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Fractionation, identification and vaccination efficacy of native antigens from the screwworm fly, Chrysomya bezziana George Riding; Sri Muharsini; Roger Pearson; Sukarsih .; Edy Satria; Gene Wijffels; Petter Willadseni
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

sources of potential protective antigens from the sheep blowfly Lucilia cuprina. Their importance in the screwworm fly Chrysomya bezziana has now been investigated. Purified serine proteases from Chrysomya bezziana were tested for their potential as vaccine antigens in sheep, efficacy being assessed by in vitro and in vivo assays with larval Chrysomya bezziana. No effect of vaccination was observed by the in vitro assay. However, in the in vivo challenge, larval weights were diminished in the vaccinated sheep, although larval recoveries increased marginally. Vaccination with Chrysomya bezziana peritrophic membrane does induce an effective immune response against the parasite resulting in a significant reduction in larval growth and considerable larval mortality in the in vitro assay. Sequential fractionation of the peritrophic membrane with various surfactants and chaotrophic agents of increasing solubilisation capacity resulted in the separation of discrete groups of proteins. The groups of fractionated proteins were tested in a vaccination trial in sheep with vaccine efficacy assessed by in vitro assays. The urea extract, guanidine-HCl extract and SDS soluble fraction each induced significant levels of protection against Chrysomya bezziana larvae but the effects were poorer than those obtained from vaccination with whole, native peritrophic membrane. Several major proteins selected from the three most protective fractions were purified by SDS polyacrylamide gel electrophoresis. Since insufficient quantities of these proteins were available for vaccination trials, they were either sequenced directly from the N-terminus or subjected to endoproteinase Lys-C digestion, followed by peptide purification and amino acid sequencing. This gave the information necessary for the expression of several of these roteins as recombinants in a form suitable for vaccination studies. Key words: Chrysomya bezziana, peritrophic membrane, vaccination, amino acid sequence, serine protease

cDNA library construction and isolation of genes for candidate vaccine antigens from Chrysomya bezziana (the Old World Screwworm fly) Tony Voucolo; Florentina Supriyanti; Sri Muharsini; Gene Wijffe
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The construction and use of cDNA libraries for the isolation of genes encoding candidate antigens for use in a recombinant vaccine against Chrysomya bezziana is described. RNA was isolated and mRNA purified from first and third instar larvae of Chrysomya bezziana and used in the synthesis of two cDNA libraries in the bacteriophage vector λ ZAP express®. These libraries were screened using Digoxigenin-labeled DNA probes obtained from two independent approaches. First, a homolog approach used probes designed from previously characterized peritrophic membrane genes identified from the related myiasis fly, Lucilia cuprina. Secondly, a de novo approach used amino-terminal and internal peptide sequence information derived from purified Chrysomya bezziana peritrophic membrane proteins to generate DNA probes. Three peritrophic membrane genes were identified and characterized. Chrysomya bezziana peritrophin-48 was identified using the homolog approach and, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42 were identified using the de novo approach. The identification of these genes as encoding candidate antigens against Chrysomya bezziana has allowed the production of recombinant proteins for use in vaccination trials. Key words: cDNA library, peritrophin, peritrophic membrane, Chrysomya bezziana, Lucilia cuprina, vaccine

Localization of the glycoprotein Cb42 in larvae of the screwworm fly Chrysomya bezziana (Diptera: Calliphoridae) Creig Eisemanni; Sri Muharsini
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The glycoprotein Cb-peritrophin-42 was localized in third instar larvae of Chrysomya bezziana using primary antibodies raised against a non-glycosylated bacterial recombinant form of this protein. Both immunofluorescent antibody techniques on unfixed whole mounts of gut tissues and immunogold electron microscopy techniques on ultra-thin sections of fixed and embedded tissues were employed. The protein was shown to be exposed over the whole of both surfaces of the peritrophic membrane and to occur throughout its thickness. Immunogold labelling indicated that Cb-peritrophin-42 was expressed in the peritrophic membrane-secreting cells of the cardia, a specialized peritrophic membrane-forming organ situated at the junction of the foregut and midgut. The accessibility of Cb-peritrophin-42 present in intact peritrophic membrane to the primary antibodies used in the immunofluorescent antibody localization indicates that this glycoprotein is a potential molecular target for vaccination of host animals against larvae of Chrysomya bezziana. Key words: Cb-peritrophin-42, Cb42, glycoprotein, peritrophic membrane, immunolocalization

Bacterial expression of larval peritrophins of Chryosomya bezziana Gene Wijffels; Tony Voucoloco; Sri Muharsini; Florentina Supriyanti
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies. Key words: Chrysomya, peritrophic membrane, peritrophin, recombinant protein, hexaHis tag, GST

Expression in yeast (Pichia pastoris) of recombinant Cb-peritrophin-42 and Cbperitrophin- 48 isolated from Chrysomya bezziana (the Old World Screwworm fly) Sri Muharsini; Tony Voucoloco
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Pichia pastoris has been investigated as a means to express recombinant forms of two putative peritrophic membrane antigens from Chrysomya bezziana, Cb-peritrophin-42 (Cb42) and Cb-peritrophin-48 (Cb48). Recombinant Cb48 was expressed as a secreted and glycosylated protein. The yield of recombinant protein was 8 mg per litre of culture. In contrast, recombinant Cb42 was not expressed at detectable levels in Pichia pastoris, probably due to A + T rich sequence which may cause premature transcriptional termination. To expedite Cb42 expression in yeast, Cb42 was divided into two domains: Cb42A and Cb42B. Cb42B was successfully expressed in Pichia pastoris, yielding 0.4 mg per litre of culture. However, Cb42A was not expressed. This work demonstrates that although Pichia pastoris offers considerable benefits as an expression system producing high level of glycosylated protein, success may vary from protein to protein. Key words: Chrysomya bezziana, peritrophin, Pichia pastoris

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana Roger Pearson; Sri Muharsini; Gene Wijffels; Tony Voucoloco
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike. Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatography

Vaccine Development Strategy for Myiasis Caused by the Larvae of Chrysomya Bezziana (The Old World Screwworm Fly) Sri Muharsini
WARTAZOA, Indonesian Bulletin of Animal and Veterinary Sciences Vol 15, No 2 (2005): JUNE 2005
Publisher : Indonesian Center for Animal Research and Development

Indonesia is an endemic area for myasis caused by the larvae of Chrysonrya bezziana . So far, the controlling method for myiasis is using insecticides such as organophosphate and coumaphos compounds . Sterile Insect Technique (SIT) was a successful method for myiasis control, however, this method is expensive, beside it needs a large amount of insecticide. Therefore, vaccinations as an alternate controlling method for myiasis which relatively cheap, environment friendly and in accordance with the sustainable agriculture concept are needed . The strategy of myiasis vaccine development has been done by the Indonesia Research Institute for Veterinary Science in collaboration with IUC-ITB, Bandung and CSIRO, Brisbane-Australia, are briefly discussed in this paper. The first step was to identify and purify the protective antigens from excretory/secretory material and peritrophic membrane. The excretory/secretory material was purified and analysed using amino-terminal sequencing and resulted trypsin and chymotrypsin at 26 kDa and 28 kDa, respectively . The peritrophic membrane was then fractionated and resulted candidate antigens of Cb 15, Cb42 and Cb48 . The three antigens were expressed into bacterial Eschericia coli and yeast Pichia pastoris resulting different yield depending on each protein character . The strategy of myiasis vaccine could be used as an alternate way for vaccine development for other parasite diseases in Indonesia. Key words: Chrysoniya bezziana, vaccine, excretory/secretory, peritrophic membrane

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