined. Genome DNA of falciparum was extracted from blood samples. Based on the different allele type sequences of MSP1 and MSP2, 4 pairs primers and 3 pairs primers were designed to amplify the block 2 and 3 of MSP1 and the variable regions (block 3) of MSP2 by nested PCR respectively. Amplification products were analyzed by 2% agarose gel electrophoresis stained with ethidium bromide and were viewed under UV transillumination. From 52 out of 55 blood samples from falciparum malaria patients, gene fragments of MSP1 were amplified,with the MAD20-type allele being dominant (84.6%), followed by Kl-type allele, and no RO33-type allele was found. On the other hand, from 53 out of 55 blood samples, gene fragments of MSP2 were amplified, of which the majority was the 3D7-type allele (83%) and the minority was the FC27-type allele. The mixed infection rate of two different allelic types of MSP1 and MSP2 was 15.4% and 17% respectively. Therefore two allelic types of MSP1 gene, MAD20- and Kl-type, and two allelic types of MSP2 gene, 3D7- and FC27-type, exist in malaria endemic areas in Hainan province. MAD20-type and 3D7-type were dominant.For the polymorphism analysis of chloroquine-resistant genes of Plasmodium falciparum, fragments of pfmdr1 and pfcrt genes were amplified by nested PCR. Then PCR products were digested with different restriction endomucleases to detect whether point mutations existed in pfmdr1 and pfcrt genes and their relations with chloroquine resistance. For the pfmdrl N86Y mutation, Apo I cut the wild type but Nsp 1 cut the mutant gene into two fragments. For the pfmdr1 D1246Y mutation, EcoRV cut mutant gene into two fragments. For the pfcrt K76T mutation, Apo I cut wild type into two fragments and for the pfcrt A220S mutation, Bgl I cut wild type into two fragments. All the restriction digests for pfmdrl and pfcrt were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide and were viewed under UV transillumination. As a result, pfmdrl gene N86Y mutation was not detected in all 45 isolates and D1246Y mutation was present in 3 of 45 isolates, one was chloroquine resistant and the other two were sensitive. The pfcrt K76T mutation was detected in 28 of 45 isolates, 20 were chloroquine resistant and the other 8 were sensitive and A220S mutation was detected in all isolates. It was clear that a significant association existed between mutations in pfcrt, especially the K76T mutation, and chloroquine resistant isolates and polymorphisms of pfmdr1 gene seemed to be no apparent association with chloroquine resistance.
|
PDF Full Text Request