Immunoprotective effect of recombinant peptidyl-prolyl cis-trans isomerase from Babesia microti against B. microti infection in mice / 中国血吸虫病防治杂志

Objective To evaluate the immunoprotective effect of active immunization with recombinant peptidyl-prolyl cis-trans isomerase from Babesia microti against B. microti infection in mice. Methods Female BALB/c mice at 6 weeks of age, each weighing approximately 20 g, were divided into the recombinant protein immunization group, the infection control group and the normal control group, of 25, 18, 15 mice in each group, respectively. Mice in the recombinant protein immunization group were given active immunization with recombinant BmPPIase protein, and 18 mice with the highest antibody titers were intraperitoneally injected with 100 μL of B. microti-infected whole blood 2 weeks after the last immunization. Mice in the infection control group were intraperitoneally injected with 100 μL of B. microti-infected whole blood, while 15 mice in the normal control group received no treatment. Blood samples were collected from mice in the recombinant protein immunization group and the infection control group on days 0 to 30 post-immunization for detection of B. microti infection, and blood samples were collected on days 0, 7, 14, 21, and 28 post-immunization for routine blood tests with a blood cell analyzer and for detection of serum cytokines using cytometric bead array. Results Anti-BmPPIase antibodies were detected in 25 mice in the recombinant protein immunization group 2 weeks after the last immunization, with titers of 5 × 103 to 8 × 104. B. microti infection rate peaked in mice in both the recombinant protein immunization and the infection control group on day 7 post-immunization, with positive infection rates of 13.3% and 50.0%, and there were significant differences between the two groups in terms of B. microti infection rate on days 3 (χ2= 113.18, P < 0.01), 5 (χ2 = 475.22, P < 0.01), 7 (χ2 = 465.98, P < 0.01) and 9 post-infection (χ2= 18.71, P < 0.01), while the B. microti infection rate tended to be 0 in both groups on day 11 post-immunization. Routine blood tests showed higher red blood cell counts [(5.30 ± 0.50) × 1012 to (9.87 ± 0.24) × 1012 counts/L)] and hemoglobin levels [(89.67 ± 22.80) to (148.60 ± 3.05) g/L)] in the recombinant protein immunization group than in the infection control group on days 0 to 28 post-immunization. Cytometric bead array detected higher serum interferon-γ [(748.59 ± 17.56) to (3 858.28 ± 1 049.10) fg/mL], tumor necrosis factor-α [(6 687.34 ± 1 016.64) to (12 708.13 ± 1 629.79) fg/mL], interleukin (IL)-6 [(611.05 ± 75.60) to (6 852.68 ± 1 554.00) fg/mL] and IL-17a [(167.68 ± 185.00) to (10 849.27 ± 355.40) fg/mL] and lower IL-10 levels [(247.65 ± 138.00) to (18 787.20 ± 2 830.22) fg/mL] in the recombinant protein immunization group than in the infection control group during the study period. Conclusions Recombinant BmPPIase protein induces up-regulation of interferon-γ, tumor necrosis factor-α and presents a high immunoprotective activity against B. microti infection in mice, which is a potential vaccine candidate protein.

Evaluation of immunoprotection against coronavirus disease 2019:Novel variants,vaccine inoculation,and complications / 药物分析学报

The strikingly rapidly mutating nature of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)genome has been a constant challenge during the coronavirus disease 2019(COVID-19)pandemic.In this study,various techniques,including reverse transcription-quantitative polymerase chain reaction,antigen-detection rapid diagnostic tests,and high-throughput sequencing were analyzed under different scenarios and spectra for the etiological diagnosis of COVID-19 at the population scale.This study aimed to summarize the latest research progress and provide up-to-date understanding of the methodology used for the evaluation of the immunoprotection conditions against future variants of SARS-CoV-2.Our novel work reviewed the current methods for the evaluation of the immunoprotection status of a specific population(endogenous antibodies)before and after vaccine inoculation(adminis-tered with biopharmaceutical antibody products).The present knowledge of the immunoprotection status regarding the COVID-19 complications was also discussed.Knowledge on the immunoprotection status of specific populations can help guide the design of pharmaceutical antibody products,inform practice guidelines,and develop national regulations with respect to the timing of and need for extra rounds of vaccine boosters.

