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Published on: May 6, 2015
[Construction and identification of recombinant adenovirus of muramidase-released protein gene fragment from
Tao Wang1, Zihao Tang, Xiaoou Zhou
1Department of Pathogenic Biology, College of Basic Medicine, Jiujiang University, China. comwangtaocom@sohu.com
Objective:
To construct and identify the recombinant adenovirus of muramidase-released protein (MRP) gene fragment from Streptococcus suis type 2 (SS2).
Methods:
The specific primers were designed based on the sequence of MRP gene fragment. The MRP gene fragment (467-1351 bp) was amplified by PCR method with genomic DNA of SS2 as a template. PCR products were cloned in pMD18-T vector. Then MRP gene fragment was linked into the adenovirus shuttle plasmid (pShuttle-CMV) to construct recombinant shuttle plasmid (pShuttle-CMV-MRP). After PmeI digestion, it was transformed into BJ5183-AD-1 competent cells containing adenoviral backbone plasmid pAdEasy-1 to construct homogeneous recombinant adenovirus plasmid (pAdeno-CMV-MRP). Then the recombinant adenovirus plasmid was linearized by PmeI and then transfected into AD-293 cells for viral packaging. Finally, the virus liquid was tested by PCR and Western blotting.
Results:
Cytopathic effect (CPE) was observed at 8 d after transfection of linear pAdeno-CMV-MRP in AD-293 cells. MRP gene fragment and protein expression were also detected in the virus liquid.
Conclusion:
The recombinant adenovirus of MRP gene fragment (rAdeno-MRP) from SS2 was constructed successfully.
Insights
Researchers successfully constructed a recombinant adenovirus carrying the muramidase-released protein (MRP) gene fragment from Streptococcus suis type 2. This advancement facilitates further study of MRP's role in SS2 infections.
Area of Science:
- Molecular Biology
- Virology
- Bacteriology
Context:
- Streptococcus suis type 2 (SS2) is a significant pathogen causing various infections in both animals and humans.
- The muramidase-released protein (MRP) is a key virulence factor in SS2.
- Understanding MRP's function requires effective tools for its study and manipulation.
Purpose:
- To construct and identify a recombinant adenovirus vector expressing the muramidase-released protein (MRP) gene fragment from Streptococcus suis type 2 (SS2).
Summary:
- The MRP gene fragment (467-1351 bp) was amplified from SS2 genomic DNA.
- The fragment was cloned into an adenovirus shuttle plasmid and subsequently used to create a recombinant adenovirus plasmid (pAdeno-CMV-MRP).
- Successful viral packaging and expression of the MRP gene fragment and protein were confirmed in AD-293 cells.
Impact:
- This study reports the successful construction of a recombinant adenovirus carrying the SS2 MRP gene fragment (rAdeno-MRP).
- This recombinant adenovirus provides a valuable tool for investigating the pathogenesis of SS2 and for developing potential therapeutic strategies.
