Construction and sequence determination of E. coli - Mycobacteria recombinant shuttle plasmid of P30 gene fragment of Toxoplasma gondii.

Aim To construct a E.coli-Mycobacteria recombinant shuttle plasmid containing P30 gene of Toxoplasma gondii. Methods Injecting mice with RH strain tachyzoites then harvesting infected ascites of mice ,genomic DNA of Toxoplasma gondii was extracted with phenol/chloroform. A pair of primers were designed according to the sequence of P30 gene, a fragment of gene coding P30 antigen was amplified by means of polymerase chain reaction(PCR).The purified PCR products and pBCG5.6 plasmid were digested by BamHI+KpnI and ligated by T4 ligase, the recombinant shuttle expression plasmid, pBCG-P30, was transferred into E.coli DH5α,positive clones were screened and identified by endonulease digestion and PCR technique. Sequence determination analysis was performed to identify the cloned gene. Results The P30 gene fragment with about 1037 base pairs were specifically amplified by using PCR technique. The positive recombinant plasmid pBCG-P30 was screened and digested by XbaI+KpnI ,a compound gene fragment of including P30 and hsp70 promoter gene was obtained , the size of compound fragment is 1170 bp and in accordance with the expected one. Sequence determination analysis showed that the cloned gene was gene fragment coding P30 antigen. Conclusion The E.coli-Mycobacteria shuttle expression plasmids containing P30 gene from Toxoplasma gondii were constructed successfully.