Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum
Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is one of the major respiratory tract infections of the poultry, resulting in significant economic loss to the poultry farmers. Diagnosis of such ailment is highly necessary for effective control measures. In addition, promis...
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| Format: | Article |
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Indian Council of Agricultural Research
2021-08-01
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| Series: | Indian Journal of Animal Sciences |
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| Online Access: | https://epubs.icar.org.in/index.php/IJAnS/article/view/113814 |
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| author | K MANIMARAN ADARSH MISHRA V HARINI SATHISH B SHIVACHANDRA T V MEENAMBIGAI G DHINAKAR RAJ |
| author_facet | K MANIMARAN ADARSH MISHRA V HARINI SATHISH B SHIVACHANDRA T V MEENAMBIGAI G DHINAKAR RAJ |
| author_sort | K MANIMARAN |
| collection | DOAJ |
| description |
Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is one of the major respiratory tract infections of the poultry, resulting in significant economic loss to the poultry farmers. Diagnosis of such ailment is highly necessary for effective control measures. In addition, promising molecular tools are warranted for efficient epidemiological tracing of the outbreaks. The study was focused on the elucidation of phase variable cytadhesin protein gene (pvpA) of MG through cloning and expression analysis. A set of primers targeting the pvpA gene of MG was designed. The complete pvpA gene was amplified and cloned into pUC-derived expression vector pRSETA. Finally, the recombinant clones were examined through colony PCR and restriction endonuclease (RE) analysis with EcoR1 and BamH1 enzymes followed by sequencing. The expression of the recombinant pvpA gene was optimized at 1.4mM/μl concentration of Isopropyl-β-D-thiogalactoside induction at 30°C. The recombinant fusion protein was purified by immobilized metal affinity chromatography and characterized by SDS-PAGE followed by confirmation of recombinant cytadhesin fusion protein through western blot analysis. The pvpA gene was successfully cloned and expressed. The deduced amino acid sequence analysis had shown the presence of two direct repeats (DR1 and DR2) along with predicted PRP motifs repeatedly with high proline encoding regions at the carboxy-terminal of pvpA gene indicating its scope for epidemiological studies.
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| first_indexed | 2024-04-10T07:51:15Z |
| format | Article |
| id | doaj.art-acdd225a786c4e7b88084797da435904 |
| institution | Directory Open Access Journal |
| issn | 0367-8318 2394-3327 |
| language | English |
| last_indexed | 2024-04-10T07:51:15Z |
| publishDate | 2021-08-01 |
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| record_format | Article |
| series | Indian Journal of Animal Sciences |
| spelling | doaj.art-acdd225a786c4e7b88084797da4359042023-02-23T10:16:22ZengIndian Council of Agricultural ResearchIndian Journal of Animal Sciences0367-83182394-33272021-08-0191210.56093/ijans.v91i2.113814Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticumK MANIMARAN0ADARSH MISHRA1V HARINI2SATHISH B SHIVACHANDRA3T V MEENAMBIGAI4G DHINAKAR RAJ5Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 IndiaTamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 IndiaTamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 IndiaTamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 IndiaTamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 IndiaTamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu 600 051 India Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is one of the major respiratory tract infections of the poultry, resulting in significant economic loss to the poultry farmers. Diagnosis of such ailment is highly necessary for effective control measures. In addition, promising molecular tools are warranted for efficient epidemiological tracing of the outbreaks. The study was focused on the elucidation of phase variable cytadhesin protein gene (pvpA) of MG through cloning and expression analysis. A set of primers targeting the pvpA gene of MG was designed. The complete pvpA gene was amplified and cloned into pUC-derived expression vector pRSETA. Finally, the recombinant clones were examined through colony PCR and restriction endonuclease (RE) analysis with EcoR1 and BamH1 enzymes followed by sequencing. The expression of the recombinant pvpA gene was optimized at 1.4mM/μl concentration of Isopropyl-β-D-thiogalactoside induction at 30°C. The recombinant fusion protein was purified by immobilized metal affinity chromatography and characterized by SDS-PAGE followed by confirmation of recombinant cytadhesin fusion protein through western blot analysis. The pvpA gene was successfully cloned and expressed. The deduced amino acid sequence analysis had shown the presence of two direct repeats (DR1 and DR2) along with predicted PRP motifs repeatedly with high proline encoding regions at the carboxy-terminal of pvpA gene indicating its scope for epidemiological studies. https://epubs.icar.org.in/index.php/IJAnS/article/view/113814Chronic respiratory disease (CRD)CloningExpressionMycoplasma gallisepticumpvpA gene |
| spellingShingle | K MANIMARAN ADARSH MISHRA V HARINI SATHISH B SHIVACHANDRA T V MEENAMBIGAI G DHINAKAR RAJ Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum Indian Journal of Animal Sciences Chronic respiratory disease (CRD) Cloning Expression Mycoplasma gallisepticum pvpA gene |
| title | Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum |
| title_full | Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum |
| title_fullStr | Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum |
| title_full_unstemmed | Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum |
| title_short | Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum |
| title_sort | cloning of cytadhesin protein gene pvpa and expression analysis of recombinant fusion protein of mycoplasma gallisepticum |
| topic | Chronic respiratory disease (CRD) Cloning Expression Mycoplasma gallisepticum pvpA gene |
| url | https://epubs.icar.org.in/index.php/IJAnS/article/view/113814 |
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