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Cystic echinococcosis (CE), caused by the larva of Echinococcus granulosus (Eg), is one of the key parasitic diseases to prevent and control in China. The Eg95 protein is a recognized vaccine candidate molecule, and the development of vector-mediated Eg95 vaccine is one of the current research hotspots in CE. This article summarizes the development status of Eg95 vaccine such as vaccinia virus, orf virus, goatpox virus, rabies virus, Salmonella typhimurium, Bacille Calmette-Guerin, Agrobacterium tumefaciens and Bifidobacteria bifidum, in order to provide valuable information for the immune prevention of CE.
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Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.
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Eimeria is the pathogen of chicken coccidiosis. Vaccine control is one of the hotspots of current study. Its surface antigen is an effective vaccine candidate molecule. This paper summarizes the development status of Eimeria surface antigen vaccine mediated by Bacille Calmettle-Guerinl (BCG), Lactococcus lactis (LL), Lactobacillus plantarum (LP), Salmonella typhimurium (St), Bacillus subtilis (Bs), Enterococcus faecalis (Efs), Escherichia coli (Ec), Cyanobacterium (Cb), Pichia, Leishmania tarentolae (Lt), viruses such as Fowlpox virus (FPV), Herpesvirus of turkey (HVT), Vaccinia virus (VV) and insect Buculovirus, in order to provide reference basis for development and application of new vaccines.
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Objective To construct a recombinant Bifidobacterium bifidum (Bb) vaccine of Pseudomonas aeruginosa,identify its protective effect in mice after immunization with the vaccine and challenged by Pseudomonas aeruginosa.Methods The OCR-amplified outer membrane protein OprH gene was cloned into vector pGEX-1λT to obtain the plasmid pGEX-OprH.The pGEX-OprH was then electroporated into Bb to obtain the rBb-pGEX-OprH vaccine.After PCR identification,the expression of rBb-pGEX-OprH was induced by Isopropylβ-D-Thiogalactoside (IPTG),and the expression products were analyzed by SDS-PAGE and characterized by Western blotting;21 BALB/c mice were subdivided by random number table into vaccine group,empty vector group and Bb group (7 mice in each group),and immunized by intragastrically administration with rBb-pGEX-OprH,rBb-pGEX-1λT or Bb,respectively.Mice were immunized once a day and three times a week for three consecutive weeks,then the mice were challenged intranasally with Pseudomonas aeruginosa at four weeks after the first immunization,once a day for three consecutive days.Two weeks after the challenge,the mice were sacrificed to detect their lung bacterial loads.Sera were collected before vaccination,four weeks after the first immunization,and two weeks after the challenge.The sera were analyzed by enzyme-linked immunosorbent assay (ELISA) to detect the level of IgG.Results OprH gene of 660 bp was amplified by PCR;PCR results showed that the rBb-pGEX-OprH was constructed successfully;SDS-PAGE confirmed that the recombinant vaccine expressed a relative molecular mass 47 × 103 fusion protein,which was consistent with the expected result;Western blot results showed that the expressed protein could be identified by sera in mice infected with Pseudomonas aeruginosa.Differences of lung bacterial colonies between the three groups were statistically significant (F =1 506.793,P ＜ 0.05).The number of bacterial colonies in the vaccine group [(7.691 ± 0.069) lgCFU/g] was lower than that of the empty vector group [(8.855 ± 0.027) lgCFU/g] or the Bb group [(8.958 ± 0.037) lgCFU/g,P ＜ 0.05].There were statistically significant differences in serum IgG levels between the 3 groups 4 weeks after the first immunization (F =77.216,P ＜ 0.05),the IgG level of the vaccine group (0.078 ± 0.005) was significantly higher than that of the empty vector group (0.054 ± 0.004) and the Bb group (0.052 ± 0.004,P ＜ 0.05).Conclusion The rBb-pGEX-OprH vaccine is constructed successfully,and it could induce certain protective effect in mice.
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Vivax malaria caused by Plasmodium vivax (Pv) is one type of parasitic diseases seriously endangering human health,it recently becomes a hotspot to control this parasite by use of the vaccine.Pv merozoite surface protein 1,Pv circumsporozoite protein,Pv apical membrane antigen 1 and Pvs25 antigens are many types of candidate vaccine molecules.This review outlines the status in the research of vaccine of Plasmodium vivax mediated by bacteria such as Mycobacteria smegmatis,Agrobacterium tumefacies,and Pichiapastoris,and by viruses such as Adenovirus and Baculovirus.
