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Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate M, Murwantoko; Widada, Jaka; Nuraini, Yani Lestari
Indonesian Journal of Biotechnology Vol 13, No 1 (2008)
Publisher : Universitas Gadjah Mada

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Abstract

Rhoptry 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, ROP2
Cloning and Expression of ORF124 Koi Herpesvirus as a Vaccine ., Murwantoko; Pratiwi, Rarastoeti; Kawaichi, Masashi
Indonesian Journal of Biotechnology Vol 17, No 1 (2012)
Publisher : Universitas Gadjah Mada

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Abstract

Koi herpesvirus (KHV) which also known as Cyprinid herpesvirus 3 (CyHV-3), Koi herpes-like virus, and carp interstitial nephritis gill necrosis virus (CNGV), caused signifi cant morbidity and mortality in koi and common carp (Cyprinus carpio). The case fatality rate of this disease is 80–100%. Glycoprotein has been used for vaccine development as sub unit vaccine against viruses. The aim of this research was to clone and express membrane glycoprotein ORF124 KHV as a candidate of recombinant vaccine. ORF124 KHV gene was successfully cloned into pBSKS and sequenced. Result showed that ORF124 KHV (isolate from Indonesia) had 100 % similarity with Cyprinid herpesvirus 3 strain TUMST1 (from Japan), 99% similarity with Koi herpesvirus strain KHV-U (from USA) and Koi herpesvirus strain KHV-I (from Israel). Prediction analysis of T and B cell epitopes showed that ORF124 KHV protein had 14 and 11 T cell epitopes (IAd, Rothbard/Taylor pattern),and had 10 B cell epitopes, suggested that the protein can be used as a vaccine candidate. ORF124 gene has been expressed in Escherichia coli under pET32-a(+)vector.
Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates ., Murwantoko; Handayani, Christina Retna; Pratiwi, Rarastoeti
Indonesian Journal of Biotechnology Vol 14, No 1 (2009)
Publisher : Universitas Gadjah Mada

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Abstract

Iridovirus 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 ALIV
Analysis of Htra Gene from Zebrafish (Danio Rerio) M, Murwantoko; Oka, Chio; Kawaichi, Masashi
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

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Abstract

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.
Limited evidence for white spot syndrome virus susceptibility associated with expression of PmVRP15 in local population of giant tiger shrimp (Penaeus monodon) Aushia Tanzih Al Haq; M. Murwantoko; T. Trijoko; Nastiti Wijayanti; Ch. Retna Handayani; Rarastoeti Pratiwi
Indonesian Journal of Biotechnology Vol 20, No 2 (2015)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (912.481 KB) | DOI: 10.22146/ijbiotech.24189

Abstract

White spot syndrome virus (WSSV) is a devastating viral disease in shrimp aquaculture. Infection ofWSSV in penaeid shrimps affects immune defense and changes gene expression. PmVRP15 has been reported as a part of the WSSV propagation pathway that is highly up-regulated in hemocytes at the acute phase of WSSV infection. This study analyzed the expression of PmVRP15 in local populations of giant tiger shrimp (Penaeus monodon) to be associated with susceptibility to WSSV. Tested populations consisted of an inbreeding population (G8) and outbreeding population (G8iA) from Jepara, Indonesia. Susceptibility was determined by cumulative mortality, median lethal time (LT50), and severity of infection at time of death. Though all populations were susceptible to WSSV, the frst mortality in G8 occurred at 18 hours post-infection (hpi) with mild infection, while frst mortality of G8iA occurred at 30 hpi with severe infection. The LT50 of G8 was signifcantly lower than that of G8iA, indicating that G8iA was less susceptible to WSSV than G8. Relative PmVRP15 transcripts of G8iA were insignifcantly down-regulated, whereas relative PmVRP15 transcripts of G8were insignifcantly upregulated. Although it’s still not conclusive, the results of this study suggest that PmVRP15 has weak potentialas a WSSV susceptibility marker in G8 and G8iA broodstock selection.
Gene Cloning and Protein Expression of Koi Herpesvirus ORF25 . Murwantoko; Cahya Kurnia Fusiyanto; . Triyanto
HAYATI Journal of Biosciences Vol. 23 No. 3 (2016): July 2016
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1424.058 KB) | DOI: 10.4308/hjb.23.3.143

