Indonesian Journal of Biotechnology
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Analysis of Toxoplasma gondii Repeat Region 529 bp (NCBI Acc. No. AF146527) as a Probe Candidate for Molecular Diagnosis of Toxoplasmosis
No full text Toxoplasmosis is a disease caused by protozoan parasite Toxoplasma gondii. The infection is commonly asymptomatic. The availability of confirmative and accurate detection system is really needed. This research was aimed to develop a molecular diagnosis based on the conserved and high copy number repeat region of Toxoplasma gondii with hibridization method. Nucleic acid was isolated from tachyzoites. The repeat region of T. gondii was amplified using PuRe Taq Ready To Go-PCR Beads (Amersham Bioscience), forward primer 5’- GAC TCG GGC CCA GCT GCG -3’ and reverse primer 5’- CCT CTC CTA CCC CTC CTC -3’. The amplicon was sequenced using ABI Prism 3100-Avant Genetic Analyzer (PT. Charoen Pokphand, Jakarta). Probe was labeled using digoxigenin-11-dUTP. Application of probe to detect it’s complementary nucloeic acid was done by hibridization method.The research concluded that probe toxo-103 bp was highly homolog with several strain of T. gondii and it has no homology either with host’s genome or other parasites which have close genetic relationship with T. gondii. Hybridization analysis showed that probe could detect the complementary nucleic acid up to 10 ng/ul concentration.
Construction and Immunogenicity Testing of Salmonella, STM1 Vaccine Vector Expressing HIV-1 Antigen
No full textObjective of this study: to determine the ability of Salmonella enterica serovar Typhimurium STM1 as a delivery vehicle for the HIV p24 gene and HIV env gene. The STM1 delivery HIV-p24 vaccination was carried out in the form of a recombinant or a DNA vaccine whereas only a DNA vaccine was used for HIV env. Naked DNA vaccination was also tested and immune responses were evaluated following immunisation in mouse model. Results: vaccination cellular immune responses induced by recombinant p24 STM1 (STM1/pHly-p24) were greater than those elicited by the p24 DNA vaccine in STM1 (STM1/VR-p24), (but statistically not significance) than those induced by oral vaccination. However, IgA responses induced by oral vaccination with either a recombinant or DNA vaccine of p24 in STM1 are higher than those induced by IP vaccination. Conclusions: This result confirmsother studies that Salmonella was able to deliver HIV antigens to the immune system and induced specific immune responses to the HIV antigen
Combination PT-PCR with in vitro Transcription-Translation System: Rapid and Simple Approach for Monoclonal Antibody Production
No full textWe have developed a simple and efficient system for screening and generation of monoclonal antibodies, which can bypass conventional monoclonal antibody production system that includes time-consuming, labor-intensive establishment and cultivation. Our method consist of: (1) cDNA synthesis from single B cells of immunized mouse, followed by (ii) PCR amplification of the Lc and Hc (Fd portion) cDNAs separately using low concentration (0,05 ìM) of the respective cDNA-specific primers with 5’ homotags in the presence of the homotag-specific primer (0,5 ìM), (iii) overlapping PCR of the amplified Lc and Hc cDNAs with the cassettes for the T7 promoter (T7P) and T7 terminator (T7T) to construct the following expression units: T7P-Lc-T7T and T7P-Hc-T7T, and (iv) in vitro expression of these units using an Escherichia coli S30 extract followed by ELISA screening withoutpurification. Light-and heavy-chain cDNA were amplified and expressed using in vitro system, thus avoiding time consuming steps of cloning and bacterial expression. Having successfully amplified and expressed Lc and Hc genes from single B cells in rapid, simple, versatile, labor-saving, and cost effective, this method seem to be usefuland applicable for the high-throughput monoclonal antibody generation.
