Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.

Objective To investigate the effects of recombinant vaccine Bifidobacterium bifidum (Bb) pGEX-Sj14-3-3 on splenocyte apoptosis in BALB/c mice.Methods Ninety-six BALB/c mice were randomly divided into two groups according to their body mass:per os group (PO) and intranasal immunization group (IN),with 48 mice in each group.All mice were orally and intranasally immunized with recombinant vaccine Bb(pGEX-Sj14-3-3).Four mice in each group were sacrificed on weeks 0,2,4,6,8,10,12,14,16,18,20 and 22,respectively,after immunization,and splenocytes were separated and cultured with or without ConA stimulation.The apoptotic rates of splenocytes were detected by flow cytometry.Results It showed that apoptotic level of splenocytes in both groups remarkably increased after 2-4 weeks without ConA stimulation (PO:0.069 ± 0.005,0.076 ± 0.010; IN:0.037 ± 0.002,0.075 ± 0.002),and the value reached the peak on the 4th week,and the differences were statistically significant compared with that of week 0(all P ＜ 0.05).Apoptotic level of splenocytes in both groups with ConA stimulation increased after 2-6 weeks(PO:0.089± 0.006,0.098 ± 0.010,0.060±0.007; IN:0.054 ± 0.001,0.093 ± 0.003,0.058 ± 0.012),and the value also reached the peak after 4 week,respectively.The differences were statistically significant compared with that of week 0 (all P ＜ 0.05).Apoptotic level of splenocytes in each group with ConA stimulation was significantly higher than that without ConA stimulation.Conclusion It is suspected that the recombinant vaccine Bb(pGEX-Sj14-3-3) may inhibit apoptosis of splenocytes in mice immunized orally or intranasally.

ObjectiveTo study the dynamic changes of IgG,IgG subclasses,IgE and IgA in sera of BALB/c mice immunized with recombinant Bifidobacterium bifidum (Bb) pGEX-Sj14-3-3 vaccine of Schistosoma japonicum.MethodsNinty six BALB/c mice were randomly divided into two groups:oral immunization group and intranasal immunization group,48 mice in each group.Mice were orally and intranasally immunized with recombinant Bb(pGEX-Sj14-3-3) vaccine,respectively.Four mice from each group were sacrificed,respectively,on weeks 0,2,4,6,8,10,12,14,16,18,20 and 22 after immunization and their sera from the eyeballs were collected.The levels of IgG,IgG subclasses,IgE and IgA were assayed with routine Enzyme-linked immunosorbent assay(ELISA).ResultsThe titers of IgG,IgG1,IgG2a,IgG2b,IgG3,IgE and IgA in both groups increased during the 2 - 22th,2 - 14th,2 - 22th,2 - 22th,2 - 20th,2 - 22th,2 - 22th weeks,respectively.The values reached the highest level on weeks 8,6,6,4,8,10 and 6,respectively,in the oral group,and the values were (0.065 ±0.001,0.021 ± 0.002,0.011 ± 0.001,0.015 ± 0.003,0.014 ± 0.002,0.011 ± 0.001,0.013 ± 0.002),respectively,as compared with the values on week 0(0.032 ± 0.001,0.015 ± 0.002,0.005 ± 0.002,0.005 ± 0.001,0.006 ± 0.001,0.006 ± 0.001,0.005 ± 0.001 ),the differences were statistically significant(P ＜ 0.05 or ＜ 0.01 ) except that of IgG1 and IgG2b.In the intranasal group these values reached the highest levels on weeks 4,6,4,4,8,10 and 8,respectively,and the values were (0.064± 0.003,0.022 ± 0.002,0.012 ± 0.003,0.019 ± 0.001,0.013 ± 0.001,0.015 ± 0.001,0.014 ± 0.003),respectively,as compared with the value on week 0,the differences were statistically significant(P ＜ 0.05 or ＜ 0.01 ) except that of IgG1 and IgA.ConclusionsTypes Th1 and Th2 mixed type immune responses can be induced in mice by immunization with the recombinant Bb(pGEX-Sj14-3-3) vaccine by early period of immunization (2th - 10th week).

