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Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans.

Hajissa K, Zakaria R, Suppian R, Mohamed Z - Parasit Vectors (2015)

Bottom Line: Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification.The entire protein was successfully expressed and purified.The results showed 100 % sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. Khalid541983@yahoo.com.

ABSTRACT

Background: Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.

Findings: To accomplish our goals, a single synthetic gene of approximately 456 bp, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene was cloned into a pET32a expression vector and transformed into BL21 E. coli. The entire protein was successfully expressed and purified. Subsequently, the preliminary diagnostic performance of expressed protein was evaluated by developing IgG enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. The results showed 100 % sensitivity and specificity.

Conclusion: A purified protein expressing multi-immunodominant epitopes of T. gondii was generated. Further studies are required to evaluate the immunogenicity in animal models and to verify the immuno-reactivity of USM.TOXO1 as a diagnostic antigen.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of synthetic gene USM.TOXO1 construction; a Oligonucleotides design, U1–U10 is sense-strand primers. L1-L9 antisense-strand primers. b Assembly PCR steps 1: Oligos mixture 2: Gene assembly 3: Gene amplification
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Fig1: Schematic diagram of synthetic gene USM.TOXO1 construction; a Oligonucleotides design, U1–U10 is sense-strand primers. L1-L9 antisense-strand primers. b Assembly PCR steps 1: Oligos mixture 2: Gene assembly 3: Gene amplification

Mentions: Full amino acid sequences of SAG1, GRA2, and GRA7 were retrieved from the GenBank database. The immunodominant epitopes expressed within these antigens were identified by the ABCpred online prediction server [19]. Subsequently, three potential epitopes with high antigenicity and immunogenicity scores from each antigen were selected (Table 1). The epitopes were then combined in a manner that facilitated the design of the complementary oligonucleotides (Fig. 1). Finally, based on the DNA sequence of the predicted epitopes, a 456 bp synthetic gene (USM.TOXO1) was designed using VNTI computer program software (Life Technologies, USA).Table 1


Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans.

Hajissa K, Zakaria R, Suppian R, Mohamed Z - Parasit Vectors (2015)

Schematic diagram of synthetic gene USM.TOXO1 construction; a Oligonucleotides design, U1–U10 is sense-strand primers. L1-L9 antisense-strand primers. b Assembly PCR steps 1: Oligos mixture 2: Gene assembly 3: Gene amplification
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465724&req=5

Fig1: Schematic diagram of synthetic gene USM.TOXO1 construction; a Oligonucleotides design, U1–U10 is sense-strand primers. L1-L9 antisense-strand primers. b Assembly PCR steps 1: Oligos mixture 2: Gene assembly 3: Gene amplification
Mentions: Full amino acid sequences of SAG1, GRA2, and GRA7 were retrieved from the GenBank database. The immunodominant epitopes expressed within these antigens were identified by the ABCpred online prediction server [19]. Subsequently, three potential epitopes with high antigenicity and immunogenicity scores from each antigen were selected (Table 1). The epitopes were then combined in a manner that facilitated the design of the complementary oligonucleotides (Fig. 1). Finally, based on the DNA sequence of the predicted epitopes, a 456 bp synthetic gene (USM.TOXO1) was designed using VNTI computer program software (Life Technologies, USA).Table 1

Bottom Line: Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification.The entire protein was successfully expressed and purified.The results showed 100 % sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia. Khalid541983@yahoo.com.

ABSTRACT

Background: Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.

Findings: To accomplish our goals, a single synthetic gene of approximately 456 bp, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene was cloned into a pET32a expression vector and transformed into BL21 E. coli. The entire protein was successfully expressed and purified. Subsequently, the preliminary diagnostic performance of expressed protein was evaluated by developing IgG enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. The results showed 100 % sensitivity and specificity.

Conclusion: A purified protein expressing multi-immunodominant epitopes of T. gondii was generated. Further studies are required to evaluate the immunogenicity in animal models and to verify the immuno-reactivity of USM.TOXO1 as a diagnostic antigen.

No MeSH data available.


Related in: MedlinePlus