Preliminary Screening Of The Congenital Toxoplasmosis Diagnostic Specimen Anddiagnostic Protein During Pregnancy

Objective:During gestation,accurate diagnosis of congenital toxoplasmosis affects the eugenics.In order to improve the diagnosis accuracy,screening specimen and specificity in the diagnosis of protein is of particularly importance.Thereforethisexperiment establish congenital toxoplasmosis mice model and screen of specimens and identify the function of the different protein,hope to screening can be used as the prenatal diagnosis of the disease specificity protein when the fetus are not birth,thus to provide research foundation for accurate diagnosis of congenital toxoplasmosis for human eugenics.Methods:Establish the congenital toxoplasmosis mouse model:There were having laid BALB/c female mice were weighed and recorded,then,with BALB/c male mice according to the proportion of 2:1 in a cage,next day checks pregnant bolt.Randomly divided 20 pregnant mice whose weight gained obviously into group A,B,C,Dand group A,B,C,D pregnant mouse intragatrically with T.gondii tachyzoites at a dose of 0、5×104、10×104、20×104/0.4m L PBS respectively on gestation day 8.Observe and record pregnant mice abnormal reaction.On gestation day 18 weighed again,and the pregnant mice were killed,then to return the specimen:the placenta and fetal mouse.Took the paraffin section of the tissue:Placenta,fetal brain and fetal body obverse the pathological change in the way of HE staining.Then to detectT.gondii RH strain highly conservative B1 gene in infected pregnant mice liver,normal group and the group of placenta,fetal mice head,fetal mice body by conventional PCR,judge the model if successfully established.The screening of diagnosiscongenital toxoplasmosis appropriate specimen:There were having laid female BALB/c mice were weighed and recorded,then,with BALB/c male mice according to the proportion of 2:1 in a cage,next day check the pregnant bolt.Randomly divided 16 pregnant mice whose weight gained obviously into group A,B,and each group every pregnant mouseintragatrically with T.gondii tachyzoites at a dose of 0、5×10~4/0.4m L PBS ongestation day 8.Then,on gestation day 18 took the specimens:serum,urine and amniotic fluid to detect TCA in the way of ELISA.To determine the differences of the urine,blood,amniotic fluid specimens in the diagnosis of congenital toxoplasmosis respectively.Screening of blood specimen in accurate diagnosis of congenital toxoplasmosis specificity protein:Established congenital toxoplasmosis mice model and take blood from pregnant mice eyes on gestation day 18,centrifugal in serum.Using two-dimensional electrophoresis separation different point in the serum protein:extract serum protein and protein quantity,isoelectric focusing,two of the sds-page,staining and image analysis results.MALDI-TOF-TOF about 2-DE difference point in the serum protein identification:respectively in each group choose two of the differences protein point which protein increased and reduced obviously in protein expression,through protease solution,point target,mass spectrum detection and related database retrieval,to differences in protein point for further biological information analysis,try to screening the specificity of the diagnosis of congenital toxoplasmosis protein.Results:Compared with the normal group A,the infection group B,C,D pregnant mice appeared different degree of abnormal reactions,such as piloerection,back,burnout,or even with ascites,abortion,etc after lavage inoculation tachyzoite.Pregnant mice of group A all survived and there were 45 fetal rats;Group B,there was 1pregnant mouse died,1 pregnant mouse have not fetal rats and the other three pregnant mice totally have26 fetal rats;Group C,there were 3 pregnant mice died and the other two pregnant mice totally have 17fetal rats;Group D,there were 2 pregnant mice died and only 1 pregnant mice have 11fetal rats.During the experiments,the weight of infected groups loss have obvious difference compared with the normal group(P<0.05).HE staining showed that the placenta and fetal mouse tissue cells are not obvious pathological change,but,the typical and atypical insect were found in placental tissue;The infected pregnant mice liver,and the group of placenta,fetal mice head,fetal mice body were all detected the B1gene.The specimens were detected TCA in the approach of ELISA,there was significant difference(P<0.05)between the normal group and infect group,whilethere was no statistical significance(P>0.05)among serum,urine and amniotic fluid.Through the result of 2-DE:the normal group and infection group electrophoresis comparison showed there were 86 differences in protein point and normal group of unknown protein increases the differences of protein expression has 52 point,the infection group unknown higher protein expression differences of protein has 34point.Choose the 4 kinds of significant difference of protein database comparison respectively:HPT_MOUSE,PLMN_MOUSE,HEMO_MOUSE,Q3UKU5_MOUSE,and the change of the 4 protein expression levels are all associated with congenital toxoplasmosis pathogenic.Conclusion:Pregnant mouse infected with T.gondiitachyzoites with adose of 5×10~4/0.4ml in the way of lavage respectively on gestation day 8 can establish the suitable congenital toxoplasmosis fetal mice model.There were no difference among blood,urine,amniotic fluidas diagnostic specimens of congenital toxoplasmosis.After the pregnant mice infected with toxoplasma,the expression change of 86 different protein points in serum specimens,and theexpression levels significantly higher or lower of the four kinds of proteins associated with the clinical pathological manifestations of toxoplasm,though,whether they can be used as accurate diagnostic of congenital toxoplasmosis standard still needs further research.If there are more suitable of protein for diagnosis congenital toxoplasmosis in 82 other protein points also need for bioinformatics analysis and research.