PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA <em>Aedes aegypti</em>

Main Article Content

Syubbanul Wathon
Fitria Mutiah
Rike Oktarianti
Kartika Senjarini

Abstract

Purification of 31 and 56 kDa Immunogenic Proteins from the Salivary Glands of Aedes aegypti

The salivary gland of arthropod vector contains various bioactive compounds and plays a role in the transmission of pathogens to the host. The host develops anti-salivary antibodies against vector saliva exposure. Our previous research has identified two immunogenic proteins with molecular weights of 31 and 56 kDa from the Aedes aegypti salivary gland protein extract. However, the role of the 31 and 56 kDa immunogenic proteins from saliva Ae. aegypti is not fully known, so it is necessary to purify two immunogenic protein fractions to better specify the target of developing a dengue vaccine. This study aimed to purify the 31 and 56 kDa immunogenic protein fractions by electroelution and dialysis methods. The purification of the two protein fractions has been successful which were confirmed by the SDS-PAGE by the existence of single-band parallel to the positive control. These results were further supported by the dot blot analysis which showed a positive reaction in the form of dark spots in the two protein fractions which were reacted with dengue patients' serum, endemic healthy people, and neonates. These results indicated that the purified 31 and 56 kDa immunogenic protein fraction can be identified by specific antibodies.

Keywords: dialysis, electroelution, immunogenic, purification, saliva

 

ABSTRAK

Kelenjar saliva vektor arthropoda mengandung berbagai senyawa bioaktif dan berperan dalam transmisi patogen ke tubuh inang. Tubuh inang mengembangkan antibodi anti-saliva terhadap paparan saliva vektor. Penelitian kami sebelumnya telah mengidentifikasi dua protein imunogenik dengan berat molekul 31 dan 56 kDa dari ekstrak protein kelenjar saliva Aedes aegypti. Namun demikian, peranan protein imunogenik 31 dan 56 kDa dari saliva Ae. aegypti belum diketahui sepenuhnya sehingga perlu dilakukan purifikasi dua fraksi protein imunogenik untuk lebih menspesifikkan target pengembangan vaksin dengue. Tujuan penelitian ini untuk melakukan purifikasi fraksi protein imunogenik 31 dan 56 kDa melalui metode elektroelusi dan dialisis. Keberhasilan purifikasi dua fraksi protein 31 dan 56 kDa terbukti dari hasil konfirmasi SDS-PAGE dengan terbentuknya pita tunggal sejajar dengan kontrol positif. Hasil tersebut diperkuat dengan analisis dot blot yang menunjukkan reaksi positif dengan munculnya noktah gelap pada dua fraksi protein tersebut ketika direaksikan dengan serum pasien DBD, penduduk sehat endemik dan neonatus. Hasil ini mengindikasikan bahwa fraksi protein imunogenik 31 dan 56 kDa hasil purifikasi dapat dikenali oleh antibodi spesifik.

Article Details

How to Cite
Wathon, S., Mutiah, F., Oktarianti, R., & Senjarini, K. (2020). PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA <em>Aedes aegypti</em>. Jurnal Bioteknologi & Biosains Indonesia (JBBI), 7(1), 59–71. https://doi.org/10.29122/jbbi.v7i1.3931
Section
Research Articles
Author Biographies

Syubbanul Wathon, Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Biology Department, Faculty of Mathematics and Natural Sciences, University of Jember

Fitria Mutiah, Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Rike Oktarianti, Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Kartika Senjarini, Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

Biology Department, Faculty of Mathematic and Natural Sciences, University of Jember

References

Ahmad R (2012) Protein purification. InTech, Janeza Trdine 9 51000 Rijeka, Croatia. doi: 10.5772/1287

Almeras L, Fontaine A, Belghazi M, Bourdon S, Boucomont-Chapeaublanc E, Orlandi-Pradines E, Baragatti M, Corre-Catelin N, Reiter P, Pradines  B, Fusai T, Rogier C (2010) Salivary gland protein repertoire from Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 10:391–402. doi: 10.1089/vbz.2009.0042

Andrew J, Bar A (2013) Morphology and morphometry of Aedes aegypti adult mosquito. Ann Rev Res Biol 3:52–69

Bano S, Lateef M, Iqbal S, Naqvi B, Iqbal L (2014) Role of protein denaturing agents and EDTA on α-amylase activity from Bacillus subtilis Kibge Has. Int J Pharm Chem Biol Sci 4:411–415

Barbosa P, Berry DL, Kary CS (2015) Insect histology: Practical laboratory techniques, 1st edn. John Wiley & Sons Ltd, West Sussex, UK. doi: 10.1002/9781118876114

