[Study on Construction of Recombinant Bb-pGEX-OprI Vaccine of Pseudomonas aeruginosa and Its Protection Effect].

OBJECTIVE: To construct the recombinant Bb-pGEX-OprI vaccine of Pseudomonas aeruginosa (Pa) outer membrane protein I (OprI) and study its protection effect in mice against Pa. METHODS: The OprI gene was amplified by PCR,and cloned into pGEX-1λT to generate pGEX-OprI. The pGEX-OprI was transformed into Bifidobacterium bifidum(Bb) to construct recombinant Bb-pGEX-OprI vaccine by electroporation. After identification with double enzyme digestion,PCR and sequencing,the vaccine was then induced with IPTG,and its expression was analyzed and identified by SDS-PAGE and Western blot respectively. Twenty-one mice were randomly divided into 3 groups and vaccinated by intragastric administration with Bb-pGEX-OprI,Bb-pGEX-1λT and Bb respectively. All mice were challenged with PA01 strain at 4 weeks after the first vaccination. At 2 weeks after the challenge,mice were sacrificed to separate their lungs,and the numbers of bacterial colonies in lungs were counted. Venous blood was collected before vaccination,at 4 weeks after the first vaccination and 2 weeks after the challenge of PA01 strain. The serum IgG,IgG subclasses and IgE were detected by routine ELISA. RESULTS: The OprI gene of 194 bp was successfully amplified by PCR. Double enzyme digestion,PCR and sequencing confirmed that the OprI gene was successfully cloned into pGEX-1λT and pGEX-OprI was successfully transformed into Bb,constructing the Bb-pGEX-OprI vaccine. SDS-PAGE indicated that Bb-pGEX-OprI vaccine expressed an OprI-GST fusion protein with the relative molecular mass of approximately 32×103. Western blot verified that the fusion protein could be specifically identified by the sera of mice infected with Pa. The number of bacterial colonies in lung of Bb-pGEX-OprI vaccine group was lower than that of Bb-pGEX-1λT or Bb control ( P<0.01). The levels of serum IgG,IgG2b,IgG3 and IgE in Bb-pGEX-OprI vaccine group rose at 4 weeks after the first vaccination and 2 weeks after the challenge successively. The levels of serum antibodies in Bb-pGEX-OprI vaccine group were higher than those in Bb-pGEX-1λT or Bb control at the same time point ( P<0.01 or P<0.05). CONCLUSION: The recombinant Bb-pGEX-OprI vaccine was successfully constructed and produced an effective humoral immune response against the Pa infection.

Identification of porcine reproductive and respiratory syndrome virus inhibitors through an oriented screening on natural products.

Porcine reproductive and respiratory syndrome(PRRS) caused by porcine reproductive and respiratory syndrome virus(PRRSV) is one of the most infectious diseases in the swine industry worldwide, causing big economic losses. Vaccines are major weapons against PRRSV, however, current available vaccines have several limitations. Developing chemical drugs as alternatives is required. On the basis of traditional medical knowledge, we purposely selected 15 natural products originated from Chinese herbs with anti-infectious effects. Their antiviral activities were evaluated by PRRSV-induced cytopathic effect(CPE) on MARC-145 cells and reverse transcription polymerase chain reaction(RT-PCR) assay. Compounds ethoxysanguinarine(EOSG) and atractylodinol were found to be the hits which could significantly reduce PRRSV-associated CPE with 50% inhibited concentration(IC50) values of 7.9 and 39.4 µmol/L, respectively. Meanwhile, compounds ethoxysanguinarine and atractylodinol significantly decreased mRNA expression of ORF7 gene in a dose-dependent manner. Study results suggest that compounds ethoxysanguinarine and atractylodinol may be useful anti-PRRSV drugs for swine industry or the hits for further lead optimization.

[Diagnosis of chronic schistosomiasis japonicum with the recombinant Sj26GST-Sj32 fusion protein by ELISA].

OBJECTIVE: To study the feasibility of rSj26GST-Sj32-IgG-ELISA for diagnosis of chronic schistosomiasis japonicum. METHODS: The Escherichia coli BL21 (DE3) with recombinant plasmid pET32alphaSj26GST-Sj32 were induced with isopropy-beta-D-thiogalactopyranosid (IPTG), and the expression product was analyzed by SDS-PAGE and purified by Ni-NTA kits. Schistosoma japonicum (Sj) adult worm antigen(SjAWA) was produced by routine method. The IgG antibodies were detected with the sera of chronic schistosomiasis japonicum by ELISA using recombinant Sj26GST-Sj32 (rSj26GST-Sj32) fusion protein and SjAWA, while the controls included the sera of patients with clonorchiasis, paragonimiasis westermani, alveolar echinococcosis, cystic echinococcosis, hepatitis B, pulmonary tuberculosis and healthy people. RESULTS: The sensitivity and specificity of rSj26GST-Sj32 fusion protein were 95.00% (38/40) and 97.67% (42/43) respectively, they were 92.50% (37/40) and 97.67% (42/43) respectively in SjAWA groups. There were no difference in sensitivity and specificity between rSj26GST-Sj32 and SjAWA (P > 0.05). There were different cross reactions in clonorchiasis sinensis and paragonimiasis westermani between the two methods. The cross reaction with SjAWA was 20.00% (2/10) in patients with alveolar echinococcosis, but no cross reaction with rSj26GST-Sj32 (P > 0.05). CONCLUSION: rSj26-Sj32-IgG-ELISA probably could be applied to immunodiagnosis for chronic schistosomiasis japonicum.

[Detection of specific IgG in the sera of patients with chronic schistosomiasis japonica by dot-ELISA with the recombinant Sj26-Sj32 fusion protein].

OBJECTIVE: To study the diagnostic value of the Dot ELISA with rSj26-Sj32 fusion protein for chronic schistosomiasis japonica. METHODS: rSj26-Sj32 fusion protein and SjAWA were used to establish the HRP-IgG-Dot-ELISA. Serum samples from patients with chronic schistosomiasis japonica (40 cases), clonorchiasis sinensis (21 cases), paragonimiasis westermani(13 cases), alveolar echinococcosis (10 cases), cystic echinococcosis(9 cases), hepatitis B(20 cases), pulmonary tuberculosis (20 cases) and healthy persons (43 cases) were examined. RESULTS: Sensitivity and specificity were respectively 92.5% (37/40) and 95.4% (41/43) for rSj26-Sj32-Dot-ELISA and 95.0% (38/40) and 93.0% (40/43) for SjAWA-Dot-ELISA, and there was no significant difference between two antigens (P > 0.05). There were different cross reactions to the sera of patients with clonorchiasis sinensis, paragonimiasis westermani or alveolar echinococcosis, but no cross reaction to the sera of patients with cystic echinococcosis, hepatitis B or pulmonary tuberculosis. The positive and negative predictive value and efficiency of diagnosis of rSj26-Sj32-Dot-ELISA for chronic schistosomiasis japonica were 84.1% (37/44), 97.7% (129/132), and 94.3% (166/176), respectively, and those of SjAWA-Dot-ELISA were 77.6% (38/49), 98.4% (125/127), and 92.6% (163/176), respectively. There was no significant difference between the two methods (P > 0.05). CONCLUSION: rSi26-Si32 fusion protein can be applied to immunodiagnosis for chronic schistosomiasis japonica.

[Study on diagnosis of chronic schistosomiasis japonicum with rSj26-Sj32 fusion protein].

AIM: To study whether rSj26-Sj32 fusion protein can be used for diagnosis of chronic schistosomiasis japonicum. METHODS: ELISA, Dot-ELISA and dipstick were performed to detect the IgG in the sera of patients with chronic schistosomiasis japonicum using the antigens of rSj26-Sj32 fusion protein and SjAWA. The control sera were taken from health donors and the patients with clonorchiasis sinensis, paragonimiasis westermani, alveolar echinococcosis and cystic echinococcosis. RESULTS: The sensitivity of the three methods was 95.00%, 92.50% and 92.50% respectively, whereas the specificity of the three methods was 97.67%, 95.35% and 97.67%, respectively. CONCLUSION: The rSj26-Sj32 fusion protein has therapeutic potential for immunodiagnosis of chronic schistosomiasis japonicum.

[Changes of spleen cytokines in mice immunized with recombinant Bb-Eg95-EgA31 vaccine against Echinococcus granulosus].

