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Original Article

Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Yonghong Hu1, Jincheng Zhang2, Shujie Yang1, Hui Wang1, Hua Zeng1, Tiantian Zhang1, Jingze Liu1
The Korean Journal of Parasitology 2013;51(3):327-334.
Published online: June 30, 2013

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Corresponding author (jzliu21@heinfo.net)
• Received: January 23, 2013   • Revised: April 24, 2013   • Accepted: April 25, 2013

© 2013, Korean Society for Parasitology and Tropical Medicine

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Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis
Korean J Parasitol. 2013;51(3):327-334.   Published online June 30, 2013
DOI: https://doi.org/10.3347/kjp.2013.51.3.327
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Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis
Korean J Parasitol. 2013;51(3):327-334.   Published online June 30, 2013
DOI: https://doi.org/10.3347/kjp.2013.51.3.327
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Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis
Image Image Image Image Image
Fig. 1 Western blot of midgut antigens from H. longicornis. M, molecular weight marker stained by amido black; Lane 1, midgut antigens incubated by rabbit negative serum; Lane 2, midgut antigens incubated by rabbit anti-H. longicornis midgut serum.
Fig. 2 SDS-PAGE analysis of midgut antigens of H. longicornis. M, molecular weight marker; Lane 1, midgut antigens of H. longicornis. The 92kDa protein (arrow) was cut for LC-MS/MS analysis.
Fig. 3 Nucleic acid and deduced amino acid sequences of Hl-Pmy. The start codon and polyadenylation signal are underlined respectively; the stop codon is marked with an asterisk. Light black shaded area indicates the peptides identified by LC-MS/MS. The predicted protective epitopes are boxed.
Fig. 4 Multiple alignment of paramyosin sequences in ticks and protective epitope of paramyosin in other invertebrates. Genbank accession numbers: Ixodes scapularis, XP_002407289; Rhipicephalus microplus, AAO20875; Haemaphysalis longicornis (Hl-Pmy), AFR32950. YX1, protective epitope (EEAEGTTDAQIDANRKRESE) of Trichinella spiralis; SP2, protective epitope (LDELSGTSSQTHDAIRRKDME) of Taenia solium. Dark black shade shows identity and light black shade shows residues conserved in 4/5 sequences.
Fig. 5 The predicted secondary structure for Hl-Pmy by using DSC algorithm. h, alpha helix; c, random coil; e, extended strand. The predicted protective epitope of Hl-Pmy are boxed.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis
Category Primer name Sequencea Hl-Pmy fragment Para-1 AGGTGCTCATTATGGACTTG Anti-1 TTGGCTTCCTCCTGCTT 3´ RACE Adaptor GTTTTCCCAGTCACGACT(15) AP GTTTTCCCAGTCACGAC FP-para2 ACGGGATGGAGATCAA 5´ RACE HBS1 ATGTCTAGCAGGAGGAGCAAGTAT HBX1 ACTTGAGGTCGATGTTGAGCTTCT Peptide Sequence MH+ -.STKDILVEEQER.- 1446.7434 -.AKELLLQTEEDHK.- 1553.8169 -.VSLDNVNHLK.- 1138.6214 -.LEEVEANALAGGKR.- 1456.7754 -.RLENERDELAAAYK.- 1677.8554 -.RCQGLQAELDEQR.- 1602.7653 -.QLQQCADQLAISQR.- 1658.8279 -.NKLESELSALQADYDELHK.- 2203.0877 -.ISEYEEQLEALLTR.- 1693.8643 -.FQAEVYELLAQVENTNK.- 1996.0022 -.VNELTTINVNIAAAK.- 1570.8799 -.SSGGAGDISIEYGTDLGALTR.- 2039.9880
Table 1. Primer pairs used for Hl-Pmy cDNA amplification by PCR

The orientation of the primer is from 5´ end to 3´ end.

Table 2 Peptides of P92 identified by LC-MS/MS from H. longicornis midguts

Table 1.

Table 2

PHD : Parasites, Hosts and Diseases

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