Berger ST, Ahmed S, Muntel J, Polo NC, Bachur R, Kentsis A, Steen J, Steen H (2015) MStern blotting–high throughput polyvinylidene fluoride (PVDF) membrane-based proteomic sample preparation for 96-well plates. Mol Cell Proteomics 14:2814–2823. doi: 10.1074/mcp.O115.049650

Bhaskar S, Dutt S, Mukherjee R (2008) A simple method of electroelution of individual protein bands from SDS polyacrylamide gels for direct study in cellular assays. J Immunoassay 21:355–375. doi: 10.1080/01971520009349542

Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farra JJ, Hay SI (2013) The global distribution and burden of dengue. Nature 496:504–507. doi: 10.1038/nature12060

Boesri H (2011) Biologi dan peranan Aedes albopictus (Skuse) 1894 sebagai penular penyakit. Aspirator 3:117–125. doi: 10.22435/aspirator.v3i2.2966

Burgess RR (2009) Elution of protein from gels. Methods Enzymol 463:565–572. doi: 10.1016/S0076-6879(09)63032-9

Chau TNB, Hieu NT, Anders KL, Wolbers M, Lien le B, Hieu LT, Hien TT, Hung NT, Farrar J, Whitehead S, Simmons CP (2009) Dengue virus Infections and maternal antibody decay in a prospective birth cohort study of Vietnamese Infants. J Infect Dis 200:1893–1900. doi: 10.1086/648407

de Melo BMS, da Silva NLL, Gomez RS, Navegantes KC, Oliveira ALB, Almeida L, Azevedo CHM, Monteiro MC (2015) Bioactive compounds of the salivary glands from Aedes aegypti with anti-hemostatic action. Ann Res Rev Biol 8:1–17. doi: 10.9734/ARRB/2015/20322  

Dong F, Fu Y, Li X, Jiang J, Sun J, Cheng X (2012) Cloning, expression, and characterization of salivary apyrase from Aedes albopictus. Parasitol Res 110:931–937. doi: 10.1007/s00436-011-2579-x

Farjana T, Tuno N (2013) Multiple blood feeding and host-seeking behavior in Aedes aegypti and Aedes albopictus (Diptera: Culicidae). J Med Entomol 50:838–846. doi: 10.1603/ME12146

Farnesi LC, Brito JM, Linss JG, Pelajo-Machado M, Valle D, Rezende GL (2012) Physiological and morphological aspects of Aedes aegypti developing larvae: Effects of the chitin synthesis inhibitor novaluron. PLoS One 7:e30363. doi: 10.1371./journal.pone.0030363

Fontaine A, Diouf I, Bakkali N, Misse D, Pages F, Fusai T, Rogier C, Almeras L (2011) Implication of haematophagous arthropod salivary proteins in host-vector interactions. Parasites Vectors 4:187. doi: 10.1186/1756-3305-4-187

Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB, Pelegrino JL, Simmons C, Yoksan S, Peeling RW (2010) Dengue: A continuing global threat. Nat Rev Microbiol 8:S7–16. doi: 10.1038/nrmicro2460

Haney P, Herting K, Smith S (2013) Molecular weight cut-off (MWCO) specifications and rates of buffer exchange with Slide-A-Lyzer dialysis devices and snakeskin dialysis tubing. Protein Biology Resource Library, ThermoFisher Scientific, Waltam

Hedhammar M, Karlström AE, Hober S (2010) Chromatographic methods for protein purification. Royal Institute of Technology, Stockholm

Hodijah DN, Prasetyowati H, Marina R (2015) Tempat perkembangbiakan Aedes spp. sebagai penular virus dengue pada berbagai tempat di Kota Sukabumi. J Ekologi Kesehat 14:1–7. doi: 10.22435/jek.v14i1.4651.1-7

Juhn J, Naeem-Ullah U, Guedes BAM, Majid A, Coleman J, Pimenta PFP, Akram W, James AA, Marinotti O (2011) Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, Aedes aegypti. Parasites Vectors 4:1. doi: 10.1186/1756-3305-4-1

Kazimírová M, Štibrániová I (2013) Tick salivary compounds: Their role in modulation of host defences and pathogen transmission. Front Cell Infect Microbiol 3:43. doi: 10.3389/fcimb.2013.00043

Londono-Renteria B, Cardenas JC, Cardenas LD, Christofferson RC, Chisenhall DM, Wesson DM, McCracken MK, Carvajal D, Mores CN (2013) Use of anti-Aedes aegypti salivary extract antibody concentration to correlate risk of vector exposure and dengue transmission risk in Colombia. PLoS One 8:e81211. doi: 10.1371/journal.pone.0081211

Luplertlop N (2014) Aedes mosquito salivary immune peptides: Boost or block dengue viral infections. J Coastal Life Med 2:163–168. doi: 10.12980/JCLM.2.2014C1219

