Objective:To construct Streptococcus suis type 2 Δ0948 complementary strain and verify its effect on suilysin (SLY) secretion and virulence. Methods:The SSU05_0948 gene sequence with promoter was amplified by PCR and ligated to pAT18 vector to construct complementary strain and verify its expression through Western blot. Growth curve was drawn to compare the growth of complementary strain against the wild-type strain and mutant strain in different periods. CD1 mice challenge model was used to verify whether complementary strain could restore the virulence of mutant. SLY hemolytic activity and Western blot were compared the effect of complementary strain and wild-type strain and mutant strain on SLY protein secretion at different time points.Results:The complementary strain was successfully constructed, but the expression of SSU05_0948 was lower than the wild-type strain. The growth rate of the complementary strain was significantly slower than the wild-type strain and mutant strain in the logarithmic growth phase, but the same in the platform phase. The CD1 mice challenge model showed the complementary strain could basically restore the virulence of the mutant strain. The hemolytic activity of SLY and Western blot showed that SSU05_0948 could inhibit the secretion of SLY protein in the early and middle logarithmic phase, but did not affect the secretion of SLY in the late logarithmic and platform phase, while the complementary strain could restore the secretion of SLY protein.Conclusions:The complementary strain CΔ0948 of Streptococcus suis can restore the virulence of mutant strain Δ0948, and SSU05_0948 affects the virulence of Δ0948, which provides a new idea for the prevention and treatment of Streptococcus suis.

The anti-hepatitis B virus (HBV) effects and its mechanisms of the ethanol extracts of Hypericum perforatum L. (EHP) in vitro were explored. HepG2 2.2.15 cells, a stable HBV-producing cell line, were cultured as the model system to observe the anti-HBV effect. The viral antigens of cellular secretion, HBsAg and HBeAg, were determined by enzyme linked immunosorbent assay (ELISA). The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR. In order to understand the mechanisms of the suppression of HBV replication, all HBV promoters (Cp, Xp, S1p, S2p and Fp) with luciferase reporter gene were transfected into HepG2 cells respectively. Then the activities of viral promoters were examined by luciferase reporter assay. It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, as well as the extracellular HBV DNA. And EHP could selectively inhibit the activity of HBV promoter Fp. Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription, which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

Acclimatization ; physiology ; Altitude ; Altitude Sickness ; prevention & control ; Humans ; Hypoxia ; physiopathology ; Male ; Military Personnel ; Mountaineering ; physiology ; Oxygen Consumption ; physiology ; Physical Fitness ; physiology ; Young Adult

OBJECTIVETo detect the serum levels of melanocyte antibodies and explore the relation between melanoma antigen recognized by T-cells (MART-1) and vitiligo in children.

METHODSThe serum samples were collected from children with vitiligo to test the autoantibodies, and divided into low- and high-titer group according to the test results. Melanocytes were incubated with the serum samples, and the changes of melanocyte surface antigen were evaluated using specific MART-1 antibody.

RESULTSThe serum melanocyte antibody levels in children with vitiligo were significantly higher than those in normal subjects. The expression level of melanocyte surface antigen MART-1 increased obviously after incubation of the melanocyte with high antibody titer serum samples, and MART-1 was found to specifically bind to specific MART-1 antibody.

CONCLUSIONMelanocytes MART-1 may correlate to the autoimmune mechanism in children with vitiligo.

