Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein 5 (Pv...

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 5 (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 5 (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 1

II Objectives 7

III Material and Methods 8

3.1 Experimental subject collection 8

3.2 Information on parasite genome 8

3.3 Genomic DNA extraction of P vivax 9

3.4 Analysis of genetic diversity of PvMSP-5 9

3.4.1 Amplifying PvMSP-5 gene 9

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 1 The whole nucleotide sequences of PvMSP-5 44

Appendix 2 Predicted amino acid comparison among the isolates 66

Acknowlegement 72

List of Tables

Table 1 P.vivax-infected blood samples used for genetic polymorphism analysis

8

Table 2 Primers used in amplification of the PvMSP-5 gene 9 Table 3 Primers used in recombinant plasmid construction of PvMSP-5 exons

11 Table 4 Insertions and deletions of isolates in exon I 19 Table 5 Nucleotide comparison among the isolates in exon I 19 Table 6 Nucleotide comparision between Type I and Type II SK in exon I 20

Table 7 Nucleotide sequence repeat in the intron of PvMSP-5 22

Table 8 Sequence similarities of exons among the isolates 24 Table 9 Estimates of average codon-based evolutionary divergence over

sequence pairs within groups 25

List of Figures

Figure 1 Amplifying and cloning of PvMSP-5 gene 17

Figure 2 Diagram of PvMSP-5 structure 18

Figure 3 Phylogenetic tree of isolates in exons 21

Figure 4 Diagram of linear B cell epitope prediction in SK 23

Figure 5 Construction of recombinant PvMSP-5 plasmids 26

Figure 6 Purification of recombinant PvMSP-5 28

Figure 7 Purified recombinant PvMSP-5 29

Figure 8 Titers of IgG antibodies to recombinant PvMSP-5 30

ABSTRACT

Genetic polymorphism and humoral immune response to Plasmodium vivax merozoite surface protein 5 (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-inositol-anchor (GPI glycosyl-phosphatidyl-inositol-anchor) in the domain This domain glycosyl-phosphatidyl-inositol-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

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

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