Research progress on optimization strategies for microencapsulated islet transplantation / 器官移植

Islet transplantation is one of the effective therapies for diabetes mellitus. Nevertheless, multiple issues still exist, such as shortage of donors and adverse reactions caused by long-term use of immunosuppressants, which limit the islet survival post-transplantation. Microencapsulated islet transplantation may overcome these difficulties to certain extent, whereas many factors, such as the destruction of immune isolation microenvironment within the microcapsules and insufficient supply of oxygen and nutrients, constrain the application of microencapsulated islet transplantation in clinical practice. In recent years, how to enhance the effect of microencapsulated islet transplantation has been gradually studied. The application of stem cells in microencapsulated islet transplantation has steadily become a research hot spot. Therefore, the optimizing strategies for microencapsulated islet transplantation and the application of stem cells in microencapsulated islet transplantation were reviewed, and the potential improvement techniques of microencapsulated islet transplantation were investigated in this article, aiming to provide reference for further clinical application of microencapsulated islet transplantation.

Research status of rhoptry protein of Toxoplasma gondii and its nucleic acid vaccine / 中国血吸虫病防治杂志

At present,there are no ideal drugs and measures in the treatment and prevention of toxoplasmosis.The develop-ment of safe,convenient,and strong protective nucleic acid vaccine is an important strategy for prevention and control of toxo-plasmosis.The rhoptry protein(ROP)is a large class of proteins secreted by Toxoplasma gondii.ROPs play an important role in the invasion of host cells,the formation of parsitophorous vacuole(PV)and the regulation of proliferation by T.gondii.Thus, ROPs become the most promising candidates of vaccine.In this paper,we summarize the important members of the ROPs,the expression vector and the immunogenicity and immunoprotection of the nucleic acid vaccine in animal experiments.

Immunoprotection of gastric mucosa in newborn mice after oral immunization with inactived H.pylori / 中国免疫学杂志

Objective:To explore the Immunoprotection of gastric mucosa in newborn mice after oral immunization with inactived H.pylori.Methods:Thirty-two BALB/c mice were randomly divided into four groups, including PBS control group ( groupⅠ) ,the group of inactivated H.pylori ( groupⅡ) , the group of inactivated H.pylori and Escherichia cole heat-labile enterotoxin B subunit( LTB) ( groupⅢ) ,and normal group( groupⅣ).Newborn BLAB/c mice within 24 hours were immunized through oral gavage with corresponding antigen,Two weeks after final immunization,the mice were challenged with 1×108CFU live H.pylori strain SS1, twice once day,for 2 days.The twenty-eighth day post-challenge,all of mice were sacrificed and stomachs were removed for grades of in-flammation and The counting of H.pylori colonizing.Results:The mean scores of stomach tissue inflammation of groupⅠ,Ⅱ,Ⅲ andⅣwere 2.25±0.46,2.12±0.35,1.5±0.53,0.25±0.46,respectively,and the score of inflammatory activity of the above four groups were 2.12±0.35,2.12±0.35,1.37±0.52,0.25±0.46.Among these four groups,the results were statistically significant between any two groups except the result between groupⅠand groupⅡ.Finally,H.pylori colonization quantity in the stomachs of groupⅢwas lower than groupⅠand group Ⅱ( P<0.05 ) , as expected, the H.pylori colonization was undetectable in group Ⅳ.Conclusion: The oral gavage with inactived H.pylori plus LTB to newborn mice may be associate with the lightening of inflammation and the decrease of H.pylori colonizing number.

