Iranian Journal of Public Health 2017. 46(10):1359-1365.

Cloning of K26 Hydrophilic Antigen from Iranian Strain of Leishmania infantum
Bibi Razieh HOSSEINI FARASH, Mehdi MOHEBALI, Bahram KAZEMI, Homa HAJJARAN, Behnaz AKHOUNDI, Reza RAOOFIAN, Abdolmajid FATA, Majid MOJARRAD, Mohammad Kazem SHARIFI-YAZDI

Abstract


Abstract

Background: Visceral leishmaniasis (VL) caused by Leishmania infantum is the most severe form of leishmaniasis in Iran, which causes a high mortality rate in the case of inaccurate diagnosis and treatment. This study aimed to clone of K26 gene from Iranian strain of L. infantum and register the sequencing results in Genbank to facilitate the preparation a new K26 antigen for the detection of L. infantum infection.

Methods: L. infantum was obtained from an infected domestic dog in Meshkin-Shahr area from northwestern Iran in 2015. Canine visceral leishmaniasis was confirmed by direct agglutination test (DAT), rK39 dipstick and parasitological methods. L. infantum was confirmed by N-acetyl glucosamine -1-phosphate transferase (nagt)–PCR and its sequencing. The band of interest for k26 form Iranian strain of L. infantum was purified by gel extraction kit after PCR amplification and then ligated into pBluescript II SK (+) and pET-32a (+), respectively. The sequences of recombinant plasmids were analyzed and submitted to Genbank.

Results: The submission of rk26 nucleotide sequence was performed to the GeneBank/NCBI Data Base under accession number KY212883. The related gene was showed a homology about 99% to L. chagasi and L. infantum k26 gene, while the level of homology in comparison with different strains of L. donovani ranged from 84-94%.

Conclusion: The successful rk26 cloning into an expression vector performed in this study could help to produce a new recombinant antigen for serodiagnosis of VL especially in areas where L. infantum is the main causative agent.

 

 


Keywords


Visceral leishmaniasis, Leishmania infantum, K26 immunodominant antigen

Full Text:

PDF

References


Bhatia A, Daifalla NS, Jen S, Badaro R, Reed SG, Skeiky YA (1999). Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi. Mol Biochem Parasitol, 20;102(2):249–61.

Dantas-Torres F (2006). Leishmania infantum versus Leishmania chagasi: do not forget the law of priority. Mem Inst Oswaldo Cruz,101(1):117–8.

Pattabhi S, Whittle J, Mohamath R et al (2010). Design, development and evaluation of rK28-Based Point-of-Care tests for improving rapid diagnosis of visceral leishmaniasis. PLoS Negl Trop Dis, 14;4(9): e822.

Mohebali M, Hajjaran H, Hamzavi Y et al (2005). Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet Parasitol, 15;129(3–4):243–51.

Mohebali M (2013). Visceral leishmaniasis in Iran: Review of the epidemiological and clinical features. Iran J Parasitol, 8(3):348–58.

Srivastava P, Dayama A, Mehrotra S, Sundar S (2011). Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg,105(1):1–6.

Badaró R, Benson D, Eulálio MC et al (1996). rK39: A cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis, 173(3):758–61.

Banu SS, Ahmed B-N, Shamsuzzaman AKM, Lee R (2016). Evaluation of recombinant K39 antigen and various promastigote antigens in sero-diagnosis of visceral leishmaniasis in Bangladesh. Parasite Epidemiol Control,1(3):219–28.

Sundar S, Rai M (2002). Laboratory Diagnosis of Visceral Leishmaniasis. Clin Diagn Lab Immunol, 9(5):951–8.

Sivakumar R, Sharma P, Chang K-P, Singh S (2006). Cloning, expression, and purification of a novel recombinant antigen from Leishmania donovani. Protein Expr Purif, 46(1):156–65.

Farahmand M, Nahrevanian H (2016). Application of recombinant proteins for serodiagnosis of visceral leishmaniasis in humans and dogs. Iran Biomed J, 20(3):128–34.

Rosati S, Ortoffi M, Profiti ME et al (2003). Prokaryotic expression and antigenic characterization of three recombinant Leishmania antigens for serological diagnosis of canine leishmaniasis. Clin Diagn Lab Immunol, 10(6):1153–6.

Farajnia S, Darbani B, Babaei H, Alimohammadian MH, Mahboudi F, Gavgani AM (2008). Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran. Parasitology, 135(9):1035–41.

Taran M, Mohebali M, Modaresi MH, Mamishi S, Mojarad M, Mahmoudi M (2007). Preparation of a K39sub recombinant antigen for the detection of Leishmania infantum antibodies in human: a Comparative study with an immu-nochromatographic test and direct agglutination. Iran J Parasitol, 2(2):25–33.

Akhoundi B, Mohebali M, Babakhan L et al (2010). Rapid detection of human Leishmania infantum infection: A comparative field study using the fast agglutination screening test and the direct agglutination test. Travel Med Infect Dis, 8(5):305–10.

Hajjaran H, Mohebali M, Teimouri A et al (2014). Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene. Infect Genet Evol, 26:203–12.

Elmahallawy EK, Martinez AS, Rodriguez-Granger J, Hoyos-Mallecot Y, Agil A, Mari JMN, et al (2014). Diagnosis of leishmaniasis. J Infect Dev Ctries, 13;8(8):961–72.

Taheri T, Seyed N, Mizbani A, Rafati S (2016). Leishmania-based expression systems. Appl Microbiol Biotechnol, 100(17):7377–85.

Rosano GL, Ceccarelli EA (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbial, 17;5:172.

Merck Millipore (2016). pET-32a (+) DNA - Novagen | 69015. Available from: http://www.merckmillipore.com/INTL/en/product/pET-32a%28%2B%29-DNA---Novagen,EMD_BIO-69015

Ali D, Abbady A-Q, Kweider M, Soukkarieh C (2016). Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein. Open Life Sci, 11(1):166–176.

Reina O (2015). Problems amplifying GC-rich regions? Problem Solved! Trinity College of Dublin, the Ireland. Available from: http://bitesizebio.com/24002/problems-amplifying-gc-rich-regions-problem-solved/

Sahdev S, Saini S, Tiwari P, Saxena S, Singh Saini K (2007). Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Mol Cell Probes, 21(4):303–7.

Godiska R, Mead D, Dhodda V et al. (2010). Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli. Nucleic Acids Res, 38(6): e88.

Hommelsheim CM, Frantzeskakis L, Huang M, Ülker B (2014). PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications. Sci Rep, 23;4:5052.

26.GeneScript (2016). Codon Optimization - Increase Protein Expression. Available from: http://www.genscript.com/codon-opt.html

Rosário EY do, Genaro O, França-Silva JC et al (2005). Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis. Mem Inst Oswaldo Cruz, 100(2):197–203.

da Costa RT, França JC, Mayrink W, Nascimento E, Genaro O, Campos-Neto A (2003). Standardization of a rapid immunochromatographic test with the recombinant antigens K39 and K26 for the diagnosis of canine visceral leishmaniasis. Trans R Soc Trop Med Hyg, 97(6):678–82.


Refbacks

  • There are currently no refbacks.


Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.