Objective To construct and identify recombinant vaccine Bifwlobacterium bifidum(Bb)pGEX-Sj14-3-3 of Schistosoma japonicum(Sj). Methods Total RNA was extracted from adult Sj, antigen encoding gene Sj14-3-3 was amplified by RT-PCR and cloned into Escherichia coli (E. coli)-Bb shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj14-3-3. The recombinant plasmid was transformed into E. coli BL21 (DE3).The plasmid was extracted and identified by using BamH I and EcoR I. Then pGEX-Sjl4-3-3 was electroporated into Bb to construct recombinant Bb (pGEX-Sj14-3-3) vaccine. The extracted plasmid of the recombinant Bb (pGEX-Sj14-3-3) vaccine was identified by PCR, and the size of the products was compared with Sj14-3-3 gene of adult worms.Results Sj14-3-3 of 399 bp in length was amplified by RT-PCR. The products were digested by BamH I and EcoR I , and the fragments length of plasmid pGEX-Sj14-3-3 vector was 4947 bp, and of Sj 14-3-3 gene was 399 bp.The product of 399 bp Sj14-3-3 gene was also amplified by PCR from template of the extracted plasmid of the recombinant Bb(pGEX-Sj14-3-3 ) vaccine. The size of the product obtained was just the same as expected.Conclusion The recombinant Bb(pGEX-Sj14-3-3) vaccine of Sj is successfully constructed.

Objective To investigate the protective immunity in mice immunized with recombinantBifidobacteria bifidum(Bb)-Eg95 vaccine of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces.Methods Fifty-six female BALB/c mice 12-14 weeks old and weighed 20-25 g were vaccinated with the recombinant Bb-Eg95 vaccine subcutaneously,intramuscularly,intranasally and orally,respectively,with blank vector,Bb and medium of solution(MRS) as control,8 mice in each group.Mice were challenged with Eg protoscoleces on week 8 after immunization and killed on week 25 after infection.The weight of hydatid cyst was measured and the decreased larva rate was calculated.Sera were collected to determine the levels of IgE,IgG and its subclasses by enzyme linked immunosorbent assay(ELISA).Splenocytes were collected and cultivated to test the proliferation of splenocytes using methyltetrazolium (MTT) assay under EgAg and concanavalin A (ConA) stimulation.The results were compared with analysis of variance and the comparison between two groups was performed with LSD-t test.Results There was significant difference in the weight of hydatid cyst between groups (F =11.062,P ＜ 0.05).Compared with MRS control group[(0.075 ± 0.019)g],the hydatid cyst weight decreased in subcutaneous group [(0.050 ± 0.013)g],intramuscular group[(0.050 ± 0.019)g],intranasal group[(0.028 ± 0.016)g] and oral group [(0.031 ± 0.018)g,all P ＜ 0.01).Compared with subcutaneous and intramuscular groups,the hydatid cyst weight decreased in intranasal and oral groups(all P ＜ 0.05).The decreased larva rate was inversely proportional to the weight of hydatid cyst.There was significant difference in the levels(obsorbancy,A) of IgG,IgG2a,IgG2b,IgG1,IgG3 and IgE between these groups(F =21.774,36.977,27.071,14.746,10.131,9.444,P ＜ 0.05 or P ＜ 0.01).Compared with MRS control group (0.015 ± 0.002,0.002 ± 0.001,0.003 ± 0.001),the levels of IgG,IgG2a and IgG2b increased in subcutaneous group(0.022 ± 0.004,0.007 ± 0.002,0.008 ± 0.002),intramuscular group (0.023 ± 0.003,0.008 ± 0.002,0.007 ± 0.002),intranasal group(0.032 ± 0.007,0.012 ± 0.002,0.013 ± 0.004)and oral group(0.028 ± 0.006,0.010 ± 0.003,0.010 ± 0.002,P ＜ 0.05 or P ＜ 0.01).Compared with subcutaneous and intramuscular groups,the levels of IgG,IgG2a and IgG2b increased in intranasal and oral groups(P ＜ 0.05 or P ＜ 0.01).Compared with MRS control group(0.009 ± 0.001,0.009 ± 0.002,0.009 ± 0.001),the levels of IgG1,IgG3 and IgE decreased in subcutaneous group(0.022 ± 0.004,0.007 ± 0.002,0.008 0.002),intramuscular group(0.004 ± 0.001,0.004 ± 0.001,0.004 ± 0.002),intranasal group(0.005 ± 0.002,0.005 ± 0.003,0.005 ± 0.002)and oral group(0.005 ± 0.001,0.004 ± 0.002,0.004 ± 0.003,all P ＜ 0.01).There was significant difference in the proliferation of splenocytes in the supernatant of cultured splenocyte,of cultured splenocyte + EgAg and of cultured splenocyte + ConA(F =63.975,359.833,167.399,P ＜ 0.01).There was significant difference in the proliferation of splenocytes inside groups(F =6741.955,4953.667,869.320,201.235,175.413,139.653,169.994,all P ＜0.01).Compared with the cultured splenocyte the proliferation of splenocytes increased in the cultured splenocyte +EgAg and splenocyte + ConA (all P ＜ 0.01).Compared with the cultured splenocyte + EgAg,the proliferation of splenocytes increased in the cultured splenocyte + ConA(P ＜ 0.01).Conclusion An effective and protective immunity is induced by the recombinant Bb-Eg95 vaccine of Eg in mice.