Studies on immunogenicity and immunoprotection induced by heat shock protein 60 kDa of Schistosoma japonicum in mice / 中国血吸虫病防治杂志

Objective To evaluate the immunogenicity and immunoprotective effect of heat shock protein 60 kDa (SjHSP60) of Schistosoma japonicum in mice after immunization and challenge infection, and explore the mechanism. Methods B cell/an?tibody?related databases and analysis tools were used to predict B?cell epitopes of SjHSP60. The mice were immunized with the recombinant SjHSP60 and challenged with S. japonicum cercariae. SjHSP60?specific antibodies in serum were detected by ELI?SA. The level of splenocyte proliferation was determined by 3H?TdR incorporation. Ex vivo suppression assay was performed to in?vestigate the effects of CD4 +CD25 + regulatory T cells (Tregs) induced by SjHSP60. Results SjHSP60 possessed multiple pre?dominant regions of B?cell epitopes. SjHSP60 induced a significant increase in both SjHSP60?specific IgG levels (P 0.05) and liver?accumulated eggs (P > 0.05) in S. japonicum?infected mice. Ex vivo assay showed that CD4+CD25+ Tregs from SjHSP60?immunized mice enhanced immunosuppressive activity. Conclusion SjH?SP60 has a dual role in host immune system, being involved in the induction of dominant humoral and cellular immune responses as well as in the enhancement of immunosuppression.

Paracoccidioidoyycosis: challenges in the develpment of a vaccine against an eendemic mycosis in the americas / Paracoccidioidomicose: desafios no desenvolvimento de uma vacina contra micose endêmica nas Américas

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.

Immunocompetence and Immunoprotection of a DNA vaccine NMB0315 against N.meningitidis serogroup B in mice / 中国免疫学杂志

Objective:To construct NMB0315 eukaryotic expression recombinant vector ,detect specific humoral and cellular immune response induced by the recombint DNA vaccine intramuscularly in female BALB /c mice,evaluate the immunocompetence and immunoprotection of the vaccine , so as to provide experimental basis for the development of a novel nucleic acid vaccine against N.meningitidis serogroup B .Methods: The whole NMB0315 gene was amplified by PCR from the standard strains MC 58 genomic DNA,cloned into a plasmid pcDNA3.1(+),identified by double digestion of the recombinant plasmid with restriction enzymes and se -quencing.The recombinant vector pcDNA 3.1 (+)/NMB0315 was transfected into eukaryotic COS-7 cells and RAW264.7 cells, the NMB0315 protein was detected by immunocytochemical method and Western blot respectively .The levels of specific humoral and cellular immune response were detected after inoculating in female BALB /c mice intramuscularly with the recombinant plasmid .The immune protective effect was investigated with the DNA vaccine and the bactericidal titer of the immune serum was deter mined by serum bactericidal assay ( SBA ) in vitro.Results: The recombinant pcDNA3.1 (+)/NMB0315 was effectively transcripted and expressed in eukaryotic cells and the specific humoral and cellular immune responses were induced in the inoculated mice .In the re-combinant pcDNA3.1(+)/NMB0315 group ,the levels of serum IgG,IgG1,IgG2a,IgG2b and IgG3 and genital tract sIgA were significantly higher than in controls ( P<0.001 ) .The stimulation index in the culture supernatant of the spleen lymphocytes of the vaccine group was higher than that of the control group (P<0.05).The ratios of serum IgG2a/IgG1 in the DNA vaccine group were less than 1.The bactericidal titer of the NMB 0315+CpG group reached 1:128 following three immunizations , the protection rate of the vaccine group was 70%against the N.meningitidis strain MC58.Conclusion:The NMB0315 nucleic acid vaccine could induce higher levels of humoral immunity and cellular immunity and showed effective protection against N .meningitidis serogroup B , the immune serum had strong bactericidal activity in vitro .

Construction of a fusion gene encoding h1a-spaO of Salmonella paratyphi A and analysis of immuno-protective effects of the recombinant protein / 中华微生物学和免疫学杂志

Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .

