5种食源性致病微生物毒力基因重组质粒的构建、克隆表达与表达产物的研究

目的构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌5种主要食源性致病微生物毒力基因重组质粒载体并鉴定其表达产物,为探讨高通量磁性荧光纳米颗粒标记相应的抗体技术快速检测带毒力基因的致病微生物的可行性奠定基础。方法分别从5种食源性致病微生物毒力岛中选择特异性的毒力基因进行引物设计,分别提取5种目的细菌DNA片段,经PCR扩增、电泳回收目的基因片段,将目的基因片段与原核表达载体pET-23a、pET-28a、pET-32a构建各自的重组质粒,将重组质粒转化入大肠杆菌DH5α内并提取质粒载体,构建后的重组质粒进行酶切和测序鉴定,再转化入表达宿主E·coliBL21（DE3）。在0.1 mmol/L的IPTG诱导下,目的质粒载体在E·coliBL21（DE3）株中表达,SDS-PAGE电泳检测表达蛋白。结果实验成功构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌等细菌的毒力基因重组质粒载体pET-23a-eaeA、pET-28a-tdh、pET-28a-enterotoxin B、pET-32a-invA和pET-28a-ipaH,并克隆出969 bp的eaeA基因片段、567 bp的tdh基因片段、798 bp的enterotoxin B基因片段、1 041 bp的invA基因片段和771 bp的ipaH基因片段。重组质粒载体经酶切和测序与目标基因序列一致。在E·coliBL21（DE3）株中有质粒表达蛋白37.5 kDa（eaeA）、26 kDa（tdh）、34.5 kDa（enterotoxin B）、41 kDa（invA）、32 kDa（ipaH）。结论本研究成功构建了5种食源性致病微生物毒力基因重组质粒并在原核细胞中高效表达,为下一步获得相应的毒力基因抗体及快速诊断试剂的研制应用奠定了基础。     

Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge

Melioidosis is a fatal disease caused by Burkholderia pseudomallei. Currently there is no vaccine available. Synthetic oligodeoxynucleotides with unmethylated CpG dinucleotide motifs (CpG ODN) can stimulate vertebrate immune cells and clear certain pathogens that are susceptible to a strong Th1 response. In our previous study, pretreatment with CpG ODN alone or CpG-ODN with cationic liposomes for 2–10 or 30 days before B. pseudomallei infection in mice conferred 80–100% protection. In the present study we investigated the protective effect of CpG-ODN together with heat-killed (HK) or paraformaldehyde-killed B. pseudomallei (PP). HK or PP were used to immunize BALB/c mice twice at 15-day intervals before intra-peritoneal challenge with 5LD50 of B. pseudomallei and observed for 30 days. We found that PP could significantly protect mice (60%) with an increased survival time (24.8 ± 11.63 days) while in the HK and PBS groups, all infected mice died within 6 days. Although either CpG ODN or PP conferred significant protection, giving them in combination did not enhance it further. Serum IFN-γ levels on day-5 (before challenge) of the PP and PP + CpG ODN groups were significantly higher than those of the PBS control group. The results further support the importance of IFN-γ in host protection against B. pseudomallei and suggest further study on paraformaldehyde-killed bacteria as a component of a future B. pseudomallei vaccine.     

Polarized immune responses modulated by layered double hydroxides nanoparticle conjugated with CpG

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.     

Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients

Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.Methods: Illumina''s GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.     

