ABSTRACT
The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.
ABSTRACT
The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirns spike protein induced hormonal and cellular immune response in Balb/c mice.
ABSTRACT
Objective To explore the relationship of the K121Q polymorphism of membrane glycoprotein PC-1 gene with the features of insulin resistance(IR) and type 2 diabetes mellitus(T2DM).Methods The K121Q polymorphism in exon 4 of PC-1 gene were determined with the technique of polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) in 133 patients with T2DM and 108 controls with normal glucose tolerance(NGT) in Shenzhen city.The clinical and laboratory data were also compared between type 2 diabetic patients with different genotypes.Results No statistically significant differences were observed in the genotype and allele frequencies between the control and the T2DM subjects.The concentrations of fasting plasma glucose(FPG),triglycerides(TG) and C-peptide were higher in T2DM patients with KQ genotype than with KK genotype(P
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)
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
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
Objective To construct the eukaryotic expression plasmids of Mycobacterium tuberculosis ESAT-6 antigen and to investigate its immunogenicity in mice. Methods The sequence encoding ESAT-6 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. Then, the amplified fragment was sub-cloned into pVAC expression vector. The eukaryotic expression plasmid pVAC-ESAT-6 was transfected into Vero E6 cells with liposome. The mRNA expression of pVAC-esat-6 in Vero E6 cells was detected by RT-PCR and its expression product was analyzed by Western-blot. The plasmid pVAC-esat-6 was introduced into the mice by gene gun injection. The specific antibody to ESAT-6 and the level of IFN-? in supernatant of spleno-lymphocyte cultures were detected by ELISA methods. Results The recombinant plasmid pVAC-esat-6 was successfully constructed, and the expression of ESAT-6 was also detected in vitro. After vaccinated three times, the mice produced specific antibody and the level of IFN-? in supernatant of spleno-lymphocyte cultures in group of pVAC-esat-6 was significantly higher than group of vector alone (P
ABSTRACT
Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.