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Objective To construct a recombinant Bifidobacterium bifidum (Bb)vaccine[Bb (pGEX-Sj26GST-Sj14-3-3)] of Schistosomajaponicum (Sj) and analyze the expression of the fusion gene Sj26GST-Sj14-3-3 of Sj in Bb.Methods The recombinant plasmid pGEX-Sj26GST-Sj14-3-3 was electroporated into Bb to construct a recombinant Bb (pGEX-Sj26GST-Sj14-3-3) vaccine.Mter induction with isopropyl-β-D-thiogalactoside (IPTG),double restriction enzymes digestion and polymerase chain reaction (PCR) were used to identify the recombinant Bb (pGEX-Sj26GST-Sj14-3-3),expression of the recombinant protein was analyzed and identified by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Results The recombinant plasmid pGEX-Sj26GST-Sj14-3-3 was successfully transformed into Bb identified by double restriction enzymes digestion and PCR.SDS-PAGE analysis showed that the relative molecular mass of the expressed recombinant protein was approximately 67 × 103.The expressed protein could be recognized by the immune sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant Bb (pGEX-Sj26GST-Sj14-3-3) vaccine of Sj is successfully constructed.The fusion gene Sj26GST-Sj14-3-3 can be expressed in recombinant Bb and the expressed target protein shows specific antigenicity.
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Toxoplasmosis caused by Toxoplasma gondii is one type of zoonotic, parasitic diseases seriously endangering human health. Vaccine recently becomes the highlight in control of this parasite. ROPs antigen is an effective candidate molecule of vaccine. This review outlines the status in the research of ROPs vaccine of Toxoplasma gondii mediated by bacteria such as Lactococcus lactis, Mycobacteria smegmatis, Bacille calmette-Guerin, Agrobacterium tumefaciens and Pichia Pastoris, and viruses such as Canine adenovirus type 2, Adenovirus serotype 5, Feline herpesvirus type-1 and Vaccinia virus.
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Objective In order to investigate the changes of T lymphocytes subsets in mice immunized with recombinant Bb-Em Ⅱ/3-Em14-3-3.vaccine of Echinococcus multilocularis (Em) and challenged with Em protoscoleces.Methods BALB/c mice were immunized with recombinant Bb-Em Ⅱ/3-Em14-3-3 vaccine by subcutaneous injection,intramuscular injection,nasal mucosa inoculation and oral administration,Bifidobacterium (Bb) and PBS were used as controls.After 12 weeks of immunization,all the mice were challenged with 50 protoscoleces of Em by intraperitoneal injection.Eighteen weeks later,mice were killed to measure alveolar hydatid weight and spleens were taken to separate spenocytes,in which the percentages of CD4+ and CD8+ T cell subsets were determined by flow cytometry.Results Alveolar hydatid weight was (0.77 ± 0.52),(0.87 ± 0.60),(2.17 ± 0.50),(3.06 ± 0.15) g in subcutaneous injection,intramuscular injection,nasal mucosa inoculation and oral administration groups,respectively,which was obviously lower than that in PBS control group [(3.54 ± 0.32) g,P ＜ 0.05 or ＜ 0.01].The level of CD49 T cell subset was (28.2 ± 2.5)％,(25.0 ± 2.7)％,(24.0 ± 1.3)％,(23.0 ± 1.8)％ in subcutaneous injection,intramuscular injection,nasal mucosa inoculation and oral administration groups,respectively,which was significantly higher than that in PBS control group [(16.1 ± 2.2)％,all P ＜ 0.01].The level of CD8+ T cell subset in the immunization groups was slightly elevated,but there was no statistical significance between groups (F =1.36,P ＞0.05).The level of CD4 + T cell subset in subcutaneous injection group was higher than that in intramuscular injection,nasal mucosa inoculation and oral administration groups (all P ＜ 0.05).Conclusion CD4+ T cell subset may play an important role in the protection of mice induced by recombinant Bb-Em Ⅱ/3-Em14-3-3 vaccine and the subcutaneous injection route appears to be a better way for immunization.
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Objective To construct and identify recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosomajaponicum (Sj).Methods The Sj32 gene amplified by PCR from template of plasmid pET28α-Sj32 that extracted from recombinant Escherichia coli(E.coli) BL21 (pET28α-Sj32) stored in our laboratory was cloned into E.coli-Bifidobacterium bifidum shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj32.The recombinant plasmid was transformed into E.coli BL21 (DE3).The plasmid was extracted and identified by restriction enzyme digestion.The recombinant Bifibacterium bifidum (pGEX-Sj32) vaccine was constructed by electroporating pGEX-Sj32 into Bb.The extracted plasmid was amplified and identified by PCR.Results The gene Sj32 of 1 270 bp in length was amplified by PCR.The restriction endonuclease digestion showed that the length of plasmid vector was 4 947 bp,and Sj32 gene was 1 270 bp.The Sj32 gene amplified by PCR from the template of pGEX-Sj32 extracted from recombinant Bb (pGEX-Sj32) vaccine was 1 270 bp in length which consistent with expected result.Conclusion The recombinant Bb (pGEX-Sj32) vaccine of Sj is successfully constructed.