幽门螺杆菌cagⅡ对胃上皮细胞IL-8基因转录的影响及机制

目的探讨HpcagⅡ对胃上皮细胞IL-8基因转录的影响及信号传导机制。方法构建 cagⅡ基因位点缺失Hp突变株及带有IL-8报告基因的人胃癌细胞系L5F11,用液体闪烁计数仪测定荧光素酶(IL8转录)活性,用ELISA法测定IL8蛋白浓度。结果所有Hp突变株诱导荧光素酶活性与IL8蛋白浓度较亲代菌株26695均降低[(0．13±0．01)×cpm比(0．59±0．05)×(P<0．01);(0．73±0．13)ng／ml比(2．22±0．65)ng／ml,(P<0．05)]。PTK抑制剂herbimycinA不仅抑制Hp诱导的荧光素酶活性[(0．71±0．18)×cpm比(1．51±0．23)×cpm,(P<0．05)],而且抑制IL-8蛋白表达[(0．83±0．41)ng／ml比(3．22±0．59)ng／ml,(P<0．05)],但herbimycinA对TNFα诱导的荧光素酶活性及IL8蛋白表达均无影响(P均>0．05);PKA抑制剂H7抑制TNFα诱导的荧光素酶活性[(0．74±0．16)×cpm比(2．62±0．26)×cpm,(P<0．001)]及IL8蛋白表达[(1．45±0．38)ng／ml比(4．12±0．43)ng／ml,(P<0．01)],而对Hp诱导的荧光素酶活性无影响(P>0．05)。结论HpcagⅡ中的多基因能够调节胃上皮细胞IL-8基因转录,且这一作用主要经蛋白酪氨酸激酶途径。     

幽门螺杆菌cag致病岛ORF10基因下调胃上皮白细胞介素-8转录

目的　幽门螺杆菌 (Hp)cag致病岛 (cagPAI)含 30多个基因 ,HpcagⅠ与cagⅡ中许多基因能增加胃上皮白细胞介素 8(IL 8)基因转录与IL 8蛋白分泌。本研究观察HpcagⅡ中ORF10基因对胃上皮细胞IL 8基因转录的影响 ,以验证HpcagPAI中某些基因下调IL 8基因转录及表达的可能性。方法　构建cagⅡORF10基因缺失 ( ORF10 )Hp突变株及带有IL 8报告基因的人胃癌细胞系L5F11,用液体闪烁计数仪测定荧光素酶 (IL 8转录 )活性 ,用ELISA法测定IL 8蛋白浓度。结果　 3株 ORF10Hp菌株 (2 8 1,2 8 2 ,2 8 3)诱导荧光素酶活性较亲代菌株 2 6 6 95显著增加 [分别为 2 8 1:(1.4 9± 0 .2 7)× 10 6cpm ,2 8 2 :(1.33± 0 .2 8)× 10 6cpm ,2 8 3:(1.33± 0 .2 9)× 10 6cpm ;亲代菌株2 6 6 95 :(0 .6 7± 0 .0 8)× 10 6cpm ;P分别 <0 .0 1,0 .0 5和 0 .0 5 ];2株 ORF10Hp菌株诱导IL 8蛋白水平较亲代菌株 2 6 6 95均显著升高 [分别为 2 8 2 :(1.2± 0 .0 8)ng/ml;2 8 3为 (1.2 4± 0 .0 7)ng/ml;亲代菌株 2 6 6 95为 (0 .33± 0 .19)ng/ml,P <0 .0 5 ,<0 .0 1]。结论　与HpcagⅠ与cagⅡ中大多数基因上调IL 8转录不同 ,cagⅡ中ORF10基因对胃上皮细胞IL 8转录起下调作用     

CpG寡核苷酸作为黏膜佐剂的研究进展

含未甲基化的CpG二核苷酸的寡聚脱氧核苷酸模体(CpG ODN)具有很强的黏膜佐剂活性,已成为近年来的研究热点之一。应用CpG ODN与各种病毒和细菌抗原进行了大量实验研究,所涉及的黏膜免疫途径包括滴鼻、口服和生殖道。CpG ODN可与传统的黏膜佐剂CT和LT产生协同效应,对人体无毒副作用,是一种极具应用前景的黏膜佐剂。目前认为,CpG ODN的佐剂效应发挥与TLR9识别和后续的一系列信号转导密切相关。