Distribution of genes encoding LPD of nontypeable Haemophilus influenzae A isolates and the immu-noprotective effects of the expressed recombinant LPD / 中华微生物学和免疫学杂志

Objective To investigate the distribution and sequence conservation of genes encoding the outer membrane lipoprotein D(LPD)of nontypeable Haemophilus influenzae(NTHi)isolates and to ana-lyze the immunogenicity and the immunoprotective effects of the expressed recombinant LPD(rLPD). Meth-ods PCR analysis was used to detect the genes encoding LPD of NTHi isolates. The PCR products were se-quenced after T-A cloning. A prokaryotic expression system for genes encoding LPD was established to ex-press the rLPD. Ni-NTA affinity chromatography was used for purification. SDS-PAGE and Bio-Rad Gel Im-age Analyzer were used to detect the expression and the yield of rLPD. The antigenicity and immunoreactivity of rLPD were detected by ELISA and Western blot assay. The immunoprotective effects of rLPD against lethal dose of NTHi were evaluated in a mouse model. Results All of the tested NTHi isolates were positive for the genes encoding LPD. They shared 98. 0% -99. 4% homologies in nucleotide sequences and 98. 5% -100% homologies in amino acid sequences. The established prokaryotic expression system expressed rLPD with a high yield. High levels of antibody in rabbits were induced by the rLPD. The anti-NTHi antiserum samples from rabbits and children could recognize and react with the rLPD. The result of ELISA indicated that 93. 6%(58 / 62)and 53. 2%(32 / 62)of the serum samples from children with NTHi infection were positive for rLPD-IgM and rLPD-IgG,respectively. The rLPD at concentrations of 100 μg and 200 μg could respectively protect 60. 0% and 73. 3% of mice from lethal NTHi infection. Conclusion The genes enco-ding LPD were extensively distributed in NTHi isolates with high sequence conservation. The expressed rLPD could be used as a potential candidate antigen in the development of genetic engineering vaccine against NTHi infection considering its high immunogenicity and immunoprotective effects.

Cloning,expression and immuno-protection analysis of a gene encoding tro-ponin T of Schistosoma japonicum(SjTnT) / 中国血吸虫病防治杂志

Objective To clone cDNA encoding troponin T of Schistosoma japonicum(SjTnT),and evaluate the protective efficacy induced by recombinant SjTnT in BALB/c mice against S. japonicum challenge infection. Methods The SjTnT gene was amplified from 28-day-schistosome cDNAs by PCR and then subcloned into pET28a(+). The recombinant SjTnT protein (rSjTnT)was expressed in Escherichia coli BL21(DE3)cells. The serum specific to rSjTnT was prepared by immunized BALB/c mice with the recombinant antigen,and the immunogenicity of rSjTnT was detected by Western blotting and ELISA. The immuno-protective efficacy induced by rSjTnT in BALB/c mice was evaluated according to the reduction in worm and egg counts. Results The cDNA encoding SjTnT was successfully cloned and expressed in E. coli. Western blotting showed that rSjTnT had a good immunogenicity. The high level of specific IgG antibodies was detected,and 33.89% worm reduction and 43.94% liver egg reduction were obtained in mice vaccinated with rSjTnT combined with Seppic 206 adjuvant compared with those in the adjuvant control group. Conclusions rSjTnT could induce partial immuno-protection against S. japonicum infec-tion in BALB/c mice. This study provided a basic for understanding the biological function of SjTnT.

The Importance of Breastfeeding in Rotaviral Diarrhoeas 103

Globally, rotaviral vaccines in use today have contributed to the reduction of the incidence of rotaviral diarrhoeas. Despite the substantial protection conferred by the current vaccines against the rotaviral strains, it is only prudent to recognise that other protective factors, like breastfeeding, also provide some degree of protection against this disease. This article has attempted to review some important mechanisms of protection in breast milk against the rotaviruses and highlight the oft forgotten non-immunoglobulin fraction in breast milk as an additional tool of protection against rotavirus disease. The adaptive capacity of breast milk to environment is another compelling reason to continue breastfeeding as it can usefully complement and be significant in the use of many vaccines. Vital immunoprotective constituents in breast milk beneficially protect the infant by initiating and strengthening many immune responses and should be borne in mind as essential tools of defence even in an era where vaccines play a pivotal role in the combat against certain diseases. It is impressive that besides nutritive advantages, the suckling infant enjoys appreciable immunoprotection via exclusive breastfeeding.

