Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system.
In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.
Bigi
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Instituto de Biotecnología, Centro de Investigaciones en Ciencias Veterinarias, Instituto Nacional de Tecnologia Agropecuaria, Moron, Argentina.
Taboga
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O
Romano
M I
MI
Alito
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A
Fisanotti
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JC
Cataldi
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AA
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D016367:Baculoviridae / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D007313:Insecta / Q000821:virology; D009163:Mycobacterium bovis / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins