| Plasmodium falciparum imposes an enormous burden upon the developing world with 500 million cases and more than 2 million deaths per year, the majority of whom are young children and pregnant women, particularly primigravid women, and there are 200,000 deaths of infants per year. Malaria has became one of the important infectious diseases in our country, which impacts to the survival and health of China. Despite extensive research efforts, there is no effect vaccine available for malaria currently, and the problem of parasite drug resistance is also seriously. However, in China, the study of malaria is mainly concentrated on the epidemiologic investigation leading to weakness in basic research, which is seriously blocked the development of effective vaccines and drugs as well as sensitivity of specific molecular diagnostic techniques.Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by about 60 var genes is an key virulence factor and a prime candidate for anti-malaria vaccines . Infected red blood cells (iRBC) adhering to endotheliums and other host cells is called sequestration, which makes the parasites escape from spleen dependent killing mechanisms and induces diseases such as cerebral malaria and pregnancy-associated malaria (PAM) .PfEMP1 participates in immune evasion and immune modulation through high variation. The variation mechanism of var gene family is the focus of the study at home and abroad.PAM is characterized by marked accumulation of parasites in the intervillous space of the placenta, and is the cause of maternal anaemia as well as low birthweight, prematurity and increased infant mortality. In endemic areas, primigravid women often infect PAM, but multiple pregnancies do not show severe symptom because of the production of specific immunity. It was known that protein which encoded by var2CSA gene is the main ligand of placental adhesion, compared to typical var genes, var2CSA has conspicuous conservatism, and it is a leading candidate for the pregnancy associated malaria vaccine. Antigenic variation of var gene in Plasmodium falciparum isolate HaiNan was studied in this experiment. 56 different var genes sequences were got from 350 samples. Since there were 59 different var genes in Plasmodium falciparum isolate 3D7, it was speculated that all var genes of isolate HaiNan had been found. These 56 var genes sequences of Plasmodium falciparum isolate HaiNan had been submitted to the GenBank(http://www.ncbi.nlm.nih.gov) , and accession numbers were FJ748609~FJ748664. NJ tree of var genes of Plasmodium falciparum isolate HaiNan and 3D7 was drawed through the Neighbor-Joining algorithm of Bootstrap Test phylogeny using MEGA 4.0 software. The result showed that var genes of them were similar in conformation. Study on var genes could reveal genetic diversity among species of Plasmodium falciparum and provide theoretical basis for effective protective measures.In order to understand the var2CSA gene of Plasmodium falciparum isolate HaiNan causing PAM, var2CSA gene was sequenced and its genetic evolution status was analyzed first time. Unexpectedly, two var2CSA named HN (1) and HN (2) respectively were found. Full-length of HN (1) gene was 10429bp, encoding 3101 amino acids; full-length of HN (2) gene was 10444bp, encoding 3106 amino acids. The nucleotide homology of HN (1) and HN (2) gene was 87.11%. TMpred analysis showed that it existed transmembrane domain at 2690 ~ 2711 amino acid in HN (1) protein sequence and at 2695 ~ 2716 amino acid in HN (2) protein sequences.There were apparent protein hydrophilic areas, better antigenicity and strong surface polarity in both HN (1) and HN (2) analyzed by DNAStar software. Moreover, there was high homology among HN(1), HN(2) and 3D7, and genetic phylogenetic tree analysis showed that the isolate HaiNan was closer to the isolate 3D7 and DD2. The homology of DBL regions of var2CSA among different isolates indicated that DBL-1x, DBL-3x,DBL-4εregions were conservative and DBL-2x, DBL-5ε,DBL-6εregions were larger variation relatively and maybe have differert on the function.In order to study the region of var2CSA genes which played a major role in the adhesion on the basic of getting whole sequence of var2CSA gene, prokaryotic express for the DBL-5εand DBL-6εregion of var2CSA gene in Plasmodium falciparum isolate HaiNan was performed to study the main pathogenic areas causing pregnancy-associated malaria, Plasmodium falciparum isolate 3D7 as a control, then recombinant proteins HN-DBL5, HN-DBL6, 3D7-DBL5, 3D7-DBL6 were obtained after purified. The results of adhesion experiment by using ELISA method showed that DBL-5εand DBL-6εregion of var2CSA gene could adhesion with the CSA, and the affinity of DBL-5εregion significantly was higher than DBL-6εregion, it preliminary indicated that DBL-5εdamain of var2CSA gene in Plasmodium falciparum isolate HaiNan was probably the main function district mediated PAM by adhesion CSA; the data of adhesion test showed that DBL-5εand DBL-6εregion of var2CSA could adhere to human IgG receptor, and the affinity of DBL-5εregion was slightly higher than DBL-6εregion, it preliminary indicated that Plasmodium falciparum isolate HaiNan binding with human non-specific immunoglobulin IgG could be one of the reasons causing PAM.This study helped us understand the diversity of evolutionary mechanisms of var gene, as well as provided valuable information for development of anti-PAM vaccine. |
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