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.

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.
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 ; SARS Virus/chemistry ; SARS Virus/*genetics ; Viral Vaccines/biosynthesis

Objective A highly feasible and reliable ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was presented for therapeutic drug monitoring of five anti-schizophrenic drugs (aripiprazole, amisulpride, olanzapine, paliperidone and ziprasidone) simultaneously and the reference range of plasma concentration was investigated.Methods establishment and evaluation.The selectivity,accuracy,precision,stability and linear range of established UPLC-MS/MS method were evaluated according to FDA and CLSI.After a ten-month clinical application, a retrospective analysis of 253 positive samples was carried out to investigate conformance of the AGNP therapeutic reference range for Chinese patients.Results The five anti-schizophrenic drugs could be simultaneously measured by the established UPLC-MS/MS within 6 minutes.The LOQs were 0.1 to 9.0 ng/ml.The correlation coefficients were all higher than 0.997 7.The intra-day and inter-day precisions were less than 11.1%,and the recoveries ranged from 86.2% to 104.8%.The clinical results suggested the good consistency in reference range with AGNP for olanzapine, aripiprazole, paliperidone and ziprasidone.While for amisulpride, the plasma concentration level(445.2 ±231.5)ng/ml was significantly higher than the AGNP-recommended range(100 -320)ng/ml.Conclusion The established UPLC-MS/MS method was very suitable for monitoring anti-schizophrenic drugs.

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.

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.

Objective To investigate the effect of dexmedetomidine pretreatment on Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway during myocardial injury induced by liver ischemia-reperfusion (I/R) in rats.Methods Twenty-four healthy adult male SpragueDawley rats,aged 8-10 weeks,weighing 220-250 g,were divided into 3 groups (n=8 each) using a random number table method:sham operation group (group S),liver I/R group (group I/R),and dexmedetomidine pretreatment group (group D).The portal vein,superior and inferior vena cava,subhepatic inferior vena cava and hepatic artery were clamped,and the liver was perfused with 4 ℃ lactated Ringer's solution for 60 min through the portal vein to establish the model of liver cold I/R in anesthetized rats.Dexmedetomidine 100 μg/kg was intraperitoneally injected at 30 min before ischemia in group D.Blood samples were collected at 8 h of reperfusion from the inferior vena cava for determination of serum cardiac troponin Ⅰ (cTnⅠ) and heart-type fatty acid binding protein (H-FABP) concentrations (using the automatic biochemistry analyzer),tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 protein (HMGB1) concentrations (by enzyme-linked immunosorbent assay).The rats were then sacrificed,andhearts were harvested for examination of histopathological changes (with a light microscope) and for determination of the malondialdehyde (MDA) content (using thiobarbituric acid method) and superoxide dismutase (SOD) activity (by xanthine oxidase method),and expression of phosphorylated STAT1 and STAT3 (p-STAT1,p-STAT3) and phosphorylated JAK2 (p-JAK2) in myocardial tissues (by Western blot).Results Compared with group S,the serum concentrations of cTnI,H-FABP,TNF-α and HMGB1 were significantly increased,the MDA content was increased,the SOD activity was decreased,and the expression of p-JAK2,p-STAT1 and p-STAT3 was up-regulated in I/R and D groups (P＜0.05).Compared with group I/R,the serum concentrations of cTnI,H-FABP,TNF-α and HMGB1 were significantly decreased,the MDA content was decreased,the SOD activity was increased,the expression of pJAK2,p-STAT1 and p-STAT3 was down-regulated (P＜0.05),and pathological changes of myocardium were significantly attenuated in group D.Conclusion The mechanism by which dexmedetomidine pretreatment mitigates myocardial injury induced by liver cold I/R may be related to inhibiting activation of JAK/STAT signaling pathway in rats.

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.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Membrane Proteins ; biosynthesis ; genetics ; isolation & purification ; Plasmids ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; chemistry ; genetics ; Viral Vaccines ; biosynthesis