Objective: To construct chloroplast expression vector, and introduce the C-terminal region of the merozoite surface protein 1 gene (msp1-42) of Plasmodium falciparum 3D7 strain into the chloroplast genome of tobacco for expression of the recombinant protein MSP1-42.
Methods: Forward and reverse primers, adjusted to tobacco chloroplast codon preferences, were used for generation of msp1-42 gene from pBluntmsp plasmid which contains msp1-42 gene. A chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a biolistic He particle delivery system. Media containing 500 mg/L spectinomycin were used for selection of spectinomycin resistant plant. PCR was carried out to check the introduction of the msp1-42 and aadA genes into the chloroplast genome. The transgenic plants with msp1-42 and aadA gene insertion were cut and cultured on the generation MS media containing 500 mg/L spectinomycin for at least 3 turns, and multiple PCR were applied to analyse their homogenization.
Results: A chloroplast expression vector LRrrmsp was constructed and confirmed with PCR and enzyme digestion analysis. Six transformants were obtained with a transformation rate 0.6/gun. The msp1-42 and aadA genes were amplified from spectinomycin resistant plants by PCR detection. Wild type chloroplast gene was detected by multiple-PCR analysis.
Conclusion: A chloroplast expression vector containing msp1-42 gene was constructed. The msp1-42 gene was introduced into chloroplast genome of tobacco and heterogeneous transgenic tobacco was obtained.