Adolescent ; Autoantibodies ; blood ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; MART-1 Antigen ; immunology ; Male ; Melanocytes ; immunology ; T-Lymphocytes ; immunology ; Vitiligo ; immunology

The anti-hepatitis B virus(HBV)effects and its mechanisms of the ethanol extracts of Hypericum perforatum L.(EHP)in vitro were explored.HepG2 2.2.15 cells,a stable HBV-producing cell line,were cultured as the model system to observe the anti-HBV effect.The viral antigens of cellular secretion,HBsAg and HBeAg,were determined by enzyme linked immunosorbent assay(ELISA).The quantity of HBV-DNA released in the supernatant was assayed by real-time PCR.In order to understand the mechanisms of the suppression of HBV replication,all HBV promoters(Cp,Xp,S1p,S2p and Fp)with luciferase reporter gene were transfected into HepG2 cells respectively.Then the activities of viral promoters were examined by luciferase reporter assay.It was found EHP effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner,as well as the extracellular HBV DNA.And EHP could selectively inhibit the activity of HBV promoter Fp.Our data suggest that EHP exerts anti-HBV effects via inhibition of HBV transcription,which helps to elucidate the mechanism underlying the potential therapeutic value of EHP.

Objective To determine whether coagulase-negative non-epidermal staphylococcus is methicillin-resistant coagulase-negative staphylococcus by mecA gene test,when the minimal inhibitory concentration(MIC)of oxacillin is between 0.5-2.0 mg/L.Methods The mecA gene was detected and analyzed by the cefoxitin disk diffusion,E-test,VITEK-2 Compact and polymerase chain reaction (PCR)purification.Results A total of 300 strains of coagulase-negative staphylococci were screened from 4032 patients(7.4％),of which 45 strains of Staphylococcus saprophyticus and 80 strains of Staphylococcus hemolyticus were identified by Compact VITEK-2.There was a statistically significant difference in the positive rate of mecA gene detection between Staphylococcus saprophyticus and Staphylococcus hemolyticus(P ＜0.05).The results of detection of cefoxitin disk diffusion(inhibitory zone diameter ≥ 25 mm),E-test(MIC of oxacillin between 0.5-2.0 mg/L)and Compact VITEK-2 (MIC of oxacillin between 0.5-2.0 mg/L)showed that there were 81 strains of coagulase-negative non-Staphylococci,of which 10 strains with positive mecA gene were confirmed by PCR.Conclusions When the minimal inhibitory concentration (MIC)of oxacillin against coagulase-negative non-Staphylococci stains is between 0.5-2.0 mg/L,the guidelines of the American clinical laboratory standardization institute(CLSI)should be strictly implemented in clinical microbiology laboratory and the mecA gene must be tested.Based on the wide dissemination of the mecA gene in Staphylococcus aureus population,if the mecA gene test is negative,it is reported as methicillin-susceptible coagulase-negative Staphylococcus(MSCNS),and the reverse result is reported as methicillin-resistant coagulase-negative staphylococcus(MRCNS).

Objective:To construct four retroviral plasmids for differential expression of green fluorescent protein (GFP) based on different terminal sequences of internal ribosome entry site (IRES) and provide reference for subsequent flow analysis or imaging.Methods:Based on the fact that the transcription efficiency of encephalomyocarditis virus IRES depends on its terminal sequence, IRES and enhanced GFP (EGFP) were fused into four fragments with different connection modes by overlapping PCR, and then cloned into retroviral plasmid pMSCV-NGFR. NGFR fragment was amplified by PCR and inserted in front of the retroviral plasmids pMSCV-IRES(1-4)-EGFP. These retrovirus plasmids pMSCV-NGFR-IRES(1-4)-EGFP were transfected into 293T cells. The expression ratio and mean fluorescence intensity (MFI) of EGFP were analyzed, and the expression of NGFR was also detected.Results:Four retroviral plasmids pMSCV-NGFR-IRES(1-4)-EGFP were successfully constructed. No significant difference in the expression efficiency of EGFP at 24 or 48 h was observed in 293T cells transfected with the four different retroviral plasmids, but there was significant difference in fluorescence intensity. Moreover, the expression of NGFR was not significantly different, indicating that the addition of different nucleotide sequences between IRES and EGFP would make a significant difference in the fluorescence intensity of EGFP.Conclusions:The expression intensity of EGFP was affected by the sequence between IRES and EGFP. Retroviral plasmids expressing EGFP of different intensity could meet different experimental requirements.