Studies On Immunodiagnosis And Biological Therapy Of Toxoplasmosis

Toxoplasma gondii infection is widespread in humans, which is a protozoan parasite that infects up to a third of the world's population, although its prevalence varies widely from place to place. In the United States and the United Kingdom, it is estimated that 16-40% of the population are infected, whereas in Central and South America and continental Europe, estimates of infection range from 50 to 80%. In China, the infection rate is about 5%, and rising year by year. Most infections in humans are asymptomatic but at times the parasite can produce devastating disease. It is necessary for earlier diagnosis and treatment, toxoplasmosis can be diagnosed by isolation of the organism, but it is time-costed and uneffective, so its diagnosis is still mainly based on serological tests. However, most of diagnostic kits produced in China have low correspondence rate compared with each other, and the sensitivity and specificity are not high enough, while the correspondence rate of imported kits are above 90%. However the imported diagnostic kits of toxoplasmosis are too expensive to be used in China, Therefore, there is an urgent need to develop an effective diagnostic kit, which will be helpful for clinical detection and epidemiological investigation. Treatment of toxoplas -mosis is another important issue. Pyrimethamine and sulfadiazine are usually used for the disease treatment in immunocompromised persons. While pregnant women have been treated with the antibiotic spiramycin or with sulfadiazine alone or the combination of pyrimethamine and sulfadiazine. However all these drugs have more or less side-effects, such as bone marrow suppression, liver lesion, et al. Many patients, such as toxoplasmic encephalitis and congenital toxoplasmosis, cannot tolerate long-term chemical therapy, but once a stop, it will result in a relapse. New drugs with potential effects against Toxoplasma gondii, such as azithromycin, atovaquone and clindamycin, require further evaluation in animal models and human studies. It's necessary to develop new medicines fortreatment and control of toxoplasmosis.In this study, we attempt to develop appropriate and new immunodiagnositic methods and explore the feasibility of the targeting biological therapy for toxoplasmosis.Part â…  Research on immunodiagnosis of toxoplasmosis1, Gene cloning and procaryotic expression of the major surface antigen gene 1 of Toxoplasma gondii RH strainTo amplify the major surface antigen gene SAG1 from genome DNA of Toxoplasma gondii RH strain, construct recombinant procaryotic expression vector and express it in E.coli. A pair of primers was designed according to cDNA sequence of SAG1, then the SAG1 gene amplified by PCR was cloned into the vector pGEX-4T-3 and proved by DNA sequencing; The SAG1 gene was subcloned into procaryotic expression vector pET-32c, its expression was induced by IPTG. The SAG1 gene was successfully amplified from genome DNA of Toxoplasma gonddi RH strain and a 37kDa fusion protein was expressed in E.coli expression system pET-32c as inclusion body. SAG1 was a major antigen in the surface of Toxoplasma, the SAG1 fusion protein has a potential value in diagnosis of toxoplasmosis.2, Expression , purification and immunoresponse evaluation of the major surface antigen SAG1 of Toxoplasma gondii RH strainTo express the major surface antigen gene SAG1 in E. coli, purify the recombinant fusion protein SAG1 and evaluate its immunoresponse. A single colony of E. coli BL21 containing the plasmid â–³SAG1/pET-32c was inoculated LB broth, then diluted 1/100 into 1000ml LB broth and induced with 1mmol/L IPTG. The recombinant SAG1 was purified with Ni²⁺ chelating HiTrap HP column. Its immunoresponse was evaluated with Western-biot. Fifty-two IgG-positive and 40 IgG-negtive human sera were tested with rSAG1. The recombinant SAG1 protein about 37kDa was expressed in E.coli as inclusion body; rSAGl was prepared with Ni²⁺ column purification; The result of western-blot showed that rSAGl could be recognized by positive serum of Toxoplasmosis; The positi...