| Trichinellosis is a serious parasitic zoonosis,which is caused by Trichinella.In the present studies,the immunodiagnosis often uses natural crude antigens,such as somatic antigens and excretory-secretory(ES) antigens.Although the natural antigens have high sensitivity,their components are extremely complex and they often cause cross-reaction with other parasitic diseases.Because there is no breakthrough of Trichinella larval culture in vitro, the natural antigens are difficult to be produced standardly.Gene recombinant protein is easy to be produced and has single component,which will has broad applications.Ts21 protein is a part of the Trichinalla muscle larval ES antigen,which is found recently.Our laboratory has cloned and expressed the Ts21 protein of Trichinalla spiralis(T1).In this paper,the Ts21 recombinant protein was purified by affinity chromatography,its character was further identified and its value of immunodiagnosis and immunoprophylaxis was studied.Materials and Methods1.Trichinella species and experimental animals:Five species of Trichinella: Trichinella spiralis(T1),T.nativa(T2),T.britovi(T3),T.pseudospiralis(T4) and T.nelsoni (T7) were obtained from International Trichinella Reference Center(ITRC).Sera from patients with trichinellosis,echinococcosis,cysticercosis,schistosomiasis and paragonimiasis were stored in our laboratory.The male BALB/c and Kunming mice with 4-6 weeks old were brought from Experimental Animal Center of Henan Province and raised in our feeding room.2.Expression and purification of Ts21 recombinant protein:The transformed E.coli TB1 bearing pMAL-c2X-Ts21 plasmid was induced by IPTG for production of fusion protein. The desired protein was purified by Amylose pre-packed column after obtaining the supernatant through treatment with ultrasonic disintegrator and centrifuging after ultra-sonication. SDS-PAGE was used for the detection of the expressed products and ultraviolet spectrophotometer was used for the concentration analysis of the recombinant protein.3.Further identification of Ts21 recombinant protein:BALB/c mice were immunized by purified Ts21 recombinant protein in order to gain the immunized mouse sera. Western blot was used to identify the immunoreaction between immunized mouse sera and Ts21 recombinant protein,T1 somatic antigens and T1 ES antigens.Immunofluorescent assay (IFA) was used to identify the reaction between immunized mouse sera and freezing section of T1 muscle larvae at 18d and 42d post infection.4.Immunodiagnostic value of Ts21 recombinant protein:Indirect ELISA was used to assay sera from mice infected with different Trichinella species and from patients with trichinellosis and other parasitic diseases after coating Ts21 recombinant protein onto the microplates.The change of serum antibody level in mice was observed from the 2nd week to the 6th week after challenged with 300 infective larvae per mouse.The antibody level was observed on the 6th week after challenged with 10 infective larvae per mouse or 5 infective larvae per mouse.5.Immunoprophylactic function of Ts21 recombinant protein:60 mice were randomly divided into 3 groups(20 mince per group):immune group(A group),adjuvant group(B group),blank control group(C group).A group was immunized with 20μg per mouse of Ts21 recombinant protein incorporated into equal complete Freund's adjuvant by hypodermic injection at the first time.This group was immunized at the same way incorporated into equal incomplete Freund's adjuvant at the second and the third time at intervals of 10 days.Venous sera were collected 10 days after the third immunization and the antibody levels were tested by ELISA.Then A group was challenged with 300 infective larvae. B group and C group were immunized with the 0.9%NaCl solution incorporated into equal Freund's adjuvant and the 0.9%NaCl solution,respectively.The method of immunity and challenge infection was the same as the A group.The above 3 groups of mice were killed on 84h after infection to observe the intestinal adult worms,killed on 6 weeks after infection to count the muscle larvae and calculate reduction rate.The reduction rate=(number of blank control group-number of test group)/number of blank control group×100%.6.ELISA:Ts21-ELISA and ES-ELISA were established by using Ts21 recombinant protein and ES antigen respectively.The microplates were coated by 5μg/ml Ts21 recombinant protein