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[Cloning and optimized prokaryotic expression of a pbmag-1 cDNA fragment from Plasmodium berghei ANKA]
1Etiology Department, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Objective:
To clone and express a novel gene cDNA fragment, pbmag-1, from Plasmodium berghei ANKA strain.
Methods:
The cDNA sequence of pbmag-1 was obtained from the GenBank of P. berghei ANKA genomic databases, with which a pair of primers was designed and RT-PCR was used to get a cDNA fragment of the gene from the parasite. The expanded cDNA 3' fragment of the gene was obtained by 3'-RACE using the oligo dT primer and a set of specific primers. The intact cDNA 3' fragment was cloned into a prokaryotic expressional vector and transformed into the BL21-(DE3)-RIL strain of Escherichia coli. The recombinant protein of PbMAg-1 was expressed with an optimized strategy and used to immunize mice.
Results:
The pbmag-1 cDNA fragment obtained was 1341 bp in length, A/T rich (73%) and with a correct 3' end sequence. By Western blot, the anti-serum of mice immunized with the recombinant protein of PbMAg-1/GST, which was expressed as inclusion bodies, specifically recognized a band with Mr 64,000 molecule from the protein extracts of P. berghei-infected mouse erythrocytes.
Conclusion:
The pbmag-l cDNA sequence with intact 3' has been obtained, which will be used for further study on its role in the immune response of P. berghei infection.
