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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| Format: | Thesis |
| Language: | English English |
| Published: |
1999
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| Online Access: | http://psasir.upm.edu.my/id/eprint/8403/ http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf |
| _version_ | 1848840860375973888 |
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| author | Ng, Perk Tsong |
| author_facet | Ng, Perk Tsong |
| author_sort | Ng, Perk Tsong |
| building | UPM Institutional Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-15T07:34:03Z |
| format | Thesis |
| id | upm-8403 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English English |
| last_indexed | 2025-11-15T07:34:03Z |
| publishDate | 1999 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-84032024-01-11T02:03:51Z http://psasir.upm.edu.my/id/eprint/8403/ Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e 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. 1999-08 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf Ng, Perk Tsong (1999) Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e. Masters thesis, Universiti Putra Malaysia. Lactococcus lactis Anemia Cloning English |
| spellingShingle | Lactococcus lactis Anemia Cloning Ng, Perk Tsong Cloning of Chicken Anaemia Virus (CUX-1) VP1 Gene Into a Lactococcus Expression Vector pMG36e |
| title | 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_short | 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 |
| topic | Lactococcus lactis Anemia Cloning |
| url | http://psasir.upm.edu.my/id/eprint/8403/ http://psasir.upm.edu.my/id/eprint/8403/1/FSMB_1999_6_IR.pdf |