Recombinant expression of Helicobacter pylori GroEL protein and immunoprotection of the expression product in mice / 中华微生物学和免疫学杂志

Objective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates,and the antigenicity,immunoreactivity and immunoprotection of recombinant expressed GroEL (rGroEL) against H.pylori-infection in mice.Methods The sequences of groEL genes from H.pylori isolates were detected by PCR and sequencing.A prokaryotic expression system of H.pylori groEL gene using pET42a and E.coli BL21DE3 was generated.SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL.The antigenicity and immunoreactivity of rGroEL were examined using ELISA and Western blot assay.The membrane location of GroEL was determined by ELISA using whole cells of H.pylorias the coated antigen.The immunoprotection of rGroEL was detected in H.pylori-infected mouse model.Results All the 35 tested H.pylori isolates possess the groEL gene with 99.4％ to 100％ sequence identities.The yield of rGroEL expression occupied 56％ of the total bacterial proteins and the extracted rGroEL showed a single band in gel after SDSPAGE.rGroEL could induce the production of high-titer IgG in rabbits or mice.The lgG against whole cell of H.pylori(Hp-IgG) and rGroEL-IgG could recognize as well as combine with rGroEL protein.GroEL is a superficial protein antigen of H.pylori.The immunization with 50,100 or 200 μg rGroEL prevented 50.0％ (6/12),75.0％ (9/12) or 91.7％ (11/12) mice from infection of H.pylori strain SS1.The immunoprotective rate ( 91.7％ ) due to the immunization of 200 μg rG roEL was significantly higher than that ( 50.0％ ) of 50 μg rGroEL ( P＜0.05 ).Conclusion The GroEL protein is a sequence-conserved,immunogenic and immunoprotective superficial antigen of H.pylori,which can be used as the immunogen candidate for developing genetically engineering vaccines.

Synthesis of multiple antigenic peptide vaccine based on predominant epitopes of Helicobacter pylori UreB protein and immunoprotection of the vaccine / 中华微生物学和免疫学杂志

ObjectiveTo generate a prokaryotic expression system of series predominant epitopes (UreB322 and UreB527) of Helicobacter pylori UreB protein,and to synthesize a multiple antigenic peptide (MAP) vaccine by linking both the two epitopes with a peptide carrier (Poly-Asp-Lys),and to determine the immunogenicity and immunoprotection of the MAP vaccine.MethodsLinking primer PCR was performed to generate an enterokinase(EK) site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system.The expressed target recombinant fusion protein 8 ×[rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column.rUreB322-EK,rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine (MAP-rUreB322/B527).The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay.An animal H.pylori strain SS1-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527.ResultsAn octuple-repeated series UreB322-UreB527 encoding gene and its prokaryotic expression system were obtained.The yield of target fusion protein 8×[rEKUreB322-EK-rUreB527-EK] was as high as 48％ of the total bacterial proteins.EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides.The linking ratio of rUreB322-EK,rUreB527-EK and Poly-Asp-Lys was as high as 92.5％.The antibody against whole cell of H.pylori and rUreB-IgG could recognize and combine with the rUreB322-EK,rUreB527-EK or MAP-rUreB322/527.The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice (P＜0.05).The immunoprotective rates(83.3％ and 91.7％ ) by immunization with50 or 100 μg MAP-rUreB322/527 in the H.pyloristrain SSI-infected mice were significantly higher that those(d1.7％ and 50.0％ ) by immunization with equal rUreB(P＜0.05).ConclusionAn gene composed for encoding a repeated series predominant epitopes of H.pylori UreB protein and its prokaryotic expression system are successfully generated in this study.MAP-rUreB322/527,the multiple antigenic peptide vaccine based on the two predominant epitopes of UreB,can noticeably increase the immunoprotection in H.py/or/infected mice.

