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Original Article

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)

Young-Ha Kim1, Mohammad Saiful slam1,2, Myung-Jo You1,*
The Korean Journal of Parasitology 2015;53(1):85-93.
Published online: February 27, 2015

1Department of Veterinary Parasitology , College of Veterinary Medicine and Biosafety Research Centre, Chonbuk National University, Jeonju 561-756, KoreaJFIFddDuckydqhttp://ns.adobe.com/xap/1.0/ Adobed     ! 1AQa"q 2#w8B36v7XRr$9bCt%u&Ws'(xy4T5fH  !1AQaq"2B Rbr#u67Ѳ3sTt5v8Sc$4ĂCÔ%UӅFV ?_Aנj- H>>,m*>fzp"TrKkr^r.|_&]|*vPuܶvoQ1mwVJUhu-I"=LniAƕ8"۲ k*ҿ[yu:.vUQ+)%F DHyVBk>Hy8jݹ q~9D4KRmzQ)^ʔ.J%k_tVi5NTjg!'ky|5asOȻ)R۸ߩFMԿ3L4j6dڜ#NIwUF]JqB/(FafJRzq3\G՛ ?~\ 6)6W4m[O^L0E&rRMض*C .]Unl-1 1r#Rj/&QɈ׉˩s6Rj=5Tg.y.·Pӡ:JJS:C8-2u]d&vUz;7p9 5VnL֢"y)">iי(IDDd| Yj0; LRfS:ktYK%*N2^m|&dğth":ey)uPQZW)gcC3Pv&MMWd&Ŵ۲mvTRoժM03*F3Yd6\8,\hݻ kߔi<k NTwSԪmljj[>->ptU%'LR>&EBH$MQAUx[$Z6vi&_a.KIQ{hyƒ j"JOC9eFҝfj;˚Ω<[3_m% lQ@4g=5$(J]Yc-OMq<Ǎ wSzڗ)k$7VIP붾ͯnV+卵*t]iЎD31~SA1éC2u)ʼnQn-Uoi3:grI8ؓWm*G zܕ)ZקJ}Y YlGeJ6cB2I NS3Q>k=KTBT]W6+SOXQgGR? telˊ%-Re\hѯ2TF"C/OJΩ6r[N.0{SpljjX1“jOsӥ;ҭhe}xu`Ք&.)yO̒ Fߑ.$Qw;9Iw2o+RVJMSOj[SoҌZ%;`d$blQ{Ro{Imڌ>3egf\O֝Uzx"䢸g+mv%Gʆ:|V[N'&ס-ޝ'kfE|K,G&˳98Juin/\\Qݿ̋v~Ǩ!rtWU d|E߫R4d}.qPw*Ӭv5YEcn~f5c%MTMkb-F>5JT,})QHg%{("ӔȸWMsYyWNRrkkJr0XドnͫT}r-jj,Ŕʍ\Q2Ri>v$5!]"JB2WɅ)]VԜUc8i|.jeRO6^V.¸ Q&#|ܶ-*uOG%JAtRZRr]FFG\۩w+?'zչSѧt jz>KW&ot{7P&2D;&\\>Q2JzܗAKSfeNn[jRrԕf6,q,F1tRfԗ>vֶևj-&R'Zi2=xv~Elbsvm8=ӛ"ū񕜈BȩlWau[]ٷBߨF~J!|Ipr3R̴#Yp)={7:G{+:\W}n|Q#%)7^-h"Ƒq:M*%J&$T軨I333׎g_- ucBwwjp[6i25$̏bU’ٱRv?G\~#Iͪb7<<}Ezt" q_Inw,7-d,G÷%T* Wg1"䥱kq/A.,_KhqŒxwvo u2ۥۧ.bQ}XκA$֣ +K״ZUNmڸII{.v{5z5ѮRme[moyƾd~cRݾK'j.\i&/S6f|b=5: p!6i_ 4j6=.si˧eƾtS^c.Y^RJVS-Vi3,esi08?H$GvZgg?gi䤟2adw릿:"۪lkSN>q-4kI܋ێe̊qۅgDoѨ9; #T.Q;7#~_Ufstb_'w~Xw1Xk,vcOt._}v}8"(4Z\ۘgk?J?bm_c!g{HZV]Fkk%~gEt)b秴vΰB|꽸}mp~E6ݹv;7P٤v+ri*3Ԣ|'O14_~7nP{7ZU\Vű[ +7󖱅o#:ǥŬ\|3r%TJX]V7ez¨Y]lc|O3V! 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*Corresponding author (tick@chonbuk.ac.kr)
• Received: May 10, 2014   • Revised: November 13, 2014   • Accepted: December 24, 2014