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Objective To construct and express a recombinant plasmid pGEX-Sj 14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli (E.Coli) BL21 (DE3).Methods Sj14-3-3 and Sj32 antigen genes were amplified by PCR from template of plasmids pGEX-Sj14-3-3 and pET28α-Sj32 which were extracted from recombinant bacteria BL21 (pET28α-Sj32) and BL21 (pGEX-Sj14-3-3) stored in Institute of Infectious and Parasitic Disease of the First Affiliated Hospital of Chongqing Medical University.Sj14-3-3-Sj32 fusion gene obtained with gene SOEing was cloned into the vector pGEX-1λT to construct pGEX-Sj14-3-3-Sj32 which was identified by double digestion.The recombinant plasmid pGEX-Sj 14-3-3-Sj32 was transformed into E.Coli BL21 (DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside (IPTG),and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Results The 1 750 bp Sj14-3-3-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into the vector pGEX-1 λT verified by restriction analysis,the recombinant plasmid pGEX-Sj 14-3-3-Sj32 was successfully constructed.The molecular mass of the expressed recombinant protein was proximately 73 × 103 as detected by SDS-PAGE.Western blotting confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosomajaponicum.Conclusion The recombinant plasmid pGEX-Sj14-3-3-Sj32 is successfully constructed and could be highly expressed in E.coli and the expressed recombinant protein has specific antigenicity.
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<p><b>OBJECTIVE</b>To observe the dynamic changes of immune responses of splenocytes in mice immunized with recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosoma japonicum and investigate the immunological mechanism of the vaccine.</p><p><b>METHODS</b>Eighty-eight BALB/c mice were randomized for immunization with 10⁶ CFU recombinant vaccine orally or with 10⁵ CFU recombinant vaccine intranasally. Four mice were selected from each group every two weeks to test the responses of the splenocytes to stimulations with SjAWA or ConA. MTT assay and flow cytometry were used to assess splenocyte proliferation and the distribution of CD4⁺ and CD8⁺ T cells, respectively; the levels of interleukin-10 (IL-10), IL-12 and tumor necrosis factor-α (TNF-α) in the cell culture supernatant were detected by ELISA.</p><p><b>RESULTS</b>Regardless of the stimulations, the splencytes showed significantly enhanced proliferation in weeks 2-16 in oral administration group and in weeks 2-18 in intranasal group (P<0.01). CD4⁺ subsets in both two groups increased obviously in weeks 2-12 (P<0.01) but CD8⁺ subsets remained stable. In oral administration group, the levels of TNF-α, IL-10 and IL-12 increased in weeks 2-14, 2-18 and 2-14, and peaked at week 8, 10 and 6, respectively; in intranasal group, the cytokines increased in weeks 2-14, 2-18 and 2-18, and peaked at week 8, 10 and 8, respectively.</p><p><b>CONCLUSION</b>The recombinant vaccine rBb (pGEX-Sj32) can induce effective immune responses in mice.</p>
Asunto(s)
Animales , Ratones , Antígenos Helmínticos , Alergia e Inmunología , Bifidobacterium , Linfocitos T CD4-Positivos , Alergia e Inmunología , Linfocitos T CD8-positivos , Alergia e Inmunología , Interleucina-10 , Alergia e Inmunología , Interleucina-12 , Alergia e Inmunología , Ratones Endogámicos BALB C , Schistosoma japonicum , Esquistosomiasis Japónica , Bazo , Biología Celular , Alergia e Inmunología , Factor de Necrosis Tumoral alfa , Alergia e Inmunología , Vacunación , Vacunas Sintéticas , Alergia e InmunologíaRESUMEN
