SURE: Shizuoka University REpository http://ir.lib.shizuoka.ac.jp/ Title Construction of spider venom peptide expressing baculovirus and its potential application as bioinsecticide Author(s) MD. PANNA, ALI Citation Issue Date 2015-06 URL http://doi.org/10.14945/00009292 Version ETD Rights This document is downloaded at: 2016-03-28T11:38:10Z THESIS Construction of spider venom peptide expressing baculovirus and its potential application as bioinsecticide MD. PANNA ALI Department of Environment and Energy System Graduate School of Science and Technology, Educational Division, Shizuoka University June 2015 THESIS CONSTRUCTION OF SPIDER VENOM PEPTIDE EXPRESSING BACULOVIRUS AND ITS POTENTIAL APPLICATION AS BIOPESTICIDES クモ毒ペプチドを発現するバキュロウイルスの構築 及びバイオ殺虫剤としての応用 モハマド パンナ アリ 静岡大学 大学院自然科学系教育部 バイオサイエンス専攻 2015 年 6 月 TABLE OF CONTENTS LIST OF FIGURES.................…................................................................................... iv LIST OF TABLES......................................................................................................... x LIST OF ABBREVIATIONS........................................................................................ xi ABSTRACT................................................................................................................... 1 CHAPTER I………………………………………………………………………….. 3 1. Introduction......................................................................................................... 4 1.1 Spider-venom as biopesticidal agent................................................................. 4 1.1.1 The insect pest problem in the world........................................................... 4 1.1.1.1 Agricultural pest insects.......................................................................... 5 1.1.1.2 Insects act as disease carrier…………………………………………… 5 1.1.2 Chemical pesticides: current challenges to control insect pest……............ 6 1.1.2.1 Health consequences and environmental impacts……………………... 7 1.1.2.2 Pesticide resistance……………………………………………………. 10 1.1.2.3 Biological pesticide…………………………………………………..... 13 1.1.2.4 Spider venom peptides as bioinsecticides for insect pest control 14 1.2 Baculovirus uses as biopesticide for insect pest management………….......... 15 1.2.1 Baculovirus: classification, structure and life cycle………………………. 17 1.2.2 Recombinat baculoviruses: current challenges and future prospects 18 1.3 Spider venom as antimicrobial agent………………………………………… 20 1.3.1 Antibiotic-resistant………………………………………………………... 20 1.3.2 Discovery of new antibiotics: current challenge………………………….. 20 1.3.3. Natural sources for antimicrobial drugs………………………………….. 21 1.3.4 Antimicrobial peptides and future prospects for drugs…………………… 21 i CHAPTER II………………………………………………………………………… 24 2. Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent……………………... 25 2.1 Introduction………………………………………………………………….. 25 2.2 Materials and methods………………………………………………………. 27 2.2.1 Construction of recombinant BmNPV bacmid…………………………… 27 2.2.2 Expression of EGFP-Cit1a fusion protein in silkworm…………………... 27 2.2.3 Confocal laser scanning microscopy……………………………………… 29 2.2.4 SDS-PAGE and western blot analysis…………………………………….. 29 2.2.5 Purification of EGFP-Cit1a fusion protein from silkworm larvae 31 and pupae 2.2.6 Mass spectrometry analysis………………………………………………. 32 2.2.7 Antimicrobial assays……………………………………………………… 32 2.3 Results……………………………………………………………………….. 34 2.3.1 Construction of an expression recombinant BmNPV bacmid…………… 34 2.3.2 Expression of EGFP-Cit1a fusion protein from silkworm larvae and 34 pupae….. 3.3.3 Purification of EGFP-Cit1a fusion protein from silkworm larvae 36 And pupae 2.3.4 Antimicrobial activity of Cit1a…………………………………………… 38 2.4 Discussion……………………………………………………………………. 41 CHAPTER III……………………………………………………………………… 44 3. Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin………………………………………………… 45 3.1 Introduction………………………………………………………………….. 45 3.2 Materials and methods……………………………………………………….. 46 3.2.1 Viruses, insects and insect cell lines……………………………………… 46 3.2.2 Construction of recombinant transfer vector…………………………….. 47 3.2.3 Recombinant virus construction and toxin expression in insect host 47 and cell…. 3.2.4 SDS-PAGE and western blot analysis……………………………………. 49 3.2.5 Purification of Polh-Cit1a fusion protein from silkworm larvae…………. 50 3.2.6 Bioassays…………………………………………………………………. 52 3.2.7 Polyhedra formation……………………………………………………… 53 3.2.8 Quantification of BmNPV and AcMNPV particles………………………. 53 3.2.9 Light and fluorescence microscopic analysis……………………………... 54 ii 3.2.10 Cell cytotoxicity assays…………………………………………………. 54 3.3. Results………………………………………………………………………. 