Measure Sequence And Analysis Of The Gene Of P.Vivax TBV New Candidate Antigen Pvs48

PrefaceMalaria constitutes a major human health threat caused by infection with protozoan parasites of the genus Plasmodium. P. vivax which represents the most widespread malaria parasite in the world. Malaria also remains one of the leading parasite diseases in China, locating in 18 provinces / autonomous regions / municipalities with a infection of 0.53 billion population. As the relapsing behaving of vivax malaria process and parasite strains resistant to anti - malarial drugs as well as mosquito vectors resistant to insecticides have emerged, new approaches for malaria control are badly needed and intensive research efforts have gone to develop malaria vaccines.Although different complementary approaches to vaccine development are being undertaken in several laboratories, the major obstacle for the development of an effective malaria vaccine is the extensive antigen diversity in natural populations infected with malaria parasites. Transmission - blocking vaccines ( TB-Vs) are becoming more attractive because of its limited genetic polymorphism. Pvs48 is the gamete surface protein, which plays a role in gamete survival in the mosquito midgut. Some results have indicated that Pvs48 can be the leading vivax malaria TBV candidate.Although these efforts have been undergone for Pvs48, the data is still limited not enough to identify the feasibility for the application of this kind of TBV. Some results have suggested that the polymorphism of target antigen exists among field isolates, even single amino acid substitution, can change the primary structure of parasite antigen. This may have an adverse impact on the efficacy of vaccines application. Therefore, it is necessary to acquire the evaluation of the pre -existing polymorphism in Pvs48 before applying it to the vivax malaria epidemic regions. Otherwise, P. vivax is prevalent in extensive areas of China, but there is no report to be published on this study. For these purposes, we provided a detailed analysis of the diversity of this malaria vaccine candidate antigen iso-lated from Hubei and Zhejiang province of China which would apply is the necessary basic data for the confirmation and development of TBVs against malaria.Materials and methodsThe parasite DNA used for the genetic polymorphism assay was obtained from dried filter paper blood spots as described by Sakihama and others. The blood samples are from 12 P. vivax infecting patients during malaria transmission season. As a positive control, genomic DNA extracted from P. vivax Sal 1 strain was used. The genes were PCR amplified and the amplified products were purified, cloned, and sequenced. Both strands were sequenced from at least two clones from two independent PCR amplifications. Computer — based algorithms were used to analyze neucleotide and amino acid sequences and the sequence a-lignments were done with Mac Vector software.Results1. The Pvs48 gene sequence for 1300 nucleotides codes 433 amino acids, which include the complete Pvs48 sequence.2. At nucleotide level, six non - synonymous mutations (G/A103, C/ A631, A/C750, G/T1003, G/A1126, A/G1253) and no synonymous mutations were identified. At arnino acid level, There are only six amino acid substitutions in 12 Chinese samples from P. vivax single endemic regions(E/K35,H/ N211, K/N250, D/Y335, A/T376, K/R418).3. These polymorphic nucleotides generated a total of 6 haplotypes ( C -?750 a 750 _ pl003 _ p 1126 _ * 1253 q103 _ p631 _ * 750 _ QH26 Q631 _ J^150 _ Q1003 _ pll26 _ * 1253 pl03 _ r>631 _ .750 _ q\126 _ ? 1253 q103 _ p631 _ a 750 _ q1003 _ qU26-A ) among the 12 sequences. Haplotypes L -A -G -G -A from Chinese isolates can be considered predominant in China.DiscussionIn this study, our report is the first one that describes the genie polymorphism of TBV candidate antigen Pvs48 from Chinese P. vivax single endemic region. We analyzed 12 complete new sequences of Pvs48 from two national parasites populations and found only 6 nucleotide changes that result in amino acid substitutions in this candidate antigen. So we can further confirm that Pvs48 is highly conservative in primary structure and will become a new and good TBV candidate antigen.Recently, Transmission - blocking vaccines are one of the strategies in controlling malaria. Protective immunity has been developed with malarial sexual stage parasites in non - human malaria models, these vaccines can induce antibodies that prohibit the sexual -stage parasites growth within the mosquito vector and consequently prevent parasite transmission from mosquito to susceptible human. As we all know, The malaria parasite is undoubtedly characterized by a broad antigenic polymorphism between different strains and isolates. These antigenic properties constitute major problems in the development of vaccines against the disease. Antigenic polymorphism limits the immunogenicity and immunore-activity of vaccines, and results in differences for efficacy of vaccines. This has been shown in developing an effective vaccine against gametophyte because of antigenic variation maybe induced by immune selection pressure. Thereby, if the problem of malarial antigenic polymorphism can be solved, the development for an efficient malarial vaccine can become true. While in contrast, the major advantages of TBVs candidate antigen Pvs48 are the strong immunogenicity and the limited pre - existing antigenic polymorphism. So, TBVs with limited pre -existing antigenic polymorphism will have a broad prospect. Therefore, incorporation with a powerful TBV and vaccines targeting other stages or anti - malarial drugs may be an important strategy to conquer the obstacle suffered from the broad antigenic polymorphism and control more and more severe malaria spread trends.Gametophyte, the only way to transmit malarial parasite from cut mosquitoto cut susceptible human, is a ideal target of TBVs. Pvs48 is the gamete surface protein and being considered as the lead TBVs candidate. At present, there is no report about it in the world, in the results, Pvs48 will become a new and ideal vaccines targeting of candidate antigen.Conclusion1. Our report is the first one that describes the gene characteristic of superficial antigen gametophyte Pvs48 from Chinese single P. vivax endemic regions.2. Genie polymorphism of TBV candidate antigen Pvs48 that from Chinese single P. vivax endemic regions were highly finie among the geographically dispersed isolates ( Hubei, Zhejiang field isolates ).