Miller N (2010) Recent progress in dengue vaccine research and development. Curr Opin Mol Ther 12:31–38

Murrell S, Wu SC, Butler M (2011) Review of dengue virus and the development of a vaccine. Biotechnol Adv 29:239–247. doi: 10.1016/j.biotechadv.2010.11.008

Oktarianti R, Senjarini K, Fatchiyah F, Aulanni’am (2014) Immunogenic protein from salivary gland of Aedes aegypti against to human sera. Adv Nat Appl Sci 8:101–107

Oktarianti R, Senjarini K, Hayano T, Fatchiyah F, Aulanni`am (2015) Proteomic analysis of immunogenic protein from salivary glands of Aedes aegypti. J Infect Public Health 8:575–582. doi: 10.1016/j.jiph.2015.04.022

Rahayu DF, Ustiawan A (2013) Identifikasi Aedes aegypti dan Aedes albopictus. Balaba 9:7–10

Raquin V, Lambrechts L (2017) Dengue virus replicates and accumulates in Aedes aegypti salivary glands. Virology 507:75–81. doi: 10.1016/j.virol.2017.04.009

Ribeiro JMC, Martin-Martin I, Arcà B, Calvo E (2016) A deep Insight into the sialome of male and female Aedes aegypti mosquitoes. PLoS One 11:e0151400. doi: 10.1371/journal.pone.0151400

Sa-Pereira P, Duarte J, Costa-Ferreira M (2000) Electroelution as a simple and fast protein purification method: Isolation of an extracellular xylanase from Bacillus sp. CCMI 966.  Enzyme Microb Technol 27:95–99. doi: 10.1016/s0141-0229(00)00185-x

Sasmono RT, Taurel AF, Prayitno A, Sitompul H, Yohan B, Hayati RF, Bouckenooghe A, Hadinegoro SR, Nealon J (2018) Dengue virus serotype distribution based on serological evidence in pediatric urban population in Indonesia. PLoS Negl Trop Dis 12:e0006616. doi: 10.1371/journal.pntd.0006616

Sattayasai N (2012) Protein purification. Intech, Rijeka, Croatia. doi: 10.5772/35425

Schmid MA, Kauffman E, Payne A, Harris E, Kramer LD (2017) Preparation of mosquito salivary gland extract and intradermal inoculation of mice. Bio-Protoc 7:e2407. doi: 10.21769/BioProtoc.2407

Sim S, Dimopoulos G (2010) Dengue virus inhibits immune responses in Aedes aegypti cells. PLoS One 5:e10678. doi: 10.1371/journal.pone.0010678

Sucipto CD (2011) Vektor penyakit tropis. Gosyen Publishing, Yogyakarta

Urdaneta-Marquez LFailloux AB (2011) Population genetic structure of Aedes aegypti, the principal vector of dengue viruses. Infect Genet Evol 11:253–261. doi: 10.1016/j.meegid.2010.11.020

Vázquez-Iglesias L, Estefanell-Ucha B, Barcia-Castro L, de la Cadena MP, Alvarez-Chaver P, Ayude-Varquez D, Rodriguez-Berrocal FJ (2017) A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum. PeerJ 5:e3407. doi: 10.7717/peerj.3407

Vogt MB, Lahon A, Arya RP, Kneubehi AR, Spencer Clinton JL, Paust S, Ricco-Hesse R (2018) Mosquito saliva alone has profound effects on the human immune system. PLoS Negl Trop Dis 12:e0006439. doi: 10.1371/journal.pntd.0006439

Wan SW, Lin CF, Wang S, Chen YH, Yeh TM, Liu HS, Anderson R, Lin YS (2013) Current progress in dengue vaccines. J Biomed Sci 20:37. doi: 10.1186/1423-0127-20-37

Wasinpiyamongkol L, Patramool S, Luplertlop N, Surasombatpattana P, Doucoure S, Mouchet F, Seveno M, Remoue F, Demettre E, Brizard JP, Jouin P, Biron DG, Thomas F, Misse´ D (2010) Blood-feeding and immunogenic Aedes aegypti saliva proteins. Proteomics 10:1906–1916. doi: 10.1002/pmic.200900626

Yung CF, Lee KS, Thein TL, Tan LK, Gan VC, Wong JGX, Lye DC, Ng LC, Leo YS (2015) Dengue serotype-specific differences in clinical manifestation, laboratory parameters and risk of severe disease in adults, Singapore. Am J Trop Med Hyg 92:999–1005. doi: 10.4269/ajtmh.14-0628

Zettel C, Kaufman P (2013) Yellow fever mosquito Aedes aegypti (Linnaeus) (Insect: Diptera: Culicidae). EENY-434. Entomology and Nematology Department, UF/IFAS Extension, Florida

Most read articles by the same author(s)