16s rRNA Sequence Analysis and Ammonium Excretion Ability of Nitrogen Fixing Bacteria Isolated from Mineral Acid Soil
No full textNitrogen fixing bacteria defined as bacteria which is capable to transform free nitrogen molecules into ammonium v (PCR). Nitrogenase activity of these selected isolates was measured using Acetylene Reduction Assay (ARA). The ability of these selected isolates in ammonium excretion was qualitatively and quantitavely measured using Nessler reagent and spectrophotometry method respectively. Taxonomic position of the selected bacteria were determined based on their 16S rRNA sequence analysis. Genetic diversity analysis of these 15 isolates of nitrogen fixing bacteria yield eight selected bacteria for subsequent analysis. Sequence of nifH gene from all of these selected bacteria were successfully amplified. Nitrogenase assay of these selected bacteria revealed 6 isolates with high nitrogen fixation capasity namely GMA3, GMA5, GMA6, GMA9, GMA12 AND GMA 13.Ammonium excretion analysis revealed 4 isolates which have remarkable ability of producing high level of ammonium namely GMA1, GMA3, GMA6, and GMA9. The 16S rRNA sequence analysis shown that isolates GMA3, GMA5, GMA11 and GMA12 had a close relationship with Brevibacillus formosus strain DSM 9885T, Flexibacter canadensis strain ISSDS-428, Rhizobium tropici strain rif 200849, and Azotobacter tropicalis strain RBS. Respectively, isolate GMA1 and GMA13 had a close relationship with Sthenotropphomonas sp. Strain MFC-C, while isolate GMA6 and GMA9 had a close relationship to Azotobacter vinelandii strain ISSDS-428.', 'string'),(105, 'en_US', 'subject', 'nitrogen fixing bacteria, ammonium excretion, identification', 'string'),(105, 'en_US', 'sponsor', '', 'string'),(107, 'en_US', 'title', 'Effect of Probiotic Lactobacillus sp. Dad13 on Humoral Immune Response of Balb/C Mice Infected with Salmonella typhimurium', 'string'),(107, 'en_US', 'abstract', 'An indigenous strain of lactic acid bacterium (LAB) identified as Lactobacillus spp. Dad13 (Dad13), isolated from traditional fermented buffalo milk, was found to be potential as probiotic. The aim of this research was to study the effect of probiotic Dad13 on humoral immune response of Balb/C mice infected with Salmonella typhimurium. Thespecific objective was to find out the effect of different Dad13 consumption time (before and along with infection of S. typhimurium) on the humoral immune response of Balb/C mice. The experiment was conducted by in vivo trial on 20males of Balb/C mice, age of 6-8 weeks, fed with AIN-93 standard diet. The mice were assigned into 4 groups. Each group received the following treatments, ie. Dad13 only, Dad13 before infection, Dad13 along with infection and Salmonella infection only. A volume of 100 μl Dad13 cell suspensions (1010 CFU/ml) were given by oral forced feeding daily for a week, at week 3 for group before infection and at week 4 for group of Dad13 only and Dad13 along with infection. Salmonella infection (109 CFU/ml) was given once orally at week 4 to all groups except group treated with Dad13 only. The humoral immune response of Balb/C mice was detected 2 weeks after infection by measuring the titers of IgG and IgA specific from serum and mucosal intestinal liquid samples using Enzyme-linked Immunosorbent Assay (ELISA) method. The result indicated that humoral immune response of Balb/C mice consuming Dad13 before and along with Salmonella infection were significantly different (p<0.05). Dad13 consumption along with Salmonella infection increased circulated IgG and IgA as well as secretory IgA. It can be concluded that Dad13 probiotic feeding along with infection increased humoral immune response more significantly compared to that before infection
Diversity of Actinomycetes at Several Forest Types in Wanagama I Yogyakarta and Their Potency as a Producer of Antifungal Compound
