The Changes and Correlation of Serum IL-17, IL-6, IL-8,CRP and TNF-α of Patients with Chronic Obstructive Pulmonary Diseases / 昆明医科大学学报

Objective To study the changes and correlation of serum interleukin-17,-6 and-8 (IL-17,IL-6 and IL-8),c-reactive protein (CRP),tumor necrosis factor alpha (TNF-α) of patients with chronic obstructive pulmonary diseases (COPD),and discuss the role and clinical significance of these factors in the occurrence and development of COPD.Methods 100 COPD patients were selected (COPD group),and 50 healthy elderly people in the same period in outpatient medical examination were served as control (control group).The levels ofIL-17,IL-6 and IL-8,CRP,TNF-α in serum were detected with ELISA method.Results The serum levels of IL-17,IL-6,IL-8,CRP and TNF-α of patients in COPD group were higher than those in the control group (P ＜ 0.01),and of patients with acute aggravating period were higher than those of patients with stable period (P ＜ 0.05 or P ＜ 0.01).Pearson correlation analysis showed there were positive correlations among IL-17,IL-6,IL-8,CRP and TNF-α (P ＜ 0.05) Conclusion The serum levels of IL-17,IL-6,IL-8,CRP and TNF-α of COPD patients have abnormal increased,and will increase with the rise of the illness severity.The above factors may be involved in the airway in patients with acute inflammatory reaction and the development process.

Production of human papillomavirus type 16 virus-like particles and its immunogenicity / 病毒学报

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.

The preliminary analysis of the recognition epitopes of anti-HEV monoclonal antibodies on HEV ORF2 / 病毒学报

Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.

Improving baculovirus transduction of mammalian cells by spinoculation / 生物工程学报

Recently many reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages. In this study, the constructed recombinant baculovirus BacV-CMV-EGFPA containing the enhanced green fluorescent protein (eGFP) gene driven by CMV promoter was used to explore the feasibility of improving the efficiencies of transduction experiment in CV-1 cells by centrifugal method. Refer to the centrifugal transduction protocol of recombinant lentivirus, CV-1 cells were incubated with the culture supematant of Sf-21 cells infected by BacV-CMV-ECFPA (moi = 30) and then centrifuged at 600g for 1 h at RT, reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM) 48h post transduction. Results showed that centrifugal method can achieve higher gene delivery and expression efficiencies than transduction by simple virus-cell mixing for 4h at 27 t with least impairment to cell viability. The centrifugal transduction protocol was further optimized by testing different centrifugal times, post-centrifugation incubation times and surrounding solutions. We found that centrifugation at 600g for 1 h at RT is sufficient to achieve the highest transduction efficiencies in target cells and PBS is more suitable than other surrounding solutions. Compared with previous protocol in which tranduction occurs for 4 - 8h at 27 degrees C, centrifugal method developed in this study could achieve more higher transduction efficiencies in more shorter time. Nine different mammalian cell lines (CV-1, 293FT, HepG2, 293T, CHO, C127, MT4, H9, Molt-4) were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the BacV-CMV-EGFPA supernatant (moi = 30) at 600g for 1h in PBS surrounding solution at RT. Results showed that most mammalian cell lines used in this study could be effectively transduced with recombinant baculoviruses by centrifugal method, and more higher and satisfactory transduction efficiencies could be achieved in primate adherent culture cells than in suspended culture cells. These results show that the baculovirus centrifugal transduction protocol have notable advantages: more rapid, efficient and nontoxic, and could be easily used in daily common experiments.