Objective To investigate the dynamic changes of splenocyte cytokines in mice immunized with the transgenic alfalfa containing Eg95-EgA31 fusion gene of Echinococcus granulosus (Eg). Methods Eighty-eight Balb/c mice were divided into 2 groups randomly according to body weights,and immunized orally or intranasally with 100μl or 10μl extracted leaf protein from the transgenic alfalfa(20 g/L) respectively once per 3 days for 2 months. Four mice randomized from each group were killed to get splenocyte on week 0(control),2,4,6,8,10,12,14,16,18 and 20 after the last immunization. The splenocyte were cultured in medium for 48 hours with EgAg or concanavalin A (ConA) stimulation to induce the interleukin (IL)-12,interferon γ(IFN-γ) and IL-10,and cultured for 72 hours with EgAg or lipopolysaccharide (LPS) stimulus to induce the tumor necrosis factor α (TNF-α). Then the supernatant was collected to measure the level of IL-12,IFN-γ,TNF-α and IL-10 by ELISA. Results In the oral immunization group,the level of IL-12,IFN-γ,TNF-α and IL-10 increased significantly from week 4 to week 6,week 2 to week 8,week 2 to week 6 and week 4 to week 12,respectively,reaching the highest level(25.0±5.8)ng/L on week 4,(575.0±28.9)ng/L on week 2,(50.0±11.5)ng/L on week 2 and (42.5±2.9)ng/L on week 8,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]; in the intranasal immunization group,it was similar about the values of IL-12,IFN-γ,TNF-α and IL-10 could be seen from week 4 to week 6,week 2 to week 10,week 4 to week 10 and week 6 to week 16,respectively,reaching the highest level(25.0±5.8)ng/L on week 6,(725.0±28.9)ng/L on week 4,(27.5±2.9)ng/L on week 6 and (60.0±11.5)ng/L on week 6,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]. The cytokine levels in the groups with EgAg,ConA or LPS stimulus were significantly higher than those in the corresponding splenocyte suspension groups(P < 0.05 or < 0.01),and the cytokine levels in the groups with ConA or LPS stimulus were obviously higher than those in the corresponding groups with EgAg stimulation (P < 0.05 or < 0.01). Conclusion The mixed responses of Th1 and Th2 types can be induced in mice immunized with the transgenic alfalfa in the early period post immunization(2-10 weeks).