No full textActinomycetes are bacterial groups that produce many secondary metabolites, which different biological activities, such as antifungi, antibacteria, antivirus, antitumor, etc. Actinomycetes are widely distributed in soil and their diversity is influenced by type of forest. The aim of this study is to investigate diversity of actinomycetes in several forest types of Wanagama I forest in Yogyakarta and their potency as a producer of antifungal compound. Soil samples under the forest of Tectona grandis, Swietenia macrophylla King, Bamboosa vulgaris, Melaleuca leucadendron, and Gliricidia maculata were used as sources of soil bacteria. Bacteria and actinomycetes communities were analyzed through culture-independent approach by RISA and nested-PCR RISA using actinomycetes spesific primer (F243), respectively. Through culture-dependent approach, isolated actinomycetes diversity were analyzed by identification of morphology (colony and cell), genetic (BOX element by rep-PCR), and secondary metabolites (thin layer chromatography). In addition, isolates were assayed for their antifungal activity against Saccharomyces cerevisae, Candida albicans, Fusarium oxysporum and Aspergillus flavus. The presence of Polyketide Synthase-I (PKS-I) and NonRibosomal Peptide Synthetase (NRPS) genes were amplified by PCR to study their correlation with antifungal activity of the actinomycete isolates. The results showed that types of forest influence diversity of rhizobacteria especially actinomycetes. According to culture-independent approach, relatively, com-munity of rhizobacteria from the highest were soil under the forest of B. vulgaris, G. maculata, T. grandis, S.macrophylla King, and M. leucadendron, respectively. Meanwhile, community of actinomycetes from the highest were soil under the forest of G. maculata, B. vulgaris, M. leucadendron, S. macrophylla King, and T. grandis, respec- tively. Fourty-three morphologically different isolates were found by using culture-dependent approach consisting of 17 isolates were found in soil under the forest of M. leucadedron, each of 9 isolates in G. maculata and T. grandis, 6 isolates in S. macrophylla King. and 2 isolates in B. vulgaris. More diversity of secondary metabolites were observed in soil actinomycetes under the forest of M. leucadendron. Of the 43 isolates, 100% were active against S.cerevisae, 37.20% against C. albicans, 95.30% against F. oxysporum, and 83.70% against A. flavus. Antifungal activity of actinomycete isolates did not always have correlation with the presence of PKS-I and NRPS
Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates
No full textIridovirus was known as agents that caused serious systemic disease in freshwater and marine fishes. The mortality up to 100% of orange-spotted grouper (Epinephelus coioides) due to iridovirus infection has been reported in Indonesia. The gene encoding capsid protein of iridovirus is supposed to be conserved and has the potency for the development of control methods. The objectives of this study are to clone the gene encoding capsid protein iridovirus and to analyze their sequences. The spleen tissues of orange-spotted grouper were collected and extracted their DNA. The DNA fragment of capsid protein of iridovirus genes were amplified by PCR using designed primers with the extraction DNA as templates. The amplified DNA fragments were cloned in pBSKSII and sequenced. The genes encoding capsid protein of iridovirus from Jepara and Bali were successfully amplified and cloned. The Jepara clone (IJP03) contained complete open reading frame (ORF) of the gene composed by 1362 bp nucleotides which encoded 453 amino acids. Those Jepara and Bali (IGD01) clones shared 99.8% similarity in nucleotide level and 99.4% at amino acid level. Based on those sequences, Indonesian iridovirus was belonged to genus Megalocystivirus and shared 99,6-99,9% similarity on nucleotide level with DGIV, ISKNV, MCIV, and ALI
Isolation and Screening of Antimicrobial Producing-Actinomycetes Symbionts in Nudibranch