Generation of spaO-ompA fusion gene of Salmonella paratyphi A and the immunoprotection of expression product of the fusion gene / 中华微生物学和免疫学杂志

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.

Distribution of Salmonella paratyphi A ompA gene and immunological identification of the recombinant expressed product / 中华微生物学和免疫学杂志

Objective To construct a prokaryotic expression vector of Salmonella paratyphi A ompA gene, and to determine immunogenieity and immonuprotection of the recombinant expressed product rOmpA and carrying frequency of ompA gene in S. paratyphi A isolates. Methods ompA gene was amplified from a clinical S. paratyphi A strain JH01 by PCR, cloned into T-A cloning vector, and then the prokaryotic expression vector was constructed. The rOmpA expression was determined by SDS-PAGE. Antigenicity and immunoreactivity of rOmpA were determined by immunodiffusion test, Micro-Widal's test and Western blot assay, ompA gene exiting in 98 S. paratyphi A isolates was detected by PCR. By using immunoprotective effect of rOmpA against the challenge of S. paratyphi A strain 50001 in mice were determinded. Results Both the nucleotide and putative amino acid sequence identities of the cloned ompA gene were 100% com-pared to the reported corresponding sequences. The expression yield of rOmpA was approximately 65% of the total bacterial proteins, rOmpA was able to induce specific antibody in rabbits, and reacted efficiently with rabbit antisortm or the antiserum against whole cell of S. paratyphi A detected by Western blot. 94.9% (93/98) of the S. paratyphi A isolates had ompA gene. 41.7% (5/12) and 58.3% (7/12) of the mice im-murtized with 100 and 200 μg rOmpA were survival after lethal challenge with S. paratyphi A strain 50001. The titer of antibody to the H antigens of S. paratyphi spp in the sera from rOmpA immunized mice and sur-viral mice in the S. paratyphi A challenge detected by Micro-Widal's test was 1:5-1:40. Conclusion ompA gene has an extensive distribution in clinical isolates of S. paratyphi A. rOmpA possesses an immunogenicity and a certain immunoprotective effect, and can be used as the candidate antigen in genetic engineering vaccine.

Study on the immunoprotections of SjRPS4 and SjRPL7 DNA vaccines against Schistosoma japonicum infection in mice / 中国人兽共患病学报

The aim of this research was to study the immunoprotections of Schistosoma japonicum (S. japonicum) DNA vaccines SjRPS4 and SjRPL7 in mice. Fourty Kunming mice were randomly divided into 4 groups (A, B, C and D), and the pcDNA3.0/SjRPS4 and pcDNA3.0/SjRPL7 plasmid DNA vaccines were prepared for experiment. Mice in group A were intramuscularly injected with 100μL normal saline, whereas mice in group B were injected with 100 (g naked plasmid pcDNA3.0 into the quadriceps. Mice in groups C and D were injected with 100μg/100μL eukaryotic recombinant plasmids pcDNA3.0/SjRPS4 and pcDNA3.0/SjRPL7 into the hind leg muscles respectively. The initial injections were followed by two sets of boosters at 2 weeks intervals. In addition, levels of the specific antibodies were detected 2 weeks after the last immunization and all mice were percutaneously infected with 20( 1) S. japonicum cercariae on abdomen. Fourty-two days after the infection, all mice were killed to detect the worm reduction rate and the egg reduction rate. Significant differences of worm burden reduction rates, LEPG reduction rates, IEPG reduction rates and intrauterine eggs reduction rates were observed in both test group (group C and D), comparing with the control groups (group A and B). Results indicated that the DNA vaccines of pcDNA3.0/SjRPS4 and pcDNA3.0/SjRPL7 could induce strong protective immunity against S. japonicum in mice.

