Pronucleus Expression And Immunogenicity Analysis Of ROP4 From RH Strain Of Toxoplasma GondⅡ

Toxoplasmosis which has two names when translated into Chinese is a kind of anthropozoonosis caused by Toxoplasma gondii. The disease which distributes all over the world is widespread in warm-blooded animals; cats are their final host and an important source of infection; intermediate hosts include a variety of mammals, birds and people, etc. Animals infected with toxoplasmosis often leads to abortion, weak child, still births and mummified, etc. so the livestock industry has been causing serious economic losses and brown to mankind a great threat to physical and mental health by the disease.In this study, a pair of primer was designed according to rhoptry protein 4 (ROP4) sequence of Toxoplasma gondii published in GenBank, and the gene was amplified by PCR and cloned; then pronucleus expression vector of Toxoplasma gondii ROP4 gene was structured and expressed in bacillus coli. The reactogenicity of the expression products were analyzed by Western blotting method. The expressed recombinant protein was purified and then mixed with Freund's adjuvant inoculate BABL/c mice. Cellular and humoral immune level were determined to detect immunogenicity of the recombinant protein.The results indicated that a gene fragment which is 1 219bp in length was amplified, the comparative analysis between the cloned ROP4 gene sequence and the sequence AY662677 showed they share 99.9% homology; that proved the gene fragment is indeed the ROP4 gene of Toxoplasma gondii. The gene was cloned into pronucleus expression vector pET-30a-c(+); the restriction fragment and PCR identification suggested the expression vector pET-30a-c(+)/ROP4 was structured successfully. The structured vector was inducted by IPTG and expressed in bacillus coli. BL21; after 4 hours the expression level reach to the highest. Detection results of SDA-PAGE and Western blotting indicated the protein which is mainly soluble protein has a good reactogenicity and its molecular weight is 44.9 kDa. After inoculate BALB/C mice the protein can improve the cellular immunity and humoral immunity level of the mice which suggests it has good immunogenicity. This study provides for the in-depth study of toxoplasmosis and its vaccine a scientific theory.