Construction of the subtractive cDNA library for tachyzoites of Toxoplasma gondii in different virulent stains

To construct the subtractive cDNA library for tachyzoites of Toxoplasma gondii in different virulent strains,the suppressive subtractive hubridization(SSH)was performed with tachyzoites of the RH strain as tester and those of the Prugniaud strain with low virulence as the driver.On the contrary,the reverse suppressive subtractive hybridization was done with tachyzoites of the Prugniaud strain as tester and those of the RH strain as driver.The products obtained from these two hybridizations were linked to vector pGEM-T and then transformed to E.coli DH5α.The positive clones screened were amplified and the inserted fragment was identified.It was found that both the forward(RH strain)and reverse(Prugniaud strain)subtractive cDNA libraries were obtained in this way,in which 384 positive clones of random recombinants were included in each group.The size of the inserted fragments were within range of 200-2 000 bps.As demonstrated by PCR amplification,the insertion rate was 98%.It is evident that two subtractive cDNA libraries have been established by suppressive subtractive hybridization technique,thus providing the basis for further screening and identification of the Toxoplasma gondii gene related to virulence.