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Title:	Molecular cloning, phylogenetic analysis, and expression of two immune related genes, serum Amyloid A and C-reactive protein from Asian seabass Lates calcarifer
Authors:	Laith Abdul Hassan Mohammed Jawad Alobaidi (P40984)
Supervisor:	Rahmah Mohamed, Prof. Dato' Dr.
Keywords:	Molecular cloning Phylogenetic analysis Expression of two immune related genes Serum Amyloid A protein Serum Amyloid C-reactive protein Asian seabass Lates calcarifer Giant perch--Immunology--Genetic aspects
Issue Date:	Feb-2012
Description:	Lates calcarifer is widely distributed throughout south-east Asia, including Malaysia. It is highly regarded as a table fish and has an eminent role in supporting the aquaculture industry in Malaysia. In this study, the two major acute-phase proteins (APPs), the serum amyloid A (SAA) and C-reactive protein (CRP) from the Asian seabass (L. calcarifer) were cloned and sequenced. A multiple sequence alignment (MSA) of the CDS and other vertebrate sequences, including the well characterized mouse and human SAA families, confirmed that L. calcarifer SAA as having not only the highest identity (60-71%) to other previously published fish SAA amino acid sequences, but also possesses a high similarity to mammalian SAAs. A phylogenetic analysis further confirmed that the L. calcarifer sequence is very closely related to available fish SAA sequences. The sequence analysis of the L. calcarifer CRP with pentraxin (PTX) sequences from other vertebrates, showed the highest identity (54-73%) to other fish CRP protein sequences. The phylogenetic analysis also showed that L. calcarifer CRP is well correlated to other available fish CRP sequences. The recombinant serum amyloid A and C-reactive proteins in E. coli were also expressed. The molecular weights of the expressed rSAA and rCRP were 14-kDa and 24-kDa, respectively. The recombinant proteins were purified by ion-exchange chromatography, yielding pure SAA and CRP at high concentrations (12.7 and 13.4 mg/L of bacterial culture), respectively, with higher purity than obtained by direct purification from blood serum. The purified rSAA and rCRP proteins were further tested to demonstrate their opsonization effect on bacterial cells. The results showed that the rSAA did not exhibit any antibacterial activity whereas rCRP inhibited the bacterial growth density at minimum inhibition concentration (MIC) of 3.5 μM rCRP, and also decreased the number of viable colonies at concentrations of 10 μM and 50 μM rCRP following 16 h incubation. The antibacterial activity of rCRP was highly effective on Gram-negative bacteria (E. coli and Aeromonas hydrophila) at highly significant statistical level of (P<0.001) and (P<0.05), respectively as compared to the control group. Activity against E. coli was much higher than against A. hydrophila (P<0.05). In contrast, the rCRP did not show any effect on Gram-positive bacteria (Staphylococcus aureus). This study showed that both the SAA and CRP genes from the L. calcarifer are important in innate immunity and are very closely related to available fish SAA and CRP gene sequences respectively, and thus are likely to have the same immune related functions. Understanding the role of SAA and CRP in the immune system of L. calcarifer will help to develop proper diagnostic tools against various pathogens that can cause significant losses to the L. calcarifer aquaculture industry.,PhD
Pages:	153
Call Number:	QR184 .A445 2012
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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