Objective To construct an Escherichia coli outer membrane protein-A (OmpA) gene-deleted strain by Red homologous recombination, and laid the foundation for subsequent research. Methods Polymerase chain reaction (PCR) primers were designed according to the known OmpA gene sequence, and plasmid pKD3 for PCR amplification and integration; the fragment was transformed into Escherichia coli by λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation OmpA protein knocked out was observed by Western-blotting. Results The knock out gene product was correspond to a expected molecular weight. The western-blotting show that OmpA protein was knocked out. The difference in growth curve between the wild type and Escherichia coli △ OmpA gene-deleted strain was not significant. Conclusion OmpA gene deletion had no significant effect on the growth of Escherichia coli, which provides a foundation for further research on live vector vaccine.
ObjectiveTo construct a prokaryotic expression strain of Staphylococcus aureus fibronectin binding protein A (FnBPA) r10-11 truncated fusion protein, and explore the immunogenicity of FnBPAr10-11. MethodsPloymerase chain reaction (PCR) amplification was carried out from the whole genome sequence of Staphylococcus aureus Newman strain by recombinant PCR technique. The amplified product was purified and transformed into Escherichia coli DH5α for cloning. The recombinant plasmid was extracted and identified by double enzyme digestion. The recovered fragment was ligated into the pET-32a plasmid and transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The FnBPAr10-11 was purified by HIS protein purification column, identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and used to immunize mice, and the mice were divided into phosphate buffered saline (PBS) group, FnBPA group, and FnBPAr10-11 group. The serum levels of immunoglobulin G (IgG) and cytokines, and the immune protection rate of the mice were detected. ResultsSDS-PAGE result showed that the relative molecular mass of the protein was about 33.1×103. The titers of IgG antibody in FnBPAr10-11 group and FnBPA group reached 1∶128 000, and were significantly different compared with PBS group (P<0.05). The cytokine level in FnBPAr10-11 group was not significantly different compared with that in FnBPA group, and they were extremely significant (P<0.01) compared with that in PBS group. The immuno-protective effect of the FnBPAr10-11 group was over 50%. ConclusionsThe prokaryotic expression strain of Staphylococcus aureu FnBPAr10-11 truncated fusion protein was successfully constructed. The truncated protein has good immunogenicity.
Objective To establish a modeling method for an animal model simulating the decline of digestive function after a large amount of tissue necrosis of the pancreas due to acute injury after severe acute pancreatitis (SAP). Methods Twenty male SD rats were randomly divided into the model group and the sham operation group according to the random number table method, with 10 rats in each group. First, the SAP model was established by retrograde bile duct injection of sodium taurocholate in the model group, whereas the sham operation group received physiological saline injection. Fluid infusion began 2 hours later, twice a day, with an 8-hour interval, for 2 days. The traditional Chinese medicine Dachengqi Decoction without Decoction Granules was formulated into a suspension in proportion and administered by gavage once at 18 hours and once at 24 hours after the operation to ensure the blood volume of rats and reduce inflammatory damage. Normal drinking water was allowed 48 hours after modeling. After 72 hours, ordinary feed was given for feeding. The feeding lasted for 14 days (the total duration of the experiment was 17 days). The body weight, vitality status and stool characteristics of the rats were observed and recorded on the day of open feed feeding and 14 days later. Fourteen days after feeding, the animals were sacrificed and samples were collected for examination of blood glucose, fecal elastase and hematoxylin-eosin staining pathological scores. Results All 10 rats in the model group were successfully modeled with a 100% survival rate. The body weight of rats in the model group 14 days after ordinary feeding was lower than that on the day of open diet [(180.80±4.39) vs. (222.90±6.14) g, P<0.001], and lower than that of rats in the sham operation group 14 days later [(180.80±4.39) vs. (221.70±7.45) g, P<0.001]. Compared with the sham operation group, inflammatory cell infiltration injury still existed in the pancreatic tissue of the model group, and some pancreatic tissues showed pathologically related changes of chronic injury. The pathological score of the model group was higher than that of the sham operation group [7.5 (7, 9) vs. 0 (0, 0), P<0.001]; the blood glucose concentration increased [(13.000±1.531) vs. (8.070±0.851) mmol/L, P<0.001]. The secretion of fecal elastase, a metabolite of trypsin in vivo, was significantly decreased [(5.451±0.936) vs. (8.593±1.105) mg/mL, P<0.001]. Conclusion The use of short-term liquid supplementation, traditional Chinese medicine anti-inflammatory treatment, and early dietary stimulation can effectively combat early severe inflammatory damage in SAP, protect the life of model rats, and enable them to survive and experience digestive dysfunction, thus establishing an experimental animal model of digestive dysfunction in the late stage of SAP.