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NguyenThiKimChi TV pdf Thesis for Master Degree Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein 5 (PvMSP 5) of Korean isolates Nguyen Thi Kim Chi Departm[.]
Thesis for Master Degree Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein (PvMSP-5) of Korean isolates Nguyen Thi Kim Chi Department of Medicine (Parasitology) Graduate School, Inje University Adviser: Prof Kho Weon–Gyu Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein (PvMSP-5) of Korean isolates Nguyen Thi Kim Chi Department of Medicine (Parasitology) Graduate School, Inje University A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Medicine Adviser: Prof Kho Weon–Gyu February, 2016 Approved by Committee of the Graduate School of Inje University in partial fulfillment of the requirements for the degree of Master of Parasitology in Medicine Chairman Committee Seo Su-Kil (signature) Committee Jang Won Hee (signature) Committee Kho Weon-Gyu (signature) Graduate School, Inje University February, 2016 Contents List of Tables List of Figures ABSTRACT i I Introduction II Objectives III Material and Methods 3.1 Experimental subject collection 3.2 Information on parasite genome 3.3 Genomic DNA extraction of P vivax 3.4 Analysis of genetic diversity of PvMSP-5 3.4.1 Amplifying PvMSP-5 gene 3.4.2 Cloning of PvMSP-5 10 3.4.3 Analysis of genetic structure of PvMSP-5 10 3.5 Recombinant PvMSP-5 expression 10 3.5.1 Construction of recombinant plasmid of PvMSP-5 10 3.5.2 Expression of recombinant PvMSP-5 12 3.5.3 Purification of recombinant PvMSP-5 12 3.5.4 Identification of recombinant PvMSP-5 13 3.6 Humoral immune response of recombinant PvMSP-5 14 IV Results 16 4.1 Amplification of PvMSP-5 gene and sequence analysis 16 4.1.1 Amplifying and cloning PvMSP-5 gene 16 4.1.2 Analysis of genetic structure of PvMSP-5 18 4.2 Recombinant PvMSP-5 expression 26 4.2.1 Construction of recombinant plasmid of PvMSP-5 26 4.2.2 Expression and purification of recombinant PvMSP-5 27 4.2.3 Identification of recombinant PvMSP-5 29 4.3 Humoral immune response of recombinant PvMSP-5 30 V Discussion 31 VI Conclusion 36 References 37 Appendix The whole nucleotide sequences of PvMSP-5 44 Appendix Predicted amino acid comparison among the isolates 66 Acknowlegement 72 List of Tables Table P.vivax-infected blood samples used for genetic polymorphism analysis Table Primers used in amplification of the PvMSP-5 gene Table Primers used in recombinant plasmid construction of PvMSP-5 exons 11 Table Insertions and deletions of isolates in exon I 19 Table Nucleotide comparison among the isolates in exon I 19 Table Nucleotide comparision between Type I and Type II SK in exon I 20 Table Nucleotide sequence repeat in the intron of PvMSP-5 22 Table Sequence similarities of exons among the isolates 24 Table Estimates of average codon-based evolutionary divergence over sequence pairs within groups 25 List of Figures Figure Amplifying and cloning of PvMSP-5 gene 17 Figure Diagram of PvMSP-5 structure 18 Figure Phylogenetic tree of isolates in exons 21 Figure Diagram of linear B cell epitope prediction in SK 23 Figure Construction of recombinant PvMSP-5 plasmids 26 Figure Purification of recombinant PvMSP-5 28 Figure Purified recombinant PvMSP-5 29 Figure Titers of IgG antibodies to recombinant PvMSP-5 30 ABSTRACT Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein (PvMSP-5) of Korean isolates Nguyen Thi Kim Chi (Adviser: Prof Kho Weon–Gyu) Department of Medicine Graduate School, Inje University Objective: PvMSP-5 plays an important role in the binding to the RBCs and is one of the vaccine candidate proteins It has two main domains A domain shows high polymorphism while an another domain shows high conservation The conserved domain contains the regions known as epidermal growth factor-like (EGF-like) domain, which can be recognized by the host antibodies Also, there is a glycosyl-phosphatidyl-inositolanchor (GPI anchor) in the domain This domain anchors to the membrane of host cells Despite of the functional importance of PvMSP-5, the study on the protein of Korean isolates has not yet been conducted The goal of the present study is to analyze the genetic structure compared with Colombian and Thai isolates and investigate the humoral immune response of the PvMSP-5 -i- Methods: The genomic DNA of P vivax was extracted from 10 of vivax malaria patients in Korea The PvMSP-5 gene was amplified by PCR and cloned into the T vector The genetic structures were compared by using DNASIS MAX 3.0 software To evaluate the humoral immune response, part of recombinant PvMSP-5 was expressed and purified with native condition Protein expression and purification was assessed by SDS-PAGE and Western blot The humoral immune response against the recombinant PvMSP-5 was tested by enzyme-linked immunosorbent assay (ELISA) The result of ELISA was evaluated by Grapad prism software Results: The exon I of PvMSP-5 showed high polymorphism in all Korean isolates (SKs) Comparing with Thai isolates (TL), T insertion was found after 51th nucleotide in all SK and A, and (G/C) insertion after 62 th and 132th respectively SKs were divided into two groups, SK type I and SK type II, depending on TAG insertion after 355 th nucleotide The nucleotide sequence in the intron and exon II was conserved However, SKs had seven or nine of 31 bp-tandem-repeat in the intron On the contrary, TL had five of the repeat in the intron The sensitivity of the recombinant PvMSP-5 against the infected sera had 96.67% and 90% of specificity showed in Korea Conclusions: The genetic polymorphism of PvMSP-5 among Korean isolates was limited in exon I There are two types of exon I within Korean - ii - isolates with the insertion of TAG nucleotide sequence Intron was conserved Exon II was highly conserved with some SNPs among CL, TL and SK Genetic distance of Type II SK PvMSP-5 was close with CL or TL The 96.7% of infected sera responded to the recombinant PvMSP-5 Limited genetic diversity of PvMSP-5 and high immune response against PvMSP-5 could explain that the PvMSP-5 has a potential to become a vaccine candidate antigen for vivax malaria More studies with various isolates from different regions in the world might be needed to evaluate the potential of this antigen for vaccine candidate Keywords: Malaria, Plasmodium vivax, Vaccine candidate antigen, Merozoite surface protein-5 - iii -