55 3.3.1 Construction of recombinant virus 55 3.3.2 Expression and purification of Polh-Cit1a in silkworm larvae…………… 55 3.3.3 Bioassays………………………………………………………………….. 57 3.3.4 Microscopic analysis of cells expressing the Polh-Cit1a fusion 60 protein 3.4 Discussion……………………………………………………………………. 63 CHAPTER IV………………………………………………………………….......... 71 4. Conclusion and future prospects……………………………………………... 72 4.1 Conclusion…………………………………………………………………… 72 4.2 Future prospects……………………………………………………………… 73 REFERENCES……………………………………………………………………… 75 ACKNOWLEDGEMENT………………………………………………………….. 96 iii LIST OF FIGURES Figure 1.1 Pesticide use is raising almost everywhere, with a few key exceptions……… 7 Figure 1.2 The variation of pesticide used by farmers in different countries…………….. 8 Figure 1.3 Pathways of environment pollution due to chemical pesticide application in agricultural farm................................................................................................. 9 Figure 1.4 Effect of chemical pesticides for boosting agricultural yields………………... 11 Figure 1.5 Mechanisms of synthetic insecticide resistance. (A) Gene duplication of carboxylesterase (COE) that discharge insecticide; (B) Transcription of P450 induces hydroxylation of the pesticide. (C) Active binding site is altered by point mutations which ultimately decrease insecticidal activity. (D) Gaining more power to transport higher amount of metabolites with excretion from the cell………………………………………………………………………. 12 Figure 1.6 Mechanism of a principal rice insect pest, Nilaparvata lugens which develop resistance against chemical insecticide at field level………………………. 15 Figure 1.7 Pesticide resistant pest species toward different pesticide that once control them…………………………………………………………………………… 16 Figure 1.8 Insecticidal toxin producing spider family with their respective peptide number………………………………………………………………………. 17 Figure 1.9 Structure of a baculovirus (AcMNPV). Baculovirus has biphasic life stages making occlusion derived virion (ODV) and budded virus (BV). The ODV is essential for primary infection and BV is responsible systematic infection……………………………………………………………………… 18 Figure 2.1 Genetic structure of cit1a and its variants…………………………………… 29 Figure 2.2 Electrophoresis analysis of colony PCR products in which positive colony showing the insert (gene of interest) into vector. PCR were performed using pFastBac insert check-F and pFastBac insert check-R primers. They bind the ~868 bp of vector plus amount of insert. Here, insert is ~900bp. M: DNA marker; lane 1 and 2 negative and positive colony…………………………. 30 Figure 2.3 Construction of recombinant plasmid. A) Recombinant plasmid with egfp- cit1a. Electrophoresis of DNA extracted from recombinant colony shows the confirmation of gene of interest. Size of recombinant vector is ~5675bp and only vector without insert is ~4775bp……………………………………….. 30 Figure 2.4 Construction of recombinant bacmid. Recombinant plasmid (pFastBac/egfp- iv cit1a) was transformed into DH10Bac E. coli cells. After transformation, the positive colony was checked using colony PCR. Positive colonies show the gene of interest. PCR were performed using M13-F and M13-R bacmid check primers. Thse primers bind the 2300bp plus insert length from positive colony. Here, insert is ~900bp……………………………………………….. 31 Figure 2.5 Construction of EGFP-Cit1a fusion gene and expression of EGFP-Cit1a fusion protein in silkworm. a Schematic representation of EGFP-Cit1a fusion gene obtained by PCR and description of EGFP-Cit1a fusion protein. Details of primers 1–5 are shown in Table 1. b Agarose gel electrophoresis of PCR products in PCR steps (PCR 1–3). Lane 1 PCR 1, lane 2 PCR 2, lane 3 PCR 3. c EGFP fluorescence analysis of the EGFP-Cit1a fusion protein expressed in silkworm on an SDS-PAGE gel. Lanes 1, 3, and 5 show the homogenates of BmNPV-CP−/EGFP-Cit1a bacmid-injected pupa, larval hemolymph, and fat body, respectively; lanes 2 and 4 show the homogenates of mock-injected pupa and larval hemolymph, respectively; and lane 6 shows the mock- injected larval fat body. Fluorescent bands were detected using Molecular Imager FX (Bio-Rad) indicated by arrows. d Western blot analysis of EGFP- Cit1a fusion protein cross-reacted with antibodies is indicated by arrows. Lane 1 shows the mock pupa homogenate; lanes 2, 4, and 6 show the BmNPV-CP−/EGFP-Cit1a bacmid-injected larval fat body, hemolymph, and pupa homogenate, respectively; lanes 3 and 5 show the mock larval hemolymph and fat body, respectively……………………………………… 35 Figure 2.6 Fluorescence detection of EGFP in silkworm larval and pupal fat bodies. a and c show BmNPV-CP−/EGFP-Cit1a bacmid-injected larval fat body and pupal fat bodies, respectively; b and d show mock-injected larval and pupal fat bodies, respectively. Cells were stained with