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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (226.702 KB) | DOI: 10.22146/ijbiotech.7850

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 koiand common carp (Cyprinus carpio). The case fatality rate of this disease is 80–100%. Glycoprotein has beenused for vaccine development as sub unit vaccine against viruses. The aim of this research was to clone andexpress membrane glycoprotein ORF124 KHV as a candidate of recombinant vaccine. ORF124 KHV gene wassuccessfully cloned into pBSKS and sequenced. Result showed that ORF124 KHV (isolate from Indonesia) had100 % 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 cellepitopes 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 hasbeen expressed in Escherichia coli under pET32-a(+)vector.
Cloning and Sequence Analysis of Capsid Protein Gene of Iridovirus Indonesian Isolates Murwantoko, M.; Handayani, Christina Retna; Pratiwi, Rarastoeti
Indonesian Journal of Biotechnology Vol 14, No 1 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (131.285 KB) | DOI: 10.22146/ijbiotech.7805

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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (646.307 KB) | DOI: 10.22146/ijbiotech.7795

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
Limited evidence for white spot syndrome virus susceptibility associated with expression of PmVRP15 in local population of giant tiger shrimp (Penaeus monodon) Al Haq, Aushia Tanzih; Murwantoko, M.; Trijoko, T.; Wijayanti, Nastiti; Handayani, Ch. Retna; Pratiwi, Rarastoeti
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.
Analysis of Htra Gene from Zebrafish (Danio Rerio) Murwantoko, M.; Oka, Chio; Kawaichi, Masashi
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (186.97 KB) | DOI: 10.22146/ijbiotech.7554

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.