Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e

Lactococcus lactis is a non-pathogenic and non-colonising bacterium, which is being developed as a vaccine delivery vehicle for immunisation by mucosal routes. To determine whether lactococci can express the Chicken Anaemia Virus capsid protein (VP1) in immunogenic fonn, we have constructed a rec...

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Main Author:	Ng, Perk Tsong
Format:	Thesis
Language:	English English
Published:	1999
Subjects:	Lactococcus lactis Anemia Cloning
Online Access:	http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf
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id	my-upm-ir.8403
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spelling	my-upm-ir.84032024-01-11T02:03:51Z Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e 1999-08 Ng, Perk Tsong Lactococcus lactis is a non-pathogenic and non-colonising bacterium, which is being developed as a vaccine delivery vehicle for immunisation by mucosal routes. To determine whether lactococci can express the Chicken Anaemia Virus capsid protein (VP1) in immunogenic fonn, we have constructed a recombinant DNA, which constitutively expresses the VP1 as N-terminal fusion protein with Open Reading Frame-32 under the control of lactococci promoter P32 in vector pMG36e. Chicken anaemia virus (CAV) is a member of the newly categorised Circoviridae, a family of circular negative single-stranded DNA viruses. CAY VP1 gene (1.4 kb) was amplified by PCR from CAV genome which was isolated from infected Marek's disease virus transformed lymphoblastoid cell line (MDCC-MSB 1) cell lysate and cloned into pCR® 2.1-TOPO vector for sequencing. The sequenced VP1 gene, lacking its own promoter, was cloned into the lactococcal expression vector pMG36e and transformed into E. coli JM109 as the intermediate host. The recombinant plasmid was sub-cloned into L. lactis MG1363 by electroporation. Several independent verifying methods such as sequencing, Southern hybridisation and restriction enzymes analysis have confirmed the insertion of VP1 gene into pMG36e. The complete sequence of VP1 gene has 99.65% homology compared to the published CAV Cux-1 VP1 gene. A total of 5 base difference was seen to result in change of 3 amino acids sequence in the polypeptide chain. The expression of fusion protein was studied until transcriptional level. Transcription of fusion gene from strains of recombinant L. lactis MG1363 were analysed by Northern hybridisation. Northern hybridisation has detected transcription of fusion gene in recombinant L. lactis MG1363 carrying pMG36e-VP1 and produced mRNA with the size of 1.4 kb. On the basis of these results, it is concluded that the recombinant DNA pMG36e was successfully constructed in bacteria E. coli JM109 and subcloned into L. lactis MG1363. The recombinant DNA in L. lactis MG1363 is capable to express the fusion protein up to transcriptional level. Therefore, recombinant L. lactis MG1363 can be used as a potential vaccine delivery system against CAV infections. Lactococcus lactis Anemia Cloning 1999-08 Thesis http://psasir.upm.edu.my/id/eprint/8403/ http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf text en public masters Universiti Putra Malaysia Lactococcus lactis Anemia Cloning Food Science and Technology Abdul Rahim, Raha English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Abdul Rahim, Raha
topic	Lactococcus lactis Anemia Cloning
spellingShingle	Lactococcus lactis Anemia Cloning Ng, Perk Tsong Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
description	Lactococcus lactis is a non-pathogenic and non-colonising bacterium, which is being developed as a vaccine delivery vehicle for immunisation by mucosal routes. To determine whether lactococci can express the Chicken Anaemia Virus capsid protein (VP1) in immunogenic fonn, we have constructed a recombinant DNA, which constitutively expresses the VP1 as N-terminal fusion protein with Open Reading Frame-32 under the control of lactococci promoter P32 in vector pMG36e. Chicken anaemia virus (CAV) is a member of the newly categorised Circoviridae, a family of circular negative single-stranded DNA viruses. CAY VP1 gene (1.4 kb) was amplified by PCR from CAV genome which was isolated from infected Marek's disease virus transformed lymphoblastoid cell line (MDCC-MSB 1) cell lysate and cloned into pCR® 2.1-TOPO vector for sequencing. The sequenced VP1 gene, lacking its own promoter, was cloned into the lactococcal expression vector pMG36e and transformed into E. coli JM109 as the intermediate host. The recombinant plasmid was sub-cloned into L. lactis MG1363 by electroporation. Several independent verifying methods such as sequencing, Southern hybridisation and restriction enzymes analysis have confirmed the insertion of VP1 gene into pMG36e. The complete sequence of VP1 gene has 99.65% homology compared to the published CAV Cux-1 VP1 gene. A total of 5 base difference was seen to result in change of 3 amino acids sequence in the polypeptide chain. The expression of fusion protein was studied until transcriptional level. Transcription of fusion gene from strains of recombinant L. lactis MG1363 were analysed by Northern hybridisation. Northern hybridisation has detected transcription of fusion gene in recombinant L. lactis MG1363 carrying pMG36e-VP1 and produced mRNA with the size of 1.4 kb. On the basis of these results, it is concluded that the recombinant DNA pMG36e was successfully constructed in bacteria E. coli JM109 and subcloned into L. lactis MG1363. The recombinant DNA in L. lactis MG1363 is capable to express the fusion protein up to transcriptional level. Therefore, recombinant L. lactis MG1363 can be used as a potential vaccine delivery system against CAV infections.
format	Thesis
qualification_level	Master's degree
author	Ng, Perk Tsong
author_facet	Ng, Perk Tsong
author_sort	Ng, Perk Tsong
title	Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
title_short	Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
title_full	Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
title_fullStr	Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
title_full_unstemmed	Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e
title_sort	cloning of chicken anaemia virus (cux-1) vp1 gene into a lactococcus expression vector pmg36e
granting_institution	Universiti Putra Malaysia
granting_department	Food Science and Technology
publishDate	1999
url	http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf
_version_	1794018743470260224

Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e

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