The Construction Of The Gene Encoding Multiepitope Of Toxoplasma Gondii And Its Expression In E.coli And Eethylotrophic Yeast

Toxoplama gOndii is an obligate ifitracellular parasite causing awidespread infection in human and animals all over the world. As anopportunistic protozoan, T gondii play a very important role in deterioratingdiseases of the immunocompromised hosts such as AIDS patients andallograft recipients, and even is life-threatening. The infected pregnantwomen could transmit it to fetus through palcenta, leading to aboftion, deathfetus, neonatal malformaiions, and melltal symptoms or eye lesions ofsurvival infants.T gOndii is also a zoonosis and may give rise to great economic loss inlivestock industry. For the prophylaxis of toxoplasmosis, the reseaches ofvaccine and diognosis are the two main themes.Vaccines of T gondii are experiencing a tranfOrmation from attenuatedvaccines to subunit and nucleic ones, which are based on the mainimmunogenic components in some protective antigens of Tgondii. The vaccines constructed with single antigen are usually difficult to elicit optimal protection for hosts due to antigenic polymorphism and genetic restriction of T-cell responses. Therefore, with multicomponents including various protective antigens and adjuvants, multiepitope vaccines might produce ideal protection by stimulating the immune-response systems at multi-direction and multilevel. The lack of specific clinical symptoms makes diagnosis of toxoplasmosis still a problem. At present the usual method for it is serological detction. But the sensitivity and specificity of the kits used for the time in clinic mostly is not good. The main problem lies in the difficulty of obtaining highly purified and specific antigens in large quantity. Recombinant antigens produced by molecular and biological methods is probably a way to solve this problem. In the present study, a multiepitope gene fragment encoding multiple epitopes of T gondii antigens and Tetanus toxin has been constructed, and expressed in E.coli and methylotrophic yeast respectively. The antigenicity of the products has been analyzed as well. 1. The construction of the multigene of Tgondii According to the reports about the epitope analysis of T.gondii, four peptides containing T and (or) B cell epitopes were chosen from SAG 1, Rop2, GRA2, which are all protective antigens of T.gondii, and a universal adjuvant peptide containing T cell epitopes from Tenus toxin, to form the multi-epitope antigen of. T.gondii. In order to maintain independant conformation of each peptide after their expression, some non polar amino acid were inserted between every two peptides by means of the analyzing results from some relevant softwares. The gene sequence was determined according to the amino acid codon favoured in eukaryotic expression systems . The gene fragment was synthesized by DNA systhesizer and cloned into a plasmid. The recombinant plasmid was proved by enzyme digestion and the sequence of the insert was identified by sequencing. 2.The preliminary expression of the multigene in E.coli and the analysis of antigencity of the expression product. The multigene of Tgondii was subcloned into a prokaryotic expresrsion vector pRESTB and the recombinant plasmid was then transformed into BL21(DE3). With the induction of IPTG, protein of interests , whose molecular wight is about 14.4 kDa, is obtained in the form of inclusion body. Improving the inducing condition, the highest quantity of the product is about 20% of total bacterial lysate, and about 150mg per liter of fermentation medium. The analysis of western-blotting show...