To observe the dynamic changes of splenocyte proliferation ,subsets and apoptosis in mice immunized with re-combinantBifidobacteriumbifidum(pGEX-Sj26GST)ofSchistosomajaponicum,themiceweresubcutaneously(SCgroup) and intranasally (IN group) immunized ,respectively .Four mice from each group were sacrificed in every 2 wk during 0-20 wk after immunization .Splenocyte proliferation was investigated by MTT colorimetric assay ,subsets of CD+4 and CD+8 T cells and apoptosis of splenocytes by FACsort flow cytometry .In SC group ,unstimulated and stimulated with S jAWA ,the level of splenocyte proliferation significantly increased at 4-20 wk after vaccination and increased markedly at 4-18 wk stimulated with ConA ,both of which peaked at 8 wk;in IN group ,the proliferation level of splenocyte cultured with SjAWA and ConA signif-icantly increased during the 4-18 wk ,2-10 wk and 14-18 wk ,2-8 wk and 12-18 wk ,respectively ,and all reached the maximum at the 4 wk after immunization (P＜0 .01 or P＜0 .05) .CD+4 subsets increased obviously during 2-14 wk ,2 wk and 6-16 wk re-spectively ,and reached the peak at 8 wk (P＜0 .01 or P＜0 .05) in both group ,while CD8+ subsets rose lightly during 2-20 wk in both group ,and reached the maximum at 8 wk (SC group) and 6 wk respectively (P>0 .05) .Whether unstimulated or stimulated with ConA ,the level of splenocyte apoptosis of which remarkably increased at 2-4 wk and 2-6 wk separately in SC group ,and both peaked at 2 wk (P＜0 .01 or P＜0 .05 );in IN group ,the level of splenocyte apoptosis all increased at 4 wk and reached the maximum at the same time .In summary ,by inducing the proliferation of splenocytes ,increasing CD4 + T cells and decreasing splenocyte apoptosis ,the rBb (pGEX-Sj26GST ) vaccine plays a critical role in the protective immune response .
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Objective To construct the recombinant plasmid pGEX-Sj32 of Schistosoma japonicum(Sj )and to research its ex-pression in Escherichia coli (E.coli)BL21.Methods Sj32 gene was amplified by PCR from template of plasmid pET28α-Sj32 ex-tracted from recombinant bacterium BL21 (pET28α-Sj32 )stored by our laboratory,and then cloned into the vector pGEX-1λT to construct pGEX-Sj32.The recombinant plasmid pGEX-Sj32 was transformed into E.coli BL21(DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside(IPTG),and the expressed products were identified by SDS-PAGE and Western blot.Re-sults Sj32 coding gene was successfully amplified by PCR and cloned into the vector pGEX-1λT,and the recombinant plasmid pGEX-Sj32 was constructed successfully.The molecular mass of the expressed recombinant protein was proximately 58 000 as de-tected by SDS-PAGE.The amount of the expressed protein was about 21% of the total bacterial protein.Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.Conclusion The recombinant plasmid pGEX-Sj32 is successfully constructed.The Sj32 protein was highly expressed in E.coli and the expressed recombinant protein possesses the specific antigenicity.
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It recently becomes highlight to control schistosomiasis by use of DNA vaccine,this review outlines the advances in the research of the DNA vaccine of glutathione-S-transferase(GST) of Schistosoma japonicum,S.mansoni and S.haematobium.
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To investigate the weight reduction of hydatid cyst and level of splenocyte subsets in mice immunized with recombinant Bifidobacteria bifidum (Bb) Bb-Eg95-EgA31 vaccine of Echinococcus granulosus (Eg) and challenged with Eg protoscoleces,BALB/c mice were immunized with the vaccine subcutaneously,intramuscularly,intranasally or orally.Then the mice were challenged with 50 Eg protoscoleces on 8~(th) week after vaccination and killed on 25~(th) week after infection.The weight of hydatid cyst was measured and the reduction of the weight was calculated.The spleens were collected for splenocytes preparation.CD_4~+ and CD_8~+ T cells were determined by flow cytometry(FCM),and the blank vector,Bb or MRS were set as control.The hydatid cyst weight was reduced by 45.33%,41.33%,70.67% and 62.67% respectively for the 4 immunization groups.Number of CD_4~+ and CD_8~+ subsets in the immunization groups was more than that in the control group.CD_4~+ and the ratio of CD_4~+/CD_8~+ in the intranasal or oral immunization group was much higher than that in the subcutaneous or intramuscular immunization group.It's concluded that CD_4~+ and CD_8~+T cells may play an important role in the protection induced by recombinant Eg95-EgA31 vaccine of Eg.In addition,intranasal and oral vaccinations could be the better ways for immunization.
