G.W. Wong¹, A.M. Ali² and H.F. Sew^1*

¹Immunology Unit, Department of Clinical Laboratory Science, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
²Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia
3400 Serdang, Selangor, Malaysia

(Received 23 March 1999 /Accepted 15 August 1999)

Abstract.
Human IL-18 cDNA which was amplified via the reverse transcription polymerase chain reaction of total RNA isolated from KU812F, a chronic myelogenous leukemic cell line, was successfully cloned into pTrcHis-2TOPO vector and expressed in E. coli. The authenticity of the human IL-18 cDNA sequence was confirmed by DNA sequencing. It exhibited 100% identity to the human IL-18 cDNA sequence as published in the database Genbank (accession no. D49950). SDS PAGE analysis of the crude lysates from pTrcHis TOPO.1L-18 cultures induced with I mM IPTG yielded a prominent protein band of the expected molecular mass of approximately 23kDa. The expressed recombinant protein was purified from the soluble fraction using the Talon metal affinity resin. A human T cell line, CEM-SS and PBMC were shown to exhibit constitutive basal levels of IL-18 mRNA expression. Con A and PMA upregulated IL-18 mRNA expression in CEM SS. Dibutyryl cyclic AMP and dexamethasone were found to downregulate IL-18 mRNA synthesis in PBMC.

Keywords: 11,18, cloning, expression, pTrcHis2 TOPO

A. Kengluecha¹, W. Noonpakdee^1*, S. Sitthimonchai¹, R. Valyasevi² and S. Panyim¹

¹Department of Biochemistry, Faculty of Science, Mahidol University, ¹National Center for Genetic
Engineering and Biotechnology, NSTDA, Ministry of Science, Technology and Environment, Thailand

(Received 22 July 1999 l Accepted 5 October 1999)

Abstract.
Catalase activity is rarely found in lactobacilli but it is a desirable characteristic for starter cultures as it prevents defects caused by hydrogen peroxide in food product. The catalase gene from Lactobacillus sake SR91II was cloned and expressed in Escherichia coli UM2, Lactobacillus sp. TISTR891 and Lactobacillus plantarumTISTR850. The Lactobacillus sp.TISTR891 and L. plantarum TISTR850 isolated from local fermented meat products were naturally devoid of catalase activity. The transformed lactobacilli were shown to decompose hydrogen peroxide and the recombinant proteins were also detected by in situ activity staining of the catalase enzyme. The catalase gene was expressed as an active enzyme in transformed Lactobacillus sp. TISTR891 by using pGKV210, a lactococcus promoter screening vector. Increase in the expression of catalase gene by about two fold was observed after cloning the gene under the strong lactococcal promotor p59 located in the expression vector pIL 1020. In transformed Lactobacillus plantarum TISTR850, catalase activity was expressed almost 6 times as that in Lactobacillus sp. TISTR891 where expression was under the control of its own promoter.

Keywords: High level expression, catalase gene, Lactobacilli

S. Lihan¹, S. Radu^1,*, G. Rusul ², M.I.A. Karim¹, O.W. Ling¹ and N. Maisin³

¹Department of Biotechnology and ²Department of Food Science, Faculty of Food Science and Biotechnology;
3Department of Biology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia.

(Received 16 April 1999 / Accepted 15 September 1999)

Abstract.
Ninety five Escherichia coli strains isolated from raw milk were analyzed for plasmid profile and antimicrobial resistance, and typed by RAPD fingerprints. All the E. coli strains were resistant to four or more of the antibiotics tested. However, none were resistant to ceftazidime, gentamicin and norfloxacin. The multiple antibiotic resistance (MAR) index values of the E. coli strains ranged from 0.25 to 0.81, indicating that all were isolated from raw milk samples that were exposed to high risk sources. Plasmids of 1.4 to 68 MDa were detected in 69 (72.6%) of the E. coli strains examined. The detection of a single plasmid of 68 MDa in 58 of 69 plasmid containing strains limits the usefulness of plasmid profiles as an epidemiological marker. RAPD fingerprints of the 95 E. coli strains were obtained using two 10 mer primers, GEN15009 and GEN15010, to differentiate or identify clonal relationship among the strains examined. The dendrogram constructed from the combined results of the RAPD patterns obtained from both primers showed that the 95 E. coli strains were grouped into six major clusters, indicating that five major clonal lineages (clusters 1, 2, 3, 4 and 5) of E. coli strains were predominant in the raw milk samples tested.

Keywords: Escherichia coli, RAPD, plasmid, antibiotic resistance, MAR

M.Y Sabri, M. Zamri Saad*, A.R. Mutalib, D.A. Israf and N. Muniandy¹

Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Veterinary Research Institute, 59 Jalan Sultan AzIan Shah, 13800 Ipoh, Perak, Malaysia

(Received 16 April 1999 / Accepted 15 September 1999)

Abstract.
The outer membrane proteins of Pasteurella haemolytica A2, A7 and A9 were subjected to SDS-PAGE and immunoblotting. The molecular weights of the polypeptide bands ranged between 33 to 97 kDa. The major polypeptide bands for P.haemolytica A2 were 33.4, 39.2 and 45 kDa while the minor polypeptide bands were 50, 58.7, 66.2, 84.7 and 97.4 kDa. Analysis of the outer membrane proteins of P. haemolytica A7 revealed two major protein bands of 33.4 and 45 kDa and three minor polypeptide of 40, 50 and 66.2 kDa. There were three major (33.4, 37.5 and 45 kDa) and one minor protein band (50 kDa) in the outer membrane proteins of P haemolytica A9. There was one major protein band from each of the P.haemolytica A2, A7 and A9, which was unique to the respective serotype and appeared to represent the respective serotype. These were the 39.2 kDa band for P.haemolytica A2, the 40 kDa band for P.haemolytica A7 and the 37.5 kDa band for P haemolytica A9. Following homologous immunoblot, all the serotypes showed pronounced antigenicity at the 30 kDa band. Heterologous immunoblot using the antiserum of P.haemolytica A2 did not reveal any antigenic band of P.haemolytica A9 but revealed antigenic bands at 30 and 31 kDa of P.haemolytica A7. Heterologous immunoblot using the antiserum of P.haemolytica A7 revealed antigenic band at 30 kDa of all the three serotypes while the antiserum of P.haemolytica A9 failed to reveal any common antigenic band between all three serotypes. Thus, the 30 kDa band of P.haemolytica A7 may be a suitable candidate for a sub unit vaccine against pneumonic pasteurellosis of sheep and goats.

Keywords: Antigenicity, cross reaction, outer membrane proteins, Pasteurella haemolytica