Overexpression of antimicrobial substance KAC5 in Lactobacillus reuteri KUB-AC5 and Its detection based on real time PCR

Name: Siriphan Sobanbua

LCSH: Kasetsart University -- Dissertations. Ph.D. (Biotechnology) 2020

Classification :.LCCS: QR1217

LCSH: Kasetsart University. -- Department of Biotechnology -- Dissertations

LCSH: Lactic acid bacteria

LCSH: Peptide antibiotics

LCSH: Lactobacillus

LCSH: Organic acids

LCSH: Bacteria

Abstract: The aims of this study were to investigate localization and overexpression of the gene coding for antimicrobial peptide (AMP) of Lactobacillus reuteri KUB-AC5 by food grade vector system, and to design the specific primer for its quantitative analysis by real time PCR. Lb. reuteri KUB-AC5 isolated from chicken intestine played an important role in promoting the growth of broiler chicken and pathogenic growth inhibition by the possible AMP named KAC5. Gene cloning technique with food grade vector pSIP609 and Lb. plantarum TLG02 as a host cell resulted that the kac5 located on the open reading frame-1 (ORF-1) of I-C46-F2.1 providing amino acid sequence of “YMLYKFLAGLFHTSIDSIYWSVTFIAPALALITYIVCWPDS”. The recombinant strain Lb. plantarum ACLP-C46-F2.1 obtained expressed lower intracellular AMP activity than the wild type KUB-AC5. While its AMP characters of proteolytic enzyme treatment, pH and temperature stability and inhibition spectrum were similar to the ones of the wild type which confirmed KAC5 character. Overexpression of kac5 into the wild type provided the recombinant Lb. reuteri ACLR-C46-F2.1 which exhibited higher inhibitory activites than the wild type for 1.6 fold without inducer. Quantitative analysis of the wild type KUB-AC5 was successful by primer P2 derived from ORF-2 which exhibited high efficacy and specificity at annealing temperature of 70°C. Hence, P2 was selected for qPCR assay by isothermal annealing and extension reaction at high temperature of 70°C resulting in linearity for its DNA sequences ranging from 102-107 target copy numbers per assay and displaying a detection limit of 6.17 log cfu/g of caecal digesta. These results suggested overexpression of kac5 and possible detection of strain KUB-AC5 in the chicken GI-tract of chicken to evaluate the efficacy and persistence of a probiotic strain which requires correct inclusion rates in the feed.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Sunee Nitisinprasert

Role: Thesis Advisor

Name: Suttipun Keawsompong

Role: Thesis CO-Advisor

Created: 2020

Modified: 2025-06-30

Issued: 2025-06-30

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2563/siriphan-sob-all.pdf

CallNumber: QR1217 .S57

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degraee

Descipline: Biotechnology

Grantor: Kasetsart University

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