OBJECTIVE: To investigate the level of cytokines in spleen from mice immunized with recombinant Bifidobacteria bifidum(Bb)-Eg95-EgA31 vaccine of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces. METHODS: 56 female BALB/c mice were randomly divided into 7 groups: Recombinant Bb-Eg9S-95-EgA31 vaccine subcutaneous injection (group A), intramuscular injection (B), intranasal immunization (C), oral administration (D), blank vector control (E), Bb control (F) and MRS control (G). Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 25th week after infection. Spleens were collected to prepare splenocytes, which were cultured under activation of crude Eg antigen (EgAg), with concanavalin A (ConA) or lipopolysaccharide (LPS) stimulation and non-stimulation as control. The splenocyte supernatants were collected to determine the levels of interferon-gamma (IFN-gamma), interleukin-12 (IL-12), tumour necrosis factor-alpha (TNF-alpha) and IL-10 by ELISA. RESULTS: The level of IFN-gamma in groups A [(137.5 +/- 23.2) pg/ml], B [(162.5 +/- 23.2) pg/ml], C [(250.0 +/- 53.5) pg/ml] and D [(215.0 +/- 37.4) pg/ml] was higher than that of group G [(50.0 +/- 10.7) pg/ml] (P < 0.01). The level of IL-12 in groups A [(27.5 +/- 4.6) pg/ml], B [(32.5 +/- 4.6) pg/ml], C [(45.0 +/- 5.4) pg/ml] and D [(35.0 +/- 5.4) pg/ml] was higher than that of group G [(15.0 +/- 5.4) pg/ml] (P < 0.01). The level of TNF-alpha in groups A [(275.0 +/- 46.3) pg/ml], B [(325.0 +/- 46.3) pg/ml], C [(450.0 +/- 53.5) pg/ml] and D [(350.0 +/- 53.5) pg/ml] was higher than that of group G [(150.0 +/- 53.5) pg/ml] (P < 0.01). The level of IL-10 in groups A [(37.5 +/- 4.6) pg/ml], B [(35.0 +/- 5.4) pg/ml], C [(25.0 +/- 5.4) pg/ml] and D [(32.5 +/- 4.6) pg/ml] was lower than that of group G [(45.0 +/- 5.4) pg/ml] (P < 0.05 or P < 0.01). CONCLUSION: The recombinant Bb-Eg95-EgA31 vaccine can induce a protective Th1 type immune response in mice

[Changes of cytokine secretion in mice by immunization with leaf protein extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa].

OBJECTIVE: To investigate the weight reduction of hydatid cyst and the changes of cytokine secretion in mice immunized by leaf protein extracted from Echinococcus granulosus (Eg) Eg95-EgA31 transgenic alfalfa and challenged by Eg protoscoleces. METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32 female BALB/c mice were randomly divided into 4 groups. Groups A and B were immunized intragastrically (100 p1) and intranasally (10 microl) respectively by leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated intranasally by 10 microl. leaf protein with pBI121, group D was given intragastrically 100 microl normal leaf protein. All mice were immunized once per 3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 24th week after infection. The weight of hydatid cysts was measured and weight-reduction rate was calculated. Spleens were collected to prepare splenocytes which were cultured under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS). The supernatant was collected to measure Objective To investigate the weight reduction of hydatid cyst and the changes of cytokine secretion in mice immunized by leaf protein extracted from Echinococcus granulosus (Eg) Eg95-EgA31 transgenic alfalfa and challenged by Eg protoscoleces. METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32 female BALB/c mice were randomly divided into 4 groups. Groups A and B were immunized intragastrically (100 microl) and intranasally (10 microl) respectively by leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated intranasally by 10 microl. leaf protein with pBI121, group D was given intragastrically 100 microl normal leaf protein. All mice were immunized once per 3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 24th week after infection. The weight of hydatid cysts was measured and weight-reduction rate was calculated. Spleens were collected to prepare splenocytes which were cultured under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS). The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA. RESULTS: The average weight of hydatid cysts in groups A, B, C, and D was (28.0 +/- 36.0), (41.0 +/- 33.0), (72.0 +/- 36.0) and (78.0 +/- 57.0) mg, respectively, the cyst weight of group A was lower than that of group D (P < 0.05), decreased by 64.1%. The levels of IFN-gamma, IL-12 and TNF-alpha in group A [(925.0 +/- 88.6), (22.5 +/- 2.7) and (82.5 +/- 11.7) pg/ml] were higher than those of group D (P < 0.01), while the IL-10 level in group A [(125.0 +/- 26.7) pg/ml] was significantly lower than that of group D (P < 0.01). The level of IFN-gamma [(750.0 +/- 100.0) pg/ml] and TNF-alpha [(80.0 +/- 13.1) pg/ml] in group B was significantly higher than those of group D (P < 0.01); but there was no significant difference in the level of IL-12 and IL-10 between the two groups (P > 0.05). No considerable difference in the cytokines was found between group C and group D (P > 0.05). The levels of the 4 cytokines in groups stimulated by EgAg, ConA or LPS were higher than those without stimulation (P < 0.05 or < 0.01), and those in groups stimulated by ConA or LPS were higher than groups stimulated by EgAg (P < 0.05 or < 0.01). CONCLUSION: Th1 response may be induced in mice by immunization with the leaf protein extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa to resist the challenge of Eg protoscoleces.

