| Objective:In order to discover new candidate diagnosis molecular for schistosomiasis, the SjTs4 protein gene of Schistosoma japonicum were cloned and expressed in the prokaryotic cell.Indirect ELISA(rSj-Ts4-ELISA) was established for schistosomiasis.Methods:The total RNA was extract from adult worms ofS.Japonicum.Primers were designed and synthesized.The target gene was amplified by RT-PCR.The product from PCR was cloned into PMD-T vector,and then was subcloned into the expression vector pGEX-5X-1.After induced by IPTG,the cells were collected to analyze by SDS-PAGE and Western blot.Then the cDNA encoding Sj-Ts4 protein was inserted into the expression vector pET28a(+).After induced by IPTG,The rSj-Ts4 were expressed in E. coli(DH5a) and purified through the column of affinity chromatography with HIS.The rSj-Ts4-ELISA,SjAWA-ELISA和SEA-IHA were subjected to the serological diagnosis in the patients with acute,chronic and advanced schistosomiasis japonica.Parallel tests were also performed using the sera of individuals with clonorchiasis,hookworm infections and normal persons those from the areas without schistosomiasis.The sensitivity,specificity and cross reaction of the recombinant antigens used in ELISA (rSj-Ts4-ELISA) were compared.Results:For RT-PCR,,a specific band of around 819bp was amplified.The gene was subcloned into the expression vector pGEX-5X-1.After sequencing,the rSj-Ts4 were induced by IPTG and expressed.The results of SDS-PAGE revealed that the molecular weight of fusion protein was approximately 58kDa.rSj-Ts4 were purified through the column of affinity chromatography.The expressed rSj-Ts4 was verified with anti-GST antibodies by Western blotting.The aim gene was inserted into the expression vector pET28a(+).The results of SDS-PAGE revealed that the molecular weight of fusion protein with HIS was approximately 32kDa.The purified rSj-Ts4 was subjected to the serological diagnosis in the patients with acute,chronic and advanced schistosomiasis japonica,clonorchiasis,hookworm infections and normal persons using indirect ELISA. Parallel tests were also performed using SjAWA-ELISA and SEA-IHA.The positive rates were 97.1%in rSj-Ts4-ELISA and 100%in SjAWA-ELISA in 34 cases of acute schistosomiasis.Statistical analysis revealed no significant difference in sensitivity (X~2=1.02,p＞0.05).The positive rates in 16 cases of chronic schistosomiasis were 100% in rSj-Ts4-ELISA and 100%in SjAWA-ELISA,statistical analysis revealed no difference of sensitivity.The positive rates were 87.5%in rSj-Ts4-ELISA and 75.0%in SjAWA-ELISA in 24 cases of advanced schistosomiasis.Statistical analysis revealed no significant difference in sensitivity(X~2=1.23,p＞0.05).The false positive reaction was found to be 6.7%in and 3.3%in two methods when detected in 30 cases of normal control sera but no statistically significant difference was noted(X~2=0.35,p＞0.05).The positive rates in 32 cases of other parasite infections(24 clonorchiasis patients and 8 hookworm infections) were 12.5%in rSj-Ts4-ELISA and 25.0%in SjAWA-ELISA, statistical analysis revealed no significant difference in sensitivity(X~2=1.64,p＞0.05). The positive rates were 97.1%in rSj-Ts4-ELISA and 82.4%in SEA-IHA in 34 cases of acute schistosomiasis,statistical analysis revealed no significant difference in sensitivity (X~2=3.98,p＞0.05).The positive rates in 16 cases of chronic schistosomiasis were 100% in rSj-Ts4-ELISA and 68.7%in SEA-IHA,statistical analysis revealed significant difference in sensitivity(X~2=5.93,p＜0.05).The positive rates were 87.5%in rSj-Ts4-ELISA and 50.0%in SEA-IHA in 24 cases of advanced schistosomiasis, statistical analysis revealed significant difference in sensitivity(X~2=7.86,p＜0.05).The false positive reaction was found to be 6.7%in and 0.0%in two methods when detected in 30 cases of normal control sera but no statistically significant difference was noted(X~2=2.07,p＞0.05).The positive rates in 32 cases of other parasite infections(24 clonorchiasis patients and 8 hookworm infections) were 12.5%in rSj-Ts4-ELISA and 9.4%in SEA-IHA,statistical analysis revealed no significant difference in sensitivity (X~2=0.10,p＞0.05).Our results demonstrate that rSj-Ts4-ELISA for the detection of the specific antibodies in both acute,chronic and advanced schistosomiasis has similar sensitivity and specificity to SjAWA-ELISA.The rSj-Ts4-ELISA seems to be more sensitive when used for detecting chronic and advanced schistosomiasis between rSj-Ts4-ELISA and SEA-IHA.The rSj-Ts4-ELISA is easy to standardize,has low crossreactions, and has the potential of practical use for the immunodiagnosis of schistosomiasis.Conclusion:The cDNA encoding Sj-Ts4 protein of S.japonicum was cloned,expressed in the prokaryotic cell and purified.The rSj-Ts4 antigens were subjected to the serological diagnosis in schistosomiasis japonica using indirect ELISA.The rSj-Ts4 are economic to prepare,easy to standardize,it may be subjected to the serological diagnosis in schistosomiasis in endemic area. |
PDF Full Text Request