The research on the immunoprotection difference among five EV71 virus / 中华微生物学和免疫学杂志

Objective To screen the 5 EV71 vaccine candidates which were isolated from MRC-5 cells to find one as the vaccine virus. Methods The ICR mother mouse were immunized by intraperitoneal injection with the 5 vaccine candidates which were made from monoclonal EV71 virus. Two weeks after booster immunization, the animals were allowed to mate, another booster was given after 2 weeks, and then attracted the milk mouse within 24 h with different types of virus by cranial cavity injection. The survival condition were recorded everyday, and the antibody titre(IgG) were detected by ELISA, the virus titre of intestine were detected by nest-PCR, and neutralizing antibodies were determined using a microassay with MRC-5 cells, and then the data were analyzed by SPSS16.0. Results The antibody titre of 5 virus immunized ICR mouse were improved with the increase in the immune times, and they got difference in neutralization capacity, the survival rate after fatal attract and the virus titre of the intestine. Conclusion It proved that the five vaccine candidates were different at the molecular level, cellular level and individual level. 123 strain was the best one in immunogenicity and immunoprotective property, which agreed with the vaccine requirement.

Prokaryotic expression of Salmonella paratyphi A nmpC gene and immunoprotective effect of the expressed product / 中华微生物学和免疫学杂志

Objective To generate a prokaryotic expression system of Salmonella paratyphi A nmpC gene that encoding an outer membrane protein(OMP),and to determine immunogenicity and immonuprotection of the recombinant expressed product rNmpC and carrying and expression frequencies of the nmpC genes in isolates of S.paratyphi A.Methods A nmpC gene clone was obtained from a clinical S.paratyphi A strain JH01 by PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Bio-Rad Agarose Image Pattern Analysis System were applied to examine the expression and yield of rNmpC.Antigenicity and immunoreactivity of rNmpC were determined by immunodiffusion test,Western blot assay and micro-Widal's test.The carrying and expression rates of nmpC genes in 98 S.paratyphi A isolates were detected by PCR and ELISA.By a mouse infection model,the immunoprotective effect of rNmpC against the lethal challenge of S.paratyphi A was determined.Results All the cloned nmpC genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The expression yield of rNmpC was approximately 30% of the total bacterial proteins.rNmpC could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains have nmpC gene as well as express NmpC protein,but no nmpC gene could be detectable in S.typhi,S paratyphi B and S paratyphi C.Immunization with 100 μg and 200 μg rNmpC contributed 41.7%(5/12) and 66.7%(8/12) immunoprotective rates in mice,respectively.The sera from rNmpC immunized mice and survival mice in the NmpC is an unique OMP antigen of S.paratyphi A with conserved sequence,extensive distribution and natural expression.This OMP can be used as one the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A based on its fine immunogenicity and certain immunoprotection.

The immunoprotective effects of new gene R049 of uropathogenic Escherichia coli / 中华微生物学和免疫学杂志

Objective To clone and express the new gene R049 of uropathogenic Escherichia coli 132.and to investigate the immunopmtective effects of the R049 recombinant protein on mice.Methods The pmkaryotic expression system of gene R049 was constructed by directed cloning.Thereafter,the R049 recombinant protein Was expressed and purified by Ni affinity chromatography.Polyclonal antibody was pre-pared by immunizing BALB/c mice with R049 recombinant protein.The R049 recombinant protein and whole bacterial proteins of UPEC132 were analyzed by SDS-PAGE and Western blot.BALB/c mice were im-munized with R049 recombinant protein before challenged by UPECl32 through urinary tract.Then the differences of urine and renal colony counts between immunization group and control group were compared.Results The recombinant strain E coli BL21(DE3)/pET32a-R049 ORF was constructed successfully,and the relative molecular mass of the R049 recombinant protein was 66.9×103 and its purity was up to 95% af-ter purification.The titer of polyclonal antibody wag≥1:102 400 analyzed by indirect ELISA.Both of the R049 recombinant protein and whole bacterial proteins of UPECl 32 were confirmed to show specffic reactions on the antiserunl throughh Western blot.The animal experiments showed the urine and renal colony counts of immunization group were significantly lower than that of the control group(P<0.01,P<0.05).Conclu-sion The new gene R049 of uropathogenic E.coli 132 had immunopmtective effects on mice and the defini-tive mechanism would be needed to further study.