[Research status on DNA vaccine against Cysticercus cellulosae infection].

DNA vaccine against Cysticercus cellulosae infection is a newly emerging vaccine in recent years. It can induce not only humoral immune response, but also a high level cellular immune response. The DNA vaccine displays prominent advantages in the prevention on cysticercosis. This article reviews the current situation on several DNA vaccines.

[The study on immune responses in BALB/c mice by immunization with the transgenic Alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus].

AIM: To investigate the immune responses and the protection induced by the transgenic Alfalfa (Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus against Eg protoscoleces. METHODS: The leaf protein was extracted from the transgenic alfalfa by heat-coagulation method, its concentration was prepared for 20 g/L. BALB/c mice were immunized intranasally or orally with the leaf protein once per 3 days for 2 months.At the same time, the leaf protein transfected with pBI-121 blank vector and the normal leaf protein without foreign antigen was served as control. The mice were then challenged intraperitoneally with Eg protoscoleces (50 protoscoleces per mouce)on week 8 after the last vaccination and sacrified on week 24 postinfection to count the rate of reduced hydatid cyst. The specific antibody (IgE, IgG and its subclasses)in the sera collected from the eyeballs was evaluated by ELISA. Splenocytes were separated and cultured in vitro with EgAg, ConA or LPS stimulus.The substes of CD4(+); and CD8(+); T cells were measured by FCM. The splenocytes' proliferation was determined by MTT method.Then the cells were collected, stained by PI and Annexin V-FITC, and analyzed by FCM to get the splenocytes' apoptotic rate.The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA. RESULTS: Compared with the control group, The hydatid cyst weight in the oral immunization group decreased by 64.1%; the splenocytes' apoptotic rate got obviously lower than that in the control group; the splenocytes' proliferation increased significantly, the CD4(+); subsets and the ratio of the CD4(+);/CD8(+); did so, and the similar trend about the specific antibody titer and the level of cytokines could be seen in this group. CONCLUSION: Apoptosis of splenocytes may be inhibited in mice by immunization with the transgenic alfalfa, splenocytes' proliferation and Th1 response can be induced in the mice against the challenge of Eg protoscoleces. CD4(+); T cell and the specific antibody(IgG, IgG2b and IgE) may play important roles in the protection induced by the transgenic alfalfa vaccine.

[Inhibition on apoptosis of splenocytes in infected mice by immunization with recombinant Bb-Eg95-EgA31 protein of Echinococcus granulosus].

