ABSTRACT
To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj14-Sj26 that contains fatty binding protein (Sj14), GST (Sj26) and paramyocin (Sj97) that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was trans-fected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and pIRES-Sj97-Sj14-Sj26 plasmid DNA, pIRES-Sj14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA,plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh.Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the pIRES-Sj97-Sj14-Sj26 group as compared with the plRES blank vector, normal saline and pIRES-Sj26 groups (P<0.01) and the pIRES-Sj14-Sj26(P<0.05). Single splenocyte suspension was prepared to detected the level of IFN-γ by ELISA and the lymphocyte stimulating index (SI) by MTT SI was significantly higher of in the pIRES-Sj97-Sj14-Sj26 group than in other groups (P<0.01), while the IFN-γ, level was significantly higher the pIRES-Sj97-Sj14-Sj26 group than in pIRES blank vector and normal saline groups (P<0.01), but no significant differences were found when compared with pIRES-Sj14-Sj26 and pIRES-Sj26 groups. Flow cytometery showed that the percent-ages of CD4+ and CD8+ T cells were much higher in the pIRES-Sj97-Sj14-Sj26 group (P<0.01,P<0.05). It was concluded that pIRES-Sj97-Sj14-Sj26 vaccine may induce stronger immune response in BALB/c mice.
ABSTRACT
In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sjl4 or pVAC-Sj26 only to get two gene fragments including Sjl4 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES,resulting in another new recombinant plasmid pIRES-Sj26-Sj 14. The expression of Sj14 and Sj26genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and pIRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
ABSTRACT
In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj14. The expression of Sj14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
ABSTRACT
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Subject(s)
Humans , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Membrane Proteins , Genetics , Plasmids , Genetics , Recombinant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus , Chemistry , Genetics , Viral VaccinesABSTRACT
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Plasmids/biosynthesis , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/genetics , Viral Vaccines/biosynthesisABSTRACT
Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.
Subject(s)
Humans , BCG Vaccine , Allergy and Immunology , Bacterial Proteins , Allergy and Immunology , Chaperonin 60 , Chaperonins , Allergy and Immunology , Cloning, Molecular , Escherichia coli , Metabolism , Genetic Vectors , Mycobacterium tuberculosis , Genetics , Allergy and Immunology , Plasmids , Genetics , Sequence Analysis, DNA , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
The BALB/c mice were immunized with rMS-Sj26GST and rBCG-Sj26GST vaccine in Schistosoma japonicum by subcutaneous injection. After they were immunized for 8 weeks, the eyeballs were removed to get blood and macrophages of abdominal cavity and spleen cells were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release was used to measure the phagocytic activity of the macrophages. By using ELISA kit, the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 10(6) CFU of rMS-Sj26GST and rBCG-Sj26GST vaccine separately by subcutaneous injection, proliferating ability of splenic lymphocytes in the mice showed no difference (P > 0.05), but both were significantly increased as compared with that in the control group (P < 0.05); The contents of NO in the intraperitoneal macrophages of rMS-Sj26GST vaccine group were significantly lower than in the control group (P < 0.001) and rBCG-Sj26GST vaccine group (P < 0.01); The levels of serum IL-2 in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.001), vector group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05); The contents of serum IFN-gamma in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05), The contents of IFN-gamma in the cultured supernatant were significantly lower than those of rBCG-Sj26GST vaccine group (P < 0.001), but were significantly increased as compared with that in the control group (P < 0.01). It was indicated that both vaccines could enhance the immune response of the mice, but rMS-Sj26GST vaccine had stronger immunogenicity than rBCG-Sj26GST vaccine.
ABSTRACT
The BALB/c mice were immunized with rMS-Sj26GST and rBCG-Sj26GST vaccine in Schistosoma japonicum by subcutaneous injection. After they were immunized for 8 weeks, the eyeballs were removed to get blood and macrophages of abdominal cavity and spleen cells were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release was used to measure the phagocytic activity of the macrophages. By using ELISA kit, the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 10(6) CFU of rMS-Sj26GST and rBCG-Sj26GST vaccine separately by subcutaneous injection, proliferating ability of splenic lymphocytes in the mice showed no difference (P > 0.05), but both were significantly increased as compared with that in the control group (P < 0.05); The contents of NO in the intraperitoneal macrophages of rMS-Sj26GST vaccine group were significantly lower than in the control group (P < 0.001) and rBCG-Sj26GST vaccine group (P < 0.01); The levels of serum IL-2 in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.001), vector group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05); The contents of serum IFN-gamma in the rMS-Sj26GST vaccine group were significantly increased as compared with that in the control group (P < 0.01) and rBCG-Sj26GST vaccine group (P < 0.05), The contents of IFN-gamma in the cultured supernatant were significantly lower than those of rBCG-Sj26GST vaccine group (P < 0.001), but were significantly increased as compared with that in the control group (P < 0.01). It was indicated that both vaccines could enhance the immune response of the mice, but rMS-Sj26GST vaccine had stronger immunogenicity than rBCG-Sj26GST vaccine.