Abstract: The LppQ N-terminal gene of Mycoplasma mycoide subsp. mycoides SC was amplified from MmmSC HVRI X strain by PCR using the primers designed according to the reported MmmSC LppQ sequence. The products of PCR were cloned into the pUC18 and pUC19 vector respectively. Mutagenesis was performed in a one-step overlap extension PCR. Sequence analysis confirmed the successful mutation from TGA to TGG in amino acid 66 in the LppQ N-terminal gene. The fragments amplified by PCR containing the mutation site were subcloned into the pET32a expression vector. Restriction enzyme, PCR and sequencing analysis indicated that the recombinant expression plasmid pET32a-LppQ was successfully constructed, which may provide a basis of expression of the LppQ N-terminal gene in vitro.