Objective　To study the effects of recombinant BCG-Sj26GST vaccine of schistosoma japonicum of TNF-α and IL-1 produced by spleen cells of mice.Methods　In experiment 1,BALB/C mice were immnized subcutaneously by 106 and 108 CFU vaccine respectively,challenged with Sj cercariae on 8 w of immunization and killed on 6 w of infection to separate spleens,PBS served as control;in experiment 2,after immunized subcutaneously and intravenously by 106 CFU vaccine respectively,4 mice were killed to separate sleens on 0,4,8,10,14 and 16 w of immunization,spleen cells were stimulated by Sj26 or mitogens,the level of TNF-α in supernatant of spleen cells were detected by ELISA,that of IL-1 by fibroblast proliferative response.Results　Levels of TNF-α and IL-1 increased obviously by immunization with the vaccine against challenge with Sj cercariae;dynamic observation showed that TNF-α and IL-1 in the subcutaneous group reached the highest level on 4～8 w;that in the intravenous group on 16 w and 4～8 w respectively.Conclusions　Recombinant BCG-Sj26GST vaccine of schistosoma japonicum may promote spleen cell of mice to secrete TNF-α and IL-1 early,these cytokines may play an important role in protective immunity against schistosomiasis.

Objective To investigate the change of splenocyte subsets in Balb/c mice immunized with transgenic alfalfa(Medicago sativa)containing Eg95-EgA31 fusion gene of Echinococcus granulosus(Eg) and challenged with Eg protoscoleces.Methods Leaf protein was extracted from transgenic alfalfa containing Eg95-EgA31 fusion gene by heat-coagulation method,and concentration of 20 g/L was used in the study.Meanwhile,leaf protein extracted from the transgenic alfalfa containing blank vector(pBI121)and the normal alfalfa was served as control.Thirty-two female Balb/c mice were randomly divided into 4 groups,8 mice in each group.Oral group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intragastrically(100μl per mouse);intranasal group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intranasally(10 μl per mouse);blank vector group was vaccinated intranasally with 10μl leaf protein with blank vector(pBI121);and normal control group was given 100μl normal leaf protein intragastrically.All mice in the above mentioned groups were immunized every 3 days for 2 months.Then,the mice were challenged intraperitoneally with Eg protoscoleces(50 protoscoleces per mouse)8 weeks after last vaccination and sacrified 24 weeks pest infection to separate the splenocytes.Flow cytometry was used to measure the percentages of CD4+ and CD8+ T ceils subsets.Resuits Compared with the normal control group(0.166±0.018,0.083±0.006,2.019 ±0.369),the percentages of CD4+(0.286±0.009)and CD8+(0.102±0.004)T cell subsets and the ratio of CD4+/CD8+(2.814±0.014)in oral group increased significantly (P<0.01 or<0.05).The percentage of CD4+ subset(0.269±0.016)and the ratio of CD4+/CD8+(2.955±0.986) in intranasal group was significantly higher than that ofthe normal control group(all P<0.01).The percentage of CD4+ subset in oral group was significantly higher than that of the intranasal group(P<0.05).No significant difference was found in the percentages of CD4+ and CD8+ T cell subsets and the ratio of CD4+/CD8+ between the blank vector group(0.169±0.018,0.093±0.019,1.852±0.188)and the normal control group(all P>0.05).Conclusions CD4+ T cell may play an important role in the protection induced by transgenic alfalfa vaccine against the challenge of Eg protoscoleces.Intragastrical immunization may be a good route.

Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj) in Escherichia coli(E.coli) BL21 (DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalton glutathione-S-transferases of Schistosomajaponicum (Sj26GST) antigen gene was amplified by real-time PCR(RT-PCR) from the total RNA,then cloned into a prokaryotic expression plasmid pGEX1λT and transformed into E.coli BL21 (DE3) to construct pGEX-Sj26GST; BL21 (pGEX-Sj26GST) was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG),and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting.Results The 676 bp Sj26GST gene was successfully amplified by RT-PCR and restriction enzyme double-digestion technique confirmed that Sj26GST antigen gene was successfully cloned into pGEX-1λT vector,the relative molecular mass of the expressed recombinant protein was approximately 52 × 103 by SDS-PAGE,and the amount of expressed protein was 20％ of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pGEX-Sj26GST is successfully constructed and highly expressed in E.coli and the expressed fusion protein shows specific antigenicity.

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