Construction of luminescent bacteriophage using CRISPR technology and its application in Escherichia coli indentification / 中华检验医学杂志

Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.

The expression characteristic of T regulatory cell, T help cell 17 and the correlation to disease progression in chronic hepatitis B patients undergoing antivirus treatment / 中国医师进修杂志

Objective To explore the expression characteristic of T regulatory cell (Treg)/T help cell (Th)17 and the correlation to disease progression in chronic hepatitis B (CHB) patients undergoing antivirus treatment,and to explore their roles in pathogenesis of CHB.Methods A total of 53 patients with CHB (CHB group) and 21 healthy controls (healthy control group) were selected,CHB patients included mild 18 cases,moderate 16 cases and severe 19 cases.The expression of Treg and Th17 were detected by flow cytometry and compared.The levels of alanine aminotransferase (ALT),total bilirubin (TBIL),cholinesterase,albumin were measured by automatic biochemical machine and for correlation analysis.Results The levels ofTh17,Treg,Treg/Th17 were (2.13 ± 0.65)％,(2.99 ± 0.68)％ and (6.07 ± 1.18)％,(5.14 ± 0.96)％ and 2.86 ± 0.67,1.73 ± 0.45 in CHB group before and after treatment for 24 weeks and (3.59 ± 0.75)％,(4.02 ± 0.77)％,1.04 ± 0.34 in healthy control group.There were significant differences (F =14.78,10.12,17.19; P ＜ 0.01).The level of Th17 in mild,moderate and severe CHB was gradually decreased,but there was no significant difference (F =1.10,P =0.337).The level of Treg in mild,moderate and severe CHB patients was gradually increased,but there was no significant difference (F =0.54,P =0.585).The level of Treg/Th17 was gradually increased with aggravation (2.58 ± 0.59,2.76 ± 0.63,3.21 ± 0.71),and there was significant difference (F =3.15,P ＜ 0.01),the level of Treg/Th17 in severe CHB patients was significantly higher than that in mild,moderate CHB patients (P ＜0.05).The level of Treg/Th17 had significantly positive correlation with ALT,TBIL (r =0.272,P=0.000; r =0.226,P=0.000).Conclusion Treg/Th17 balance not only relates with the pathogenesis of CHB,but also with the related immune inflammatory of liver tissue.

Optimization and application of dye real-time fluorescent polymerase chain reaction for detecting αβT lymphocyte clones in human peripheral blood / 中华传染病杂志

Objective To investigate the optimized parameters of dye (SYBR Green Ⅰ) realtime fluorescent polymerase chain reaction (RF-PCR) for detecting αβT lymphocyte clones in the peripheral blood and its application in monitoring specific T cell clone in the peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis B (CHB). Methods The total RNA was extracted from the PBMC of six healthy donors, and was reversely transcripted into cDNA. Then the cDNA was amplified using RF-PCR with the primers specific for T cell receptor β viable region (TCRBV) gene families as upstream primers and the primer for T cell receptor (TCR) β constant region (TCRBC) as downstream primer. The annealing temperature,concentration of primers and the total number of cycles were comparatively analyzed. The optimized PCR was performed to investigate the 24 TCRBV gene families from 12 patients with CHB, and the PCR products were monitored by melting curve analysis, and the clone expansion of peripheral blood T cell was detected by peak-motif of melting curve analysis. Results The optimized annealing temperature, final premier concentration,the number of cycles were 60.6 ℃, 0.5 μmol/L and 40 cycles, respectively. The begin temperature for melting curve analysis was better as 80 ℃ compared to 75 ℃. There was mono-peak on melting peak chart for TCRBV gene families in PBMC from patients with CHB, and PCR products of the single peak were determined as monoclonal T cell by sequencing. Conclusions The optimized reaction parameters of RF-PCR for monitoring 24 TCRBV gene families are determined. The melting peak chart could be used to monitor the clone expansion of the peripheral lymphocytes and to detect the clone-specific T cells in the peripheral blood from patients with CHB.

Study on Expression of Fas and Fas Ligand on Peripheral Blood Lymphocyte and Soluble Fas in Pa-tients with Vitiligo / 中华皮肤科杂志

Objective To investigate the role of Fas antigen,Fas ligand(FasL)and soluble Fas(sFas)in the pathogenesis of vitiligo.Methods The expression of Fas and FasL on peripheral blood lym-phocyte(PBLC)was detected by flow cytometry.Serum sFas was quantitated using a dual antibody sandwich enzyme-linked immunosorbant assay(ELISA).Results The expression of Fas and FasL was significantly de-creased in patients with vitiligo vulgaris(43.45%and58.40%)than those in normal controls(58.30%and64.65%)(P

Expression of Costimulatory Molecules CD80and CD86on Peripheral Blood Lymphocytes in Patients With Systemic Lupus Erythematosus / 中华皮肤科杂志

Objective To investigate the expression of costimulatory molecules CD80and CD86on peripheral blood lymphocytes(PBLC)in patients with systemic lupus erythematosus(SLE)and its significance.Methods The expression of CD80and CD86on PBLC was measured by flow cytometric assay in30patients with SLE and25normal controls.Results Expression of CD86on PBLC in patients with SLE was significantly lower than that of normal controls(P

Changes of Circulating Platelet-Derived Growth Factor and Granulocyte Colony-Stimulating Factor in Patients with Psoriasis Vulgaris / 中华皮肤科杂志

Objective To investigate the relationship between platelet-derived growth factor(PDGF)and granulocyte colony-stimulating factor(G-CSF)and psoriasis vulgaris.Methods Sera were taken from60patients with psoriasis vulgaris and42healthy controls.The serum levels of PDGF and G-CSF were measured by a dual antibody sandwich enzyme-linked immunosorbent assay(ELISA).Clinical severity of psoriasis vul-garis was assessed by psoriasis area and severity index(PASI)score.Results The serum PDGF levels were significantly higher in patients with psoriasis vulgaris than those in normal controls(P