Investigation the Existence of H2A Gene in Leishmaniatropica

Leishmaniases are infectious diseases caused by several species of the intracellular protozoan of Leishmania, which are transmitted by sandfly [1]. Leishmaniasis has different clinical manifestations, ranging from self-healing cutaneous ulcers to potentially fatal visceral infection [1]. These diseases represent a global public health problem, with an estimated prevalence of 12 million human beings infected. Syria is one of 9 countries have the majority of cutaneous Leishmaniasis cases in the world [2]. Current control is based on chemotherapeutic treatments which are expensive, toxic and associated with high relapse and resistance rates. Vaccination remains the best hope for control of all forms of the disease, so the development of a safe, effective and affordable anti Leishmanial vaccine is a critical global public-health priority. Immunization with naked DNA is a relatively new approach, which promises to revolutionize the prevention and treatment of infectious diseases [3]. Many studies have been made to find a protective antigen against Leishmaniasis and many candidates have been studied forth is aim, including histones. Histones are structural proteins found in the nucleus, where they play an important role in the organization and function of chromatin [4]. These highly-conserved proteins that elicit strong immune responses are generally designated as pan antigens [5]. Specific antibodies for parasite histones, obtained from dogs with visceral Leishmaniasis, react against Leishmania H2A [6], H3 [7], H2B and H4 [8] but do not recognize mammalian histones. In the right conditions,these panantigens can provide the immune modulatory properties needed for vaccine design [9]. So these histones and their genes are good candidates for vaccine.H2A gene was sequenced in many species of Leshmania, but not in L. tropica, and their immunogenicity was tested [10,11]. We chose H2A gene, to design a DNA vaccine against the infection with Leishmaniatropica which is responsible of the most cutaneous cases in Syria. The existence of H2A gene wasn’t proved in L. trpoica. In this study; A methodical study was first made to know if this gene exists in the genome of a Syrian strain of Leishmania tropica, to prove if there is an expression of H2A Gene in these parasites, and to sequence this gene.

Primer design for PCR

Sequence of H2Agene in Leishmaniatropica is not known so we made an alignment for the sequences of this gene in other species using CLC free workbench 7 and Vector NTI Express programs and theclustalw2 and emboss_needle tools. Primers were designed using http://www.idtdna.com/calc/analyzer and http://www.geneinfinity.org/sms/sms_primanalysis.html tools. High purified Primers were ordered from VBC- Biotech, Vienna. Final sequences for primers were: [forward, 5’-ATTAGAATTC ATG GCT ACTCCTCGCAGCG-3’; reverse, 5’-CT TCTAGA CTAAGCGCTCGGTGTCGCCTTG-3’]. Underlined are the ECORI and XBAI restriction sites included for direct cloning in the PCI mammalian expression vector [Promega, United States].

DNA extraction

DNA was extracted from the promastiogotes of a Syrian strain Leishmaniatropica [LCEBS,Damascus, Syria]. DNA was isolated from Leishmania promastigotes, cultured on RPMI-1640 medium [Lonza, Switzerland] supported with Fetal bovine serum [Cytogen, GmbH, Germany], with DNA extraction kit [Thermoscientific, Lithuania] according to manufacturer’s instructions.

RNA isolation and cDNA synthesis

Total RNA was extracted from harvested Leishmaniatropica promastigotes using TRI reagent [Sigma, St Louis, MO, USA]. RNA was quantified by NanoDrop1000 [Thermo Fisher Scientific MA, USA]. RNA-integrity was checked by separating in 1.5% agarose gel. First strand cDNAs were synthesized using Revert Aid First Strand cDNA Synthesis kit [Thermoscientific, Lithuania] according to manufacturer’s instructions.

Polymerase chain reaction

DNA and cDNA of H2A gene were amplified by thermal cycler [Bio-Rad, USA] using PCR master mix [Thermo scientific, Lithuania] and the primers mentioned above. PCR protocol was optimized using gradient annealing temperatures. Reactions were performed in 25 μl of a mixture containing 1μl of 10μM from each primer, 2, 5 μl of template DNA or cDNA [110 ng\μl], 12, 5 μl of PCR master mix and 8 μl of PCR water. Thermal cycling conditions were 95°C for 5min followed by 35 cycles of 95°C for 1min, 61.6°C for 45sec and 72°C for 1 min finally 72°C for 5 min. PCR products were loaded on a 2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.