OBJECTIVE: To investigate the weight reduction of hydatid cysts and apoptosis of splenocytes in infected mice by recombinant Bifidobacteria bifidum (Bb)-Eg95-EgA31 protein of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces. METHODS: 56 female BALB/c mice were randomly divided into 7 groups. Groups A and B were injected subcutaneously and intramuscularly respectively with 5 x 10(6) colony-forming unit (CFU) recombinant Bb-Eg95-EgA31 protein, group C was immunized intranasally by 5 x 10(5) CFU protein, group D was vaccinated transgastrically by 5 x 10(8) CFU protein, groups E and F were injected subcutaneously with 5 x 10(6) CFU blank vector [Bb (pGEX-1 lambda T)] and Bb respectively, and group G was injected subcutaneously with 100 microl MRS. Mice in all groups were challenged with 50 Eg protoscoleces on the 8th week after vaccination and sacrificed on the 25th week after infection. The weight of hydatid cysts was measured and weight-reduction rate was calculated. Spleens were collected to prepare splenocytes which were cultured under stimulation with concanavalin A (ConA). The apoptotic rate was determined by flow cytometry (FCM). RESULTS: The average weight of hydatid cysts in groups A [(41.0 +/- 23.0) mg], B [(44.0 +/- 22.0) mg], C [(22.0 +/- 21.0) mg], and D [(28.0 +/- 16.0) mg] was lower than that of group G [(75.0 +/- 33.0) mg] (P<0.05, P<0.01), and there was no significant difference among groups A, B, C and D (P>0.05); no significant difference was found between group G and groups E [(63.0 +/- 30.0) mg], F [(69.0 +/- 22.0) mg] (P>0.05). The apoptotic rate of splenocytes cultured with no ConA in groups A (0.14 +/- 0.01), B (0.14 +/- 0.01), C (0.13 +/- 0.01), and D (0.141 +/- 0.01) was lower than that of group G (0.21 +/- 0.01) (P<0.05); that of group C was lower than groups A, B, and D (P<0.05); there was no significant difference between groups D and A, between groups A and B, and between groups E (0.20 +/- 0.01), F (0.20 +/- 0.01) and group G. The apoptotic rate of splenocytes cultured with ConA in group A (0.19 +/- 0.01), B (0.20 +/- 0.00), C (0.17 +/- 0.01), and D (0.19 +/- 0.01) were lower than that of group G (0.26 +/- 0.01) (P<0.01), that of group C was lower than groups A and B (P<0.01), group C was lower than group D (P<0.05), group D was lower than group B (P<0.05); there was no significant difference between groups A and B, and between groups A and D, and between groups E (0.25 +/- 0.01), F (0.25 +/- 0.01), and group G (P>0.05). The apoptotic rate of splenocytes cultured with ConA was higher than those cultured without ConA (P<0.01). CONCLUSIONS: Apoptosis of splenocytes may be induced by infection of Echinococcus granulosus protoscoleces in mice, while the recombinant Bb-Eg95-EgA31 protein may inhibit the apoptosis of splenocytes in mice challenged with Eg, and induce certain protective immunity in the host.

[Dynamic observation on immune responses in mice administrated with a recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus].

AIM: To dynamically observe the immune responses in mice administrated with a recombinant Bifidobacteria bifidum (Bb)-Eg95-EgA31 vaccine of Echinococcus granulosus. METHODS: BALB/c mice were immunized orally or intranasally by the vaccine. At indicated times after vaccination, the levels of serum IgG, IgG subclass and IgE were determined by ELISA. The levels of IL-12, IFN-gamma, TNF-alpha and IL-10 were measured by ELISA in the supernatant of cultured splenocytes. Proliferation of splenocytes was tested by MTT. The percentage of spleen CD4+ and CD8+ cells was determined by flow cytometry(FCM). RESULTS: The orally immunized mice achieved IgG and IgG3 peaks on 8th week post vaccination, IgG2a peak on 2nd week, IgG2b and IgG1 peaks on 6th week, and IgE peak on 10th week. IFN-gamma and TNF-alpha reached highest level on 4th week, IL-12 on 2nd week, and IL-10 on 6th week. Splenocytes proliferated fastest on 6th week. The percentage of CD4+ cells was peaked on 6th week, while the percentage of CD8+ cells had no obvious change. The intranasally immunized group achieved peaks of IgG, IgG2b and IgE on 10th week, IgG2a peak on 6th week, and peaks of IgG1 and IgG3 on 8th week. IFN-gamma and IL-12 reached highest level on 2nd week, TNF-alpha on 4th week, and IL-10 on 8th week. Splenocytes proliferated fastest on 6th week. The percentage of CD4+ cells was peaked on 6th week, while the percentage of CD8+ cells had no obvious change. CONCLUSION: The recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus induced effective immune responses in mice by either oral vaccination or intranasal inoculation. Our data showed the better immunogenicity of oral vaccination.