Abstract

Koi herpesvirus (KHV) caused significant morbidity and mortality in koi and common carp (Cyprinus carpio). Glycoprotein has been used for vaccine development as sub unit vaccine against many viruses. KHV ORF25 is one of koi herpesvirus genes which encode a glycoprotein. The objectives of this research are to clone gene KHV ORF25and express its protein. The common carp showing necrosis and white patches of gill which was collected from Magelang was used in this research. Primers were designed to amplify partial ORF25 based on KHV J strain. KHV ORF25 was successfully amplified and cloned in pET32a. Sequence analysis showed that this KHV ORF25 has 99% homology with the sequences of KHV genotype KHV-J, KHV-I, and KHV-U. This ORF was predicted has 3, 23, and 8 B-cell epitopes based on Emini scale, Karplus and Schulz scale, and ElliPro respectively. The KHV ORF25 recombinant protein has been successfully produced in Escherichia coli as an insoluble protein with approximately 45 kDa in size. The high protein production was achieved when the protein induction was done at bacterial density at OD600 as 1.0 with 1-mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 37°C for 18 hours. The protein predicted has immunogenicity and the potency as a vaccine is needed to be evaluated.
Life Cycle of Marine Leech from Cultured Cantik Hybrid Grouper (Ephinephelus sp.) and Their Susceptibility Against Chemicals Murwantoko, Murwantoko; Condro, Sri Laksono; Isnansetyo, Alim; Zafran, Zafran
Aquacultura Indonesiana Vol 18, No 2 (2017)
Publisher : Indonesian Aquaculture Society (MAI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (726.881 KB) | DOI: 10.21534/ai.v18i2.91

Abstract

Grouper is an important fish species due to high price both in domestic and international market. Several hybrid groupers have been released and can be accepted by farmers. A major production constraint in grouper culture is mortality due to disease. Leech is an ectoparasite for grouper which may cause significant loss, so that controlling this parasite is important as one of aquaculture management tools. The objectives of this study was to know the life cycle of leech on hybrid grouper and determine the efficacy of chemicals to kill leech under laboratory condition with diffferent dossage and immersing time. Life cycle was observed by reared an adult leech, counted the fecundity and observed the development of eggs until reach adult stages. The efficacy of formalin, albendazole, oxfendazole, levamisole, H2O2, CuSO4, ivermectin, vermizyn and freshwater on several concentration were assessed to kill adult leeches. All chemicals tested (except for freshwater), are dissolved in saline water salinity of 35 ppt at concentrations of 1000, 500, 250, 125 and 62.5 ppm. During 3 days of rearing, the adult leech could deposit of 11 eggs in average, with 600 µm – 800 µm in diameter. Twelve days were needed for the new egg inside the cocoon to hatch and develop into larvae under 24-25 °C at 34 ppt of salinity. It took another 9 days for the leeches larvae to grow reach mature stage. Five chemicals were  able to kill leeches (Zeylanicobdela arugamensis), which were fresh water, formalin, levamisol, ivermectin, and CuSO4. Treatment by exposure leech to freshwater for 30 minutes shows effective to kill leech.  Treatment with formalin with a dose of 250 ppm was able to kill leech after 1 hour immersion. 
Cloning ORF2 Membrane Protein of Koi Herpesvirus Lake Toba, Indonesian Isolate MURWANTOKO MURWANTOKO
HAYATI Journal of Biosciences Vol. 16 No. 2 (2009): June 2009
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (88.532 KB) | DOI: 10.4308/hjb.16.2.49

