| Toxoplasmosis is caused by Toxoplasma gondii,an obligate intracellular protozoan parasite.From the statistics by WHO in 2002,it was estimated that 600 million people were at risk of becoming infected and 300 million people were already infected.People are infected by inadvertent ingestion of oocysts or sporozoites in cat feces,or tissue cysts in undercooked infected meat or contaminated foods,and less frequently infected by direct recipience of tissue or blood from other contaminated humans,and vertical transmission from acutely infected mothers.Infection of immunocompetent humans is usually asymptomatic.Its clinical disease is largely confined to certain risk groups.Toxoplasmosis is an opportunistic disease,which is of ten lethal for immunocompromised patients such as those with AIDS,bone marrow or heart transplant recipients.Furthermore,toxoplasmosis may cause abortion or neonatal malformations such as birth defects,retinitis,and brain damage if acutely infected during pregnancy.For effective control of toxoplasmosis,efforts should be directed to develop a sensitive vaccine to limit congenital infectionIn this paper,theeukaryotic expression recombinants containing GRA4 gene was constructed and expressed GRA4 protein in vitro.BALB/c and C57BL/6 mice were directly immunized with the eukaryotic recombinant plasmid to observe and test the cellular and humoral immune responses induced by the DNA immunization.The immunogenicity of the expression protein and its expression protein in the muscle tissue from the immunized mice were analyzed.The immune effects of liposome-encapsulated GRA4 against infection of Toxoplasma gondii was analyzed to explore the new way for DNA vaccine of T.gondii and provide the scientific basis for the immunity prevention from T.gondii infection.The recombinant plasmid pcGRA4 was constructed and transformed into murine COS-7 cell line.SDS-PAGE,Westem-blot analysis indicted that the molecular weight of the expressed protein of GRA4 was about 40 kDa,similar to the predicted value.ELISA of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4.Production of primary interferon(IFN)-γand interleukin(IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7%of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived challenge with 80 tissue cysts of ME49 strain,whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%.When the immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain,the survival time of the mice immunized with liposome-encapsulated pGRA4 was markedly longer than other groups.Our observations showed that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T.gondii.To evaluate a modified rapid ELISA method for detecting circulating antigen(CAg) during Toxoplasma gondii infection,we analyzed specificity and sensitivity of the ELISA method by using experimental toxoplasma infection in lab and also tested this method in clinical human samples including 5,428 serum,548 cerebrospinal fluid and 2 breast milk samples.The result shows that 100%sensitivity,100%specificity,0%false negativity,0%false positivity.No cross-reaction was found with antigens of protozoa(Cryptosporidium parvum,Plasmodium falciparum),trematode (Schistosoma japonicum,Paragonimus sp) and nematode(Brugia malayii, Ancylostoma duodenale,Ascaris lumbricoides and rrichinella spiralis).Among human serum samples,the positive rate of CAg was 2.11%in cerebral paralysis patients,while it was 0.22%or 0.71%in patients without neurological symptoms or in uncomplicated pregnant women.The difference among these three groups was statistically significant(p＜0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%.There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (p＜0.01).In the retrospective analysis,CAg was detected in accordance with the onset time of clinic symptoms,suggesting that CAg could reflect the clinical course in humans.Together with those,CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T.gondii. |