[Construction and expression of recombinant plasmid pET28alpha-Sj26GST of Schistosoma japonicum in Escherichia coli BL21].

OBJECTIVE: To construct and express recombinant plasmid pET28a-Sj26GST of Schistosoma japonicum (Sj) in Escherichia coli BL21 (DE3). METHODS: The total RNA was extracted from Sj adult worms by ultrasound-breaking. The Sj26GST antigen gene was amplified by RT-PCR from the total RNA, and then cloned into prokaryotic expression plasmid pET28alpha and transformed into E. coli BL2 (DE3). The BL21(pET28a-Sj26GST) was induced with isopropyl-beta-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified with SDS-PAGE and Western blot. RESULTS: The 676 bp Sj26GST gene was successfully amplified by RT-PCR and cloned into pET28alpha. The recombinant plasmid pET28a-Sj26GST was successfully constructed, with a relative molecular weight of expressed recombinant protein at approximately 36 x 10(3) as determined by SDS-PAGE. The amount of the expressed protein comprised 26% of the total bacterial proteins. The fusion protein could be recognized by the sera of rabbits infected with Sj. CONCLUSION: The recombinant plasmid pET28alpha-Sj26GST is successfully constructed and highly expressed in E. coli in a fused form with His-tag. The expressed fusion protein shows specific antigenicity.

[Dynamic observation on splenocyte subsets in mice immunized with recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus].

OBJECTIVE: To dynamically observe changes of subsets of splenocytes in mice immunized with recombinant Bifidobacteria bifidum (Bb)-Eg95-EgA31 vaccine of Echinococcus granulosus (Eg). METHODS: BALB/c mice were vaccinated by 5 x 10(8) colony forming unit (CFU) orally and 5 x 10(5) CFU intranasally respectively. Mice were killed on week 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after immunization respectively, and spleens were separated for cell preparation. CD4+ and CD8+ T cells were determined by flow cytometry (FCM), with MRS as control. RESULTS: In the oral immunization group, CD4+ cells showed a significant increase during the 4th-10th week after vaccination, and reached the highest level on the 6th week, whereas no obvious changes in CD8+ cells numbers were observed. In the intranasal immunization group, CD4+ cells showed an obvious increase during the 4th-8th week after vaccination, and reached the highest level on the 6th week, CD8+ subsets had no obvious changes. CONCLUSION: CD4+ T cell cells may play a key role in immune response in mice immunized with the recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus.

[Research progress in antigens for the diagnosis of alveolar echinococcosis].

Specific antigens of Echinococcus multilocularis is essential for the diagnosis of alveolar echinococcosis and vaccine development. Because of the limited source of nature antigen, its application is restricted. The development of recombinant antigens can provide large amount of antigen under effective quality control. This review summarizes the recent progress in antigen research, especially the recombinant antigen used for diagnosis of the disease.

[Changes of cytokines in mice immunized with recombinant Bb-EmII/3-Em14-3-3 vaccine of Echinococcus multilocularis].

AIM: To study the effects of recombinant Bb-EmII/3-Em14-3-3 vaccine on cytokine secretion in mice challenged with protoscoleces of Echinococcus multilocularis. METHODS: BALB/c mice were immunized with rBb-EmII/3-Em14-3-3 vaccine by subcutaneous injection, intramuscular injection, nasal mucosa inoculation or oral administration. After 12 weeks of immunization,all the mice were challenged with 50 protoscoleces of Echinococcus multilocularis by intraperitoneal injection. 18 weeks later, the mice were sacrificed and the splenocytes were stimulated with EmAg and ConA or LPS in vitro, then IL-12, IL-10, IFN-gamma and TNF-alpha in the culture supernatant were measured by ELISA. RESULTS: The level of IFN-gamma, IL-12 and TNF-alpha were greatly higher than that of PBS control group (P<0.05 or P<0.01), and the level of IL-10 was lower. The level of IFN-gamma, IL-12, TNF-alpha and IL-10 in each group with EmAg and ConA or LPS stimulation was greatly higher than that in the group without stimulation(P<0.05 or P<0.01). CONCLUSION: Th1 response is induced in mice challenged with Echinococcus multilocularis by rBb-EmII/3-Em14-3-3 vaccine, and the vaccine may enhance protective response against the challenge with protoscoleces of Echinococcus multilocularis.