and ES antigen respectively,then blocked by 2%BSA-PBST at 37℃for 2h.Sera were diluted into 1:100 and were added into each well.Conjugate was goat anti-mouse IgG(1:6000) or goat anti-human IgG(1:500) and the substrate was OPD.Positive control,negative control and blank were tested in each procedure.The absorbance of each well was tested after stopping the reaction on 492nm wavelength.The result was positive when the absorbance was equal and higher than 2.1 times of absorbance of negative control.Results1.Character of Ts21 recombinant protein:The results of SDS-PAGE and Western blot showed that sera of mice immunized with Ts21 recombinant protein was reacted only with the band with Mr 21 000 in soluble and ES antigen.It was discovered by IFAT that sera of mice immunized with Ts21 recombinant protein reacted with encapsulated larvae by displaying yellow-green fluorescence in section,but had negative reaction with pre-encapsulated larvae of 18 days after infection.The results demonstrated that the expression stage of Ts21 recombinant protein was in encapsulated larvae.2.Diagnosis sensitivity and specificity of Ts21 recombinant protein:Positive rates of sera from T1,T2,T3,T4,T7 infected mice were 86.0%(37/43),16.7%(3/18),7.1%(1/14), 36.4%(4/11) and 36.4(4/11) respectively by Ts21-ELISA,while the positive rates were 88.4%(38/43),88.9%(16/18),92.9%(13/14),63.6%(7/11) and 81.8%(9/11) respectively by ES-ELISA.There was no significant difference of sensitivity between Ts21-ELISA and ES-ELISA(x~2=0.104,P＞0.05),but the cross-reaction rate of ES antigen was significantly higher than that of Ts21 recombinant protein(x~2=17.069,P＜0.05).The antibody positive rate of sera from cases with trichinellosis assayed by Ts21-ELISA were 94.7%(18/19), cross-reaction rates with sera from cases with echinococcosis,cysticercosis,schistosomiasis and paragonimiasis were 10.5%(3/19),7.7%(1/13),16.7%(1/6) and 0%(0/4),respectively. Serum positive rate of trichinellosis patients assayed by ES-ELISA were 100%(19/19),serum cross-reaction rates of cases with echinococcosis,cysticercosis,schistosomiasis and paragonimiasis were 10.5%(2/19),23.1(3/19),0%(0/6) and 0%(0/4),respectively.There was no obvious difference of the sensitivity and specificity between Ts21 recombinant protein and ES antigen(x~2=0.000,P＞0.05,x~2=0.010,P＞0.05).3.Detection of infected mice with different dose at different time by Ts21-ELISA: The serum antibody positive rate of mice infected with 300 T1 muscle larvae was 20%and 60%at 2 and 3 weeks post-infection,and up to 100%at 4～6 weeks post-infection when assayed by Ts21-ELISA,while the positive rate was 20%and 40%at 2 and 3 weeks,and 100%at 4～6 weeks post infection when assayed by ES-ELISA.Serum positive rate of mice infected with 10 T1 muscle larvae were 90%and 100%when assayed by Ts21-ELISA and ES-ELISA,respectively,while the positive rate was 100%of mice infected with 5 T1 muscle larvae assayed by both methods.4.Immunoprophylaxis of Ts21 recombinant protein:Serum antibody titer of mice was 1:10~6 10 days after last immunization with Ts21 recombinant protein.At 84h after infection,intestinal adult worms of A,B and C group were 43.60±20.07,68.70±28.54 and 76.10±17.67,respectively.There was significant difference among A,B and C group (P＜0.05).The reduction rates of intestinal adult worms of A and B group were 42.71%and 9.72%,respectively.Muscle larvae of the 3 groups on 42d after infection were 16973.80±7111.06,32205.00±12145.33 and 33812.50±24703.42,respectively There was significant difference in A,B and C group(P＜0.05).The reduction rates of muscle larvae of A and B group were 49.80%and 4.75%,respectively.Conclusions1.Ts21 protein is one part of ES antigen of Trichinella spiralis muscle larvae and its expression stage is in encapsulated larvae.2.There is no obvious difference of sensitivity between Ts21-ELISA and ES-ELISA, but the cross-reaction rate of ES antigens is significant higher than that of Ts21 recombinant protein.There is no significant difference of sensitivity and specificity between Ts21-ELISA and ES-ELISA when they are used to assay the sera from patients with trichinellosis.Ts21 recombinant antigen can be applied to the serodiagnosis for trichinellosis. 3.Ts21 recombinant antigen has the partial immune protection against challenge infection with T.spiralis muscle larvae. |
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