Plasmid construction and purification

PCR product containing H2Acoding sequence was purified using Gene Jet PCR Purification kit [Thermoscientific, Lithuania] according to manufacturer’s instructions. Purified cDNA and the PCI mammalian expression vector [Promega, United States] were double digested with ECORI plus XBAI. Digested cDNA was ligated with the digested PCI mammalian expression vector using t4 DNA ligase [Thermoscientific, Lithuania]. PCI-H2A clones were transformed in to E-coli TOP 10. Recombinant plasmid was extracted from colonies and purified using GF-1 plasmid DNA extraction kit [Vivantis, Malaysia].

Sequences Analysis

Full-length double-stranded sequence analysis was performed on purified Recombinant plasmid PCI-H2Aby sequencing, using T7 [forward] and PCR 3’[reverse] vector primers on automated sequencers [ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit, Perkin-Elmer, Foster City,USA].

Evaluation DNA and RNA quality and purity

Extracted DNA electrophoresison a 1% agarose gel only showed one band which proves that there isn’t any degradation in the extracted DNA [Figure 1]. The purity of the DNA was 1.8 which showed a high degree of purity. Extracted RNA electrophoresis on a 1.5% agarose gel showed a standard profile and there was no contamination with genomic DNA [Figure 2]. The purity of the RNA was 2.1 which showed a sufficient degree of purity.

Figure 1: Electrophoresis of extracted Leishmaniatropica DNA: Lane 1 DNA ladder 1Kb, Lane 2,3 extracted Leishmaniatropica DNA.

Figure 2: Electrophoresis of extracted Leishmaniatropica RNA: Lane1 Leishmaniatropica RNA, Lane 2 extracted Leishmaniatropica DNA.

Polymerase chain reaction

PCR protocol was optimized using gradient annealing temperatures and we chose the most suitable annealing temperature which was 61.6°C.Gel electrophoresis of H2APCR product on the level of DNA and cDNA showed only one bandabout400 base pair which proves the specificity of our primers. So we proved the existence of H2A gene in the genome of a Syrian strain of Leishmaniatropica [Figure 3] and in the cDNA of this strain [Figure 4].

Figure 3: Amplification products of Leishmaniatropica H2Aon 2% agarose gel electrophoresis stained with ethidium bromide. Lane 1 DNA ladder 50bp, lane 2 H2A- DNA amplification product about 400bp.

Figure 4: Amplification products of Leishmaniatropica H2A on 2% agarose gel electrophoresis stained with ethidium bromide. Lane 1 DNA ladder 50bp, lane 2 H2A-cDNA amplification product about 399 bp.

Cloning and sequencing of the H2A cDNA

In an effort to clone and characterize the Leishmaniatropica H2A gene, a cDNA clone was identified and sequenced. The sequence was submitted to the Gene bank underKT956066.1 Accession number. The complete nucleotide sequence of the 399bp cDNA insert, and the deduced amino acid sequence in comparison with other species were showed in [Figures 5 & 6] respectively. The deduced amino acid sequence predicts a protein containing 132 amino acids. The H2A predicted amino acid sequence was compared with the sequences of proteins coded by cDNA of H2A in other species of Leishmania such as L.infantum, L.major, L.donovania, L.mexicana and L.braziliensis using Vector NTI Program. This search revealed that H2A belongs to the histone H2A family. Interestingly, L.tropicaH2A shows significant similarity with H2A histones of L.infantum [92.4%], L.major [91.7%], L.donovania [92.4%], L.mexicana [91.7%] and L.braziliensis [78%]. The last high similarity shows that a good H2A DNA vaccine could protect from infection by the different species of Leishmania.

Figure 5: The complete nucleotide sequence of the H2A cDNA.

Figure 6: Nucleotide sequences of H2A cDNA for L.tropica, L.donovani, L.infantum, L.major and L.mexicana. The deduced amino acid sequence is indicated below the cDNA sequence.

Our results proved the existence of H2A gene in the genomic DNA and the cDNA of a Syrian Leishmaniatropica strain. We proved for the first time the existence of H2A gene in Leishmaniatropica, and defined the sequence of the cDNA of this gene and submitted it to the Gene bank under KT956067 accession number. PCI-H2A clones could now be tested to evaluate their immunogenicity as DNA vaccine against Leishmania species.

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