Prokaryotic Expression Of Toxoplasma Gondii SAG1and GRA4and Construction Of Recombinant Feline Herpes Virus Type I Transfer Vector

Toxoplasmosis is parasite in humans and a variety of animals zoonoses caused by a obligate intracellular parasite Toxoplasma gondii. It not only brought huge economic livestock losses, while the risk of maternal abortion, stillbirth etc. It can be vertically transmitted to the fetus, resulting in fetus congenital diseases; It is also an important reason that caused by AIDS, cancer and other immune deficiency of death in patients. Cats are the definitive hosts of Toxoplasma gondii, therefore the development of vaccines against toxoplasmosis in cats is particularly important. Virus vaccine vectors to get a good immune effect, cat herpes virus type I s a good vaccine candidate vectors, TK gene is non-essential proliferation and major virulence genes of herpesvirus gene, allowing exogenous gene insertion and expressionToxoplasma gondii RH strain SAG1and GRA4gene were amplified by RT-PCR to construct pMD-18T-SAG1/GRA4cloning vector, pColdⅢ-SAG1/GRA4prokaryotic expression vector, pBluescipt-TK transfer vector and recombinant herpes cats virus shuttle plasmid pBS-TK-SAG1/GRA4. pColdⅢ-SAG1/GRA4prokaryotic expression vector were transformed into E.coli BL21competent cells’ to express TgSAGl/GRA4protein by IPTG induction, SDS-PAGE and Western-blot analysis of expression products, and apply Freund’s adjuvant to prepare TgSAG1/GRA4protein subunits vaccine used to immune BALB/c mice and prepare anti rTg SAG1/GRA4of the serum. The recombinant herpes cats virus shuttle plasmid pBS-TK-SAG1/GRA4were transfected into293T cells by liposome method. The expressed products in293T cells were analyzed by IFAT and western blot. The results showed that the amplified T. gondii RH strain SAG1and GRA4gene length were960bp and1038bp, prokaryotic expression plasmid pColdⅢ-SAG1/GRA4were constructed successfully, and the recombinant proteins with good reactogenicity molecular weight were52ku and57ku. The molecular weight of expressed productions that SAG1/GRA4proteins in293T cells recognized by mouse anti-rTg SAG1/GRA4serum were35ku and45ku. The recombinant cat herpes virus shuttle plasmid pBS-TK-SAG1/GRA4with good reactogenicity were successfully constructed...