Abstract

Koi herpesvirus (KHV) caused significant morbidity and mortality in koi and common carp. KHV which showed strong antigenic property implied that KHV virion or proteins may be used as antigen to raise antibody or vaccine to increase the resistance. The objectives of this research were to (i) clone KHV membrane protein ORF2, (ii) analysis on immunogenicity, and (iii) genetic tracing. Based on genbank data, one pair of primers was designed to amplify KHV ORF2. The KHV ORF2 can be amplified using infected fish DNA which originally from Toba Lake, Sumatera, Indonesia. The KHV ORF2 composed of 699 nucleotides encoded for 292 amino acids. BLAST analysis showed that KHV ORF2 had 100% homology with KHV-J and KHV0301 strains from Japan; 98 and 91% homology on nucleotides and amino acids respectively with both KHV-U strain from Unites State and KHV-I strain from Israel. KHV in Indonesia was most likely to have originated from Japan via spreading directly or not directly to China or Hongkong. Based on T- and B-cell epitopes prediction, membrane protein ORF2 was proposed has a potency to be used in development vaccine and immunodetection. Key words: genetic tracing, koi herpesvirus (KHV), membrane protein, ORF2
Pengaruh Salinitas terhadap Perkembangan Parasit pada Benih Gurami, Osphronemus goramy Rahayu, Nani S.; Susanti, Dewi; Lantiani, Dwi; Wibowo, Sutopo A.; Diana, Roosita; Murwantoko, Murwantoko
Jurnal Perikanan Universitas Gadjah Mada Vol 11, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (34.127 KB) | DOI: 10.22146/jfs.10

Abstract

Research was conducted to know the effect of salinity on the reduction of parasites in Gouramy (Osphronemus goramy). The Research was performed using completely Randomized design with 5 salinity treatments (water pond, water with salt concentration 0, 2, 4 dan 6 g/L) with 4 replications. The 5-7 cm of gouramy fingerling were reared as on treatments and the parasites were counted  using skin scrapping dan wet mount methods. Observation was performed every 3 day for 15 days on parasite intensity, prevalence, mortality, symptoms and water quality.The result showed that several parasites including Trichodina sp., Apiosoma sp., Ichthyophthirius sp., Oodinium sp., dan Henneguya sp were observed. The highest intensity and prevalence was found in Trichodina sp. The Salinity and lenght administration were significantly decrease number parasites.  Salinity treatments (2, 4 dan 6 g/L ) for 3 days could totally removing Trichodina sp. from fishes. Meanwhile the data of other parasite species were unsignificant due to the low level of its intensity and prevalence. Salinity administration in level 6 g/L could reduce mortality.
Short-term Response in Molecular and Biochemical Adaptation of White Shrimp (Litopenaeus vannamei) Postlarvae Reared in A Biofloc System Yasa, Ngurah Sedana; Anshory, Lutfi; Triastutik, Gemi; Murwantoko, Murwantoko; Isnansetyo, Alim; Lusiana, Lusiana
Aquacultura Indonesiana Vol 20, No 2 (2019)
Publisher : Indonesian Aquaculture Society (MAI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (745.262 KB) | DOI: 10.21534/ai.v20i2.155

Abstract

Up regulation of heat shock proteins (HSPs) in organisms can be detected in response to many kinds of stressor. Recently there were few studies have focused on the three kinds of heat shock proteins (HSPs) and antioxidant enzyme activity after biofloc stress. The objective of this study was to investigate the effect of different biofloc volume on the expressions of (LvHSP60, LvHSP70, and LvHSP90), the activity of superoxide dismutase (SOD) and phenoloxidase (PO) of the L. vannamei after biofloc addition. PL10 L. vannamei were divided into three groups: precondition, biofloc stress (5mL/L, 10mL/L, 15mL/L), and recovery. The gene expression results showed that the expressional levels of LvHSP60, LvHSP70, and LvHSP90 were increased significantly in 6h treatment and tend return to normal conditions after 48h treatment. Superoxide dismutase activity was reduced during treatment and phenoloxidase activity was elevated slightly after 12h to 24h and tend to pretreatment level at recovery periods. All of these HSPs expression reverted to normal levels 6h after the recovery period. The results indicated that different expression patterns of the three HSPs. HSP60 have a longer and higher protection expression after 12h treatment than HSP70 and HSP90. HSP90 was more sensitive in 6h at all treatments than HSP60 and HSP70. It is concluded that supplementation of biofloc with the volume of 5-15mL/L caused Hsp protection in L. vannamei PLs at the first 6h to 48h treatment and increased phenoloxidase activity at 24-48h treatment and reduced survival rate of the white shrimp.