No full text The aims of this study were to isolate and to screen actinomycetes associated with sea slug which have the ability to produce antimicrobial compound, especially against MDR strains. Actinomycetes were isolated from nudibranchs collected from Bandengan coastal waters and the Panjang island, Jepara, Central Java. Actinomycete isolates were assayed for their antimicrobial activity against MDR strains (MDR 6 E. coli, MDR 7 Enterobacter sp., MDR 13 Proteus sp., MDR 14 Staphylococcus sp.). The genetic diversity of the active isolates was analyzed by using repetitive DNA fingerprinting. Antimicrobial activity was also performed on the ethyl acetate bacterial extract. The amplification of Polyketide Synthase-I (PKS-I) and Non-Ribosomal Peptide Synthetase (NRPS) genes was carried out to estimate the genetic potency of actinomycetes. The most active actinomycete isolate was sequenced based on 16S rDNA approach. General profile of antimicrobial substances was analyzed by using Thin Layer Chromatography (TLC). A total 27 isolates were obtained from nudibranchs Jorunna sp. and 12 isolates from Chromodoris sp. Ten isolates exhibited antimicrobial activity. Five representative isolates were selected based on rep-PCR analysis. Three ethyl acetate extracts exhibited antimicrobial activity against MDR 7, MDR 13, and MDR 14, except MDR 6. NPC 8 isolates significantly inhibited the growth of the tested strain and amplified NRPS gene fragment. Molecular identification revealed that isolate NPC 8 closely related to Streptomyces sp with a high homology of 96%
Isolation and Analysis of DNA Fragment of Genes Related to Kopyor Trait in Coconut Plant
No full textKopyor coconut is a natural mutant that has abnormal endosperm development. For the first time several genes that were suspected to be related to kopyor trait were identified based on the chemical compounds of the endosperm that different from that of normal coconut. Sucrose synthase (SUS), Stearoyl acyl carrier protein desaturase (SACPD), and Absicid acid insensitive (ABI) genes were isolated and analyzed. Four DNA fragments with length of 746, 738, 780, and 687 bp (CnSus1A, CnSus1B, CnSus2A, and CnSus2B) were obtained from SUS gene. Sequence analysis at DNA and amino acid level showed that CnSus1A, CnSus1B, CnSus2A, and CnSus2B were classified into monocot SUS group with nongrass SUS type. Isolation of SACPD gene resulted in one DNA fragment with DNA length of 716 bp. CnSacpd shared a high homology with SACPD gene of oil palm and soybean. Isolation of ABI gene resulted in two DNA fragments, CnAbi3A and CnAbi3B, with DNA length of 760 and 728 bp, respectively. CnAbi3A and CnAbi3B showed a high homology with ABI3 gene of several plants. All DNA fragment obtained from SUS, SACPD, ABI genes were used as templates to design spesific markers for each corresponding gene. There were 7 specific primer sets designed, i.e., CnSUS1A, CnSUS1B, CnSUS2A, CnSUS2B, CnSACPD, CnABI3A, and CnABI3B.
Effects of Dissolved Oxygen Tension and Ammonium Concentration on Polyhydroxybutyrate Synthesis from Cassava Starch by Bacillus cereus IFO 13690
No full textAttempting to get low price of raw material for producing polyhydroxybutyrate is always studied. Tapioca starch is one of the raw material with low price. The objective of this research was to study the effects of initial ammonium concentration and dissolved oxygen tension (doT) on producing PHB by Bacillus cereus IFO 13690 with tapioca starch as the carbon source. This fermentation was carried out in 5 L fementors with a 2 L working volume, temperature of 30 oC, and agitation of 500 rpm. The pH medium was controlled at 5.6 after it came down from the initial pH of 6.8. Meanwhile, the initial doT was 100 % air saturation and also came down to and maintained at doT of experiment, i.e. 1 , 5 , or 10 % air saturation. The best result was obtained when the initial ammonium concentration was 5 g/L and the doT value maintained at 5 % air saturation. By this conditions, the cell growth reached 5,457 g cell dry weight/L containing PHB of 2.42 % cell dry weigh after 29 hours fermentation
Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate
No full textRhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory and secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the cloned gene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.', 'string'),(99, 'en_US', 'subject', 'Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP