Construction And Immunoscreening Of The CDNA Library Of Toxoplasma Gondi And Analysis Of The Positive Colones

Objective: Constructing a cDNA library of Toxoplasma gondii tachyzoite stage ( RH strain) .and screening it using seral of mouse infected with bradyzoites (Prugniaud strain) in order to look for new vaccine molecular and earlier period diagnosis reagents. Methods: T. gondii tachyzoite stage ( RH strain) were collected. Total RNA was extracted from T. gondii tachyzoite stage and cDNA was synthesized by RT-PCR. The cDNA was recombinant oriented into phage vector with sfi A.B adaptor . A Toxoplasma cDNA library was constructed after packaging. The seral of mouse (Prugniaud strain) were collected and eliminated anti-E-coli antibodies by E-coli lysate, used as antibody to screen cDNA Library constructed. The positive clones from rescreening three times were sequenced. The obtained genes were compared against GenBank datebase by BLASTN and BLASTP. A sequenced gene which have complete open reading frame was analysed by bioinformatics method .Result: The cDNA molecule weight range from 400~2500bp was identified with agar electrophoresis. The recombination rate were 90% measured by plate with IPTG and X-Gal. seven recombinant clones were pick out randomly and were self-cycled into plasmid. The plasmid were digested by two enzymes, the inserted fragments range from350 to 1500bp. About 7.2×10~4 recombinant phages were screened by using the seral of mouse(Prugniaud strain), thirty-eight positive clones were obtained in which fifteen were rescreened until the four continuing positive clones were confirmed. The sequence data of positive clones were analyzed in GenBank datebase. Sequence of WT1 clone is T. gondii P30 gene and that of WT4 clone is phosphatidylinositol N-acetylglucosaminyltransferase subunit C gene. The sequence of WT9 has no significant similarity found. Sequence of insert of WT12...