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To observe changes on subsets of splenocytes in mice immunized with the transgenic alfalfa (Medicago sativa) vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus dynamically,the leaf protein was extracted from the transgenic alfalfa by heat-coagulation method and prepared for a solution with a concentration of 20 g/ L.The 132 BALB/c mice were divided into 3 groups randomly and immunized intranasally or orally with the leaf protein solution once per 3 days for 2 months.At the same time,the group of intranasal immunization with leaf protein from the non-transgenic alfalfa was set as control group.4 mice randomized from each group were killed to get the spleens at Week 0,2,4,6,8,10,12,14,16,18 and 20 after last immunization,and the splenocytes were separated to measure CD_4~+and CD_8~+T cell subsets by FCM.In the oral group,CD_4~+subset increased significantly from Week 6 to Week 10 after the last immunization and reached the peak at Week 6.While CD+ 8 subset increased obviously from Week 4 to Week 12 and reached the highest level at Week 8.In the intranasal group,the significant increase of the CD_4~+subset was observed from Week 4 to Week 6 and also reached the peak at Week 6.The similar trend of CD_8~+ subset was observed from Week 4 to Week 10 and reached the highest level at Week 8.It was suggested that CD+ 4and CD+ 8subsets played an important role in the protection induced in mice immunized with the transgenic alfalfa vaccine.
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Objective:To dynamically observe changes of subsets of splenocytes in mice by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em).Methods:BALB/C mice were intranasally vacci- nated with the vaccine and killed to get spleen at 0,2,4,6,8,10,12,14,16 and 18w of immunization respectively.Splenocytes were.separated to measure subsets of CD_4~+ and CD_8~+T cells by FACsort,PBS served as control.Results:In the groups of im- munization,CD_4~+ and CD_8~+subsets increased obviously at 2～12w and 2-18w respectively,which reached the highest level at 6 and 10w respectively.Conclusion:The number of CD_4~+ subsets of splenocytes increases remarkably in the early stage (2～6week)by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine.
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Objective To dynamically observe changes of cytokines of splenocytes in mice by immunization with recombinant BCG-EmII/3 vaccine of Echinococcus multilocularis(Em).Methods BALB/C mice were intranasally vaccinated and then killed to get spleen on 0、2、4、6、8 and 10w of immunization respectively, splenocytes were isolated to culture with stimulation of EmAg or ConA,their supernatants were gathered to measure the level of IL-2,IFN-?,TNF-? and IL-4 by ELISA Kits. Results In the groups of immunization, levels of IL-2,IFN-?,TNF-? and IL-4 obviously increased on 2～6、2～6、2～10 and 8～10w respectively, reached the highest level on 4、4、8 and 10w respectively. Conclusion TH1 response was induced in mice by rBCG-EmII/3 vaccine by early immunization(2～8w).
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Objective To investigate the protection by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em) and against challenge of Em protoscolexes.Methods BALB/C mice were subcutaneously or intranasally vaccinated with mixed recombinant BCG vaccines respectively.Eight weeks later,the immunized mice were challenged with Em protoscolexes.On the 18th week after vaccination,the mice were killed to count the rate of reduced alveolar echinococcus weight and to examine the levels of IgG and its subclasses,IgE;the spleen cells were separated to measure subsets of CD4+ and CD8+ T cells by FACsort.Blank vector,BCG and PBS served as control.Results In the immunized groups,the rate of reduced alveolar echinococcus weight was 45.29%-76.47%;the levels of IgG,IgG2a,IgG2b and IgE increased apparently while IgG1 and IgG3 decreased remarkably;CD4+ subset and the ratio of CD4+/CD8+ increased obviously,but CD8+ subset had no change.Conclusion Intranasal vaccination with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine is a good way for immunization.IgG,IgG2a,IgG2b,IgE and CD4+ T cell subset may play an important role in protecting against challenge of Em protoscolexes.
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Objective To evaluate effect of the non-fused recombinant antigen in immuno diagnosis of echinococcosis. Methods The coding sequence of the target gene was cloned into prokaryotic non-fusion expression vector pQE30 ( + ) and expressed in E. coli. The recombinant protein was identified by SDS-PAGE and Western Blot, purified with affinity chromatography and used in ELISA to detect specific antibody in patients' sera. Results SDS-PAGE and Western blot demonstrated that Echinococcus multilocularis immunodominant major surface antigen gene was highly expressed in E. coli. The result of ELISA showed that out of 68 echinococcosis sera, 66 samples (97.1% ) were detected positively by the assay. Only 2 samples (2% ) were positive in 100 sera from cases with other infective diseases including cysticercosis or healthy persons. The sensitivity and specificity of the assay were 97.1% and 98% respectively. Conclusion Echinococcus multilocularis immunodominant major surface antigen gene has been highly expressed in E. coli in non-fusion form. ELISA with the recombinant protein is highly sensitive and genus-specific for echinococcosis diagnosis.