© 2015, Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)
Korean J Parasitol. 2015;53(1):85-93.   Published online February 27, 2015
DOI: https://doi.org/10.3347/kjp.2015.53.1.85
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Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)
Korean J Parasitol. 2015;53(1):85-93.   Published online February 27, 2015
DOI: https://doi.org/10.3347/kjp.2015.53.1.85
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Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)
Image Image Image Image Image
Fig. 1. Comparison of Coomassie brilliant blue R250-stained proteins on 1-DE gel from unfed and partially fed female ticks. F, proteins from partially fed female ticks; M, relative molecular weight and position of standards; U, proteins from unfed female ticks. Arrows mark differences in protein amounts between unfed and partially fed female ticks.
Fig. 2. The results of 1-DE immunoblot SDS/12.5% PAGE comparing proteins extracted from unfed and partially fed female ticks. F, proteins from partially fed female ticks; M, relative molecular weight and position of standards; U, proteins from unfed female ticks. Fifteen μg of whole body proteins from unfed and partially fed female ticks were used in the experiment. Representative images are based on 3 independent experiments. Arrows mark differences in protein amounts between unfed and partially fed female ticks. White arrow indicates a decrease in immune reaction in fed female ticks.
Fig. 3. Representative silver-stained 2-DE images of whole body proteins from female ticks. (A) Proteins from unfed female ticks. (B) Proteins from partially fed female ticks. Proteins (240 μg) were focused on pH 4-7 IPG strips (13 cm) and were then separated by SDS/12.5% PAGE. Reference molecular masses are indicated on the right of the gel. Representative images are based on 3 independent experiments. (C, D) Analysis of A and B using ImageMaster 2-D Platinum Software. The total number of detected spots (red crossed) was 818 for unfed female ticks and 670 for partially fed female ticks. The numbers of spots detected below 20 kDa (blue crossed) were 186 and 370 spots for unfed and partially fed ticks, respectively.
Fig. 4. Representative 2-DE images of the whole body proteins from unfed female ticks of H. longicornis. (A) Silver stained gel. (B) Immunoblot probed with polyclonal antibodies against H. longicornis. Proteins (240 μg) were focused on pH 4-7 IPG strips (13 cm) and were then separated by SDS/12.5% PAGE. Reference molecular masses are indicated on the right. 2-DE immunoblot shows the antigenic spots revealed by a pool of sera from rabbits experimentally infested with H. longicornis. The proteins of interest are marked in the image (rectangles). (C, D) Magnifications of panels A and B. In D, antigenic spots are indicated by black numbered circles and are equivalent to the proteins identified in panel C.
Fig. 5. Representative 2-DE images of the whole body proteins from partially fed female ticks of H. longicornis. (A) Silver stained gel. (B) Immunoblot probed with polyclonal antibodies against H. longicornis. Proteins (240 μg) were focused on pH 4-7 IPG strips (13 cm) and were then separated by SDS/12.5% PAGE. Reference molecular masses are indicated on the right of the gel. 2-DE immunoblot shows the antigenic spots revealed by a pool of sera from rabbits experimentally infested with H. longicornis. The proteins of interest are marked in the image (rectangles). (C, D) Magnifications of panels A and B. In D, antigenic spots are indicated by black numbered circles and are equivalent to the proteins identified in panel.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)
Molecular weight (kDa) of antigenic proteins
Unfed female ticks 1-DE 20, 60, 90
2-DE (pI 4-7) 35 (no. 10), 60 (no. 9), 70-90 (no. 1-8)
Partially fed female ticks 1-DE 16-20, 50-60, 90
2-DE (pI 4-7) 18-20 (no. 6-8), 20-23 (no. 2-5), 32 (no. 1)
Spot no. Protein ID (GenBank accession no.) Protein scores Sequence coverage (%) No. peptides P < 0.05 Calculated MW/PI
Partially fed female ticks 1 Calreticulin [Amblyomma scutatum] (AAR29938) 56 28 1 47.15 / 4.50
Calreticulin [Amblyomma rotundatum] (AAR29937) 54 27 12 47.27 / 4.52
Calreticulin [Rhipicephalus annulatus (Boophilus annulatus)] (AAR29939) 54 30 13 47.724 / 4.48
Calreticulin [Haemaphysalis longicornis] (AAQ18695) 49 27 10 47.22 / 4.52
Calreticulin [Haemaphysalis leporispalustris] (AAR29947) 45 23 9 43.25 / 4.54
6 Putative secreted WC salivary protein [Ixodes scapularis] (AAY66524) 29 31 4 12.56 / 6.54
8 Conserved hypothetical protein [Ixodes scapularis] (XP_002435538) 32 31 4 9.73 / 6.54
Table 1. Comparison of molecular weights of the antigenic proteins from unfed and partially fed female ticks
Table 2. Summary of antigenic proteins identified in the whole body of H. longicornis by MALDI-TOF

Table 1.

Table 2.

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