[Cytokines of splenocytes in mice by different vaccination with recombinant BCG-Em14-3-3 vaccines against Echinococcus multilocularis].

OBJECTIVE: To detect the changes of cytokines of splenocytes in mice immunized by recombinant BCG-Em14-3-3 vaccines against Echinococcus multilocularis (Em). METHODS: BALB/c mice were subcutaneously and intranasally vaccinated by the BCG-Em14-3-3 vaccines respectively, with PBS as a control. The mice were challenged with Em protoscoleces eight weeks after the vaccinations. The spleens of the mice were collected 18 weeks after the infections. The splenocytes were separated and cultured with the stimulation of EmAg or ConA. The IL-2, IFN-gamma, TNF-alpha and IL-4 in the supernatants were measured with ELISA. RESULTS: Significant increase of IFN-gamma and TNF-alpha were found in the immunized mice. But the IL-4 was decreased after the vaccinations. The levels of TNF-alpha were higher in the intranasal vaccinated mice than in the subcutaneous vaccinated mice. CONCLUSIONS: TH1 response is induced in mice by rBCG-Em14-3-3 vaccines to fight against the challenge of Em protoscoleces. Intranasal vaccinations are preferable.

[Changes of cytokines of splenocytes in mice immunized by mix recombinant BCG-EmII/3 and BCG-Em14-3-3 vaccine of Em].

AIM: To investigate the changes of cytokines of splenocytes in mice immunized by mix recombinant BCG-EmII/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em) and challenged by Em protoscoleces. METHODS: BALB/c mice were vaccinated subcutaneously and intranasally by the vaccine respectively. In the eighth week of vaccination they were Challenged by Em protoscoleces.In the eighteenth week of infection they were killed for spleen. After splenocytes were separated,their culture was stimulated by EmAg or ConA and their supernatants were gathered. The levels of IL-2, IFN-gamma, TNF-alpha and IL-4 were measured by ELISA kit. Blank vector, BCG and PBS were served as control. RESULTS: In the groups of vaccine immunization, the levels of IFN-gamma and TNF-alpha increased obviously but the level of IL-4 decreased. The level of TNF-alpha in subcutaneous group was higher than that in intranasal group. CONCLUSION: Th1 response was induced in mice immunized by mix recombinant BCG-EmII/3 and BCG-Em14-3-3 vaccine of Em against the challenge by Em protoscoleces. Subcutaneous injection with this vaccine may be a good way of vaccination.

[Research progress in immunopathogenesis of cystic echinococcosis].

This article reviews the immunopathogenesis of cystic echinococcosis in the following seven aspects: innate immunity, establishment phase immunity, cystic phase immunity, influencing factor of CD4+ T cell polarization, cytokine function in infected host, Echinococcus granulosus infection and allergy, and immune evasion mechanism.

[Change of splenocyte lymphokines in mice induced by recombinant BCG-Eg95 vaccine against Echinococcus granulosus].

OBJECTIVE: To investigate the reduction of hydatid cyst weight and change of splenocyte lymphokines in mice immunized with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg). METHODS: BALB/C mice were subcutaneously, intranasally, orally and intramuscularly vaccinated respectively, with BCG and PBS served as controls. The mice were challenged with Eg protoscolices 8 weeks after vaccination and sacrificed in 18 weeks after infection. The weight of hydatid cyst was measured and reduction of the weight was obtained, spleens were used to separate splenocytes which were cultured under stimulation with EgAg or ConA. The supernatant was collected to measure the level of IL-2, IFN-gamma, TNF-alpha and IL-4 by ELISA. RESULTS: The hydatid cyst weight reduced by 45.77%, 18.20%, 88.05% and 92.46% respectively in the 4 immunization groups. In comparison with PBS control, the level of IL-2, IFN-gamma and TNF-alpha was (30.0+/-0) pg/ml, (65.0+/-0) pg/ml and (425.0+/-10.7) pg/ml respectively in the intramuscular group with a significant increase, but that of IL-4 decreased, with a value of (10.0+/-0) pg/ml. CONCLUSION: The recombinant BCG-Eg95 vaccination induces Thl response in mice against challenge infection.