A Study Of Polymorphism Of Lactate Dehydrogenase(LDH) Gene Of Plasmodium Vivax From Jianghuai Regin

Objective: To analyze the nucleotide sequences features and genetic polymorphisms of lactate dehydrogenase of Plasmodium vivax (JiangHuai Region, China), and to evaluate its scientific significance and prospective application as a target antigen in the diagnosis of malaria and new drug screening.Methods: 1. The peripheral blood of patients with Plasmodium vivax from Bengbu, Wuhe and Lixin, Anhui province, was collected and made into malarial parasite thick film blood slides and filter paper with dried blood spot (DBS) specimens. Thirty nine samples of Plasmodium vivax genomic DNA were extracted from DBS specimens by using QIAamp DNA minikit (QIAGEN, Germany). All the samples were proved to be simple Plasmodium vivax by blood smear dyed with microscopic examination and PCR test. 2. A pair of primers were picked according to the nucleotide sequences of lactate dehydrogenase from Plasmodium vivax Belem (Accession No:DQ060151); then the LDH gene of Plasmodium vivax (JiangHuai Region) was amplified, electrophoresed, purified and cloned into pGEM-T vectors. The vectors were transformed into E.coli DH5α; the positive clones were tested by PCR and sequencing. The sequences were searched with NCBI BLAST program in the web.Results: All the 39 blood samples were proved to be simple Plasmodium vivax by blood smear dyed with microscopic examination and PCR test, simple infection or concurrent infection with plasmodium falciparum or other plasmodium were not detected. The positive clones of PvLDH were sequenced and the length was 951bp, no changes were found among 39 samples. The results of NCBI BLAST showed that the gene homology of LDH among Plasmodium vivax JiangHuai Region, Sal-I, Belem, Salvador, EJEU60134, MIMO612514, ASAO6G29643, ASAO6R09632 and MIAO61042 was up to 99.7%-99.9%, only 1-3 bases were changed. The amino acids sequences of Plasmodium vivax (JiangHuai Region) were also analyzed, the results showed that homology among JiangHuai Region, Sal-I and Belem was 100%, 99.7% (V195I) between JiangHuai Region and EJEU60134, and 99.7% (D69N)between JiangHuai Region and MIAO61251, respectively.Conclusion: The nucleotide sequences of LDH gene from Plasmodium vivax (JiangHuai Region) are highly conserved. The genetic homology among Sal-I and Belem were up to 99.7%-99.9%, and were mostly synonymous mutations in amino acids. Only 0.3% variation existed between EJEU60134 and MIAO61251. This study provides a certain theoretical foundation for the development of Plasmodium vivax diagnostic kit.