Development and verification of an FLP/FRT system for gene editing in Bacillus licheniformis / 生物工程学报

Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.

Pathological observation and serum biochemical indexes of traumatic osteoarthritis rat / 中华地方病学杂志

Objective To observe the relationship between serum hydroxyproline,nitric oxide (NO) and cartilage oligomeric matrix protein (COMP) and severity of traumatic osteoarthritis in rats,so as to provide scientific basis for prevention and treatment of Kaschin Beck disease.Methods Thirty-six healthy six-week-old male Wistar rats were divided into 3 groups according to weight via the random digital table method,including control group,ligamentum cutting group,ligamentum cutting + exercise group,with 12 rats in each group.Except the control group,the other two groups of rats were treated by anterior cruciate ligament transaction (ACLT).After 7 days,the ligamentum cutting + exercise group was tested at a 6-animal-treadmills for 20 min per day for 4 weeks.At the tenth week after operation,the surgical unilateral knee joints were taken;under a biological optical microscope,the gross joint morphology of each group was compared,which was scored according to C.Colombo articular cartilage pathological evaluation parameter standard.Rat serum was separated,and the contents of hydroxyproline,NO and COMP were determined.Results There were statistically significant differences among the 3 groups in the pathological evaluation of knee cartilage in rats (F =38.80,P ＜ 0.01).The scores of pathological evaluation of ligamentum cutting group,ligamentum cutting + exercise group were significantly higher than that of the control group (14.82 ± 6.19,18.25 ± 5.71 vs 1.58 ± 0.99,P ＜ 0.01).There was no statistical significant difference in comparison of ligamentum cutting group and ligamentum cutting + exercise group (P ＞ 0.05),and there was no statistical significant difference in serum hydroxyproline content of the 3 groups (F =2.15,P ＞ 0.05).There were statistical significant differences in serum NO and COMP of the 3 groups (F =36.13,16.77,P ＜ 0.01).Conclusions Traumatic osteoarthritis is caused by combining ACLT with movement,and serum levels of NO and COMP are closely related to the severity of osteoarthritis.Detections of NO and COMP are introduced into the research field of Kaschin-Beck disease,which will provide experimental basis for monitoring and treatment of Kaschin-Beck disease.

The value of 64-row helical CT multi-phase enhancement scan combined with angiography in the diagnosis of pulmonary mass / 重庆医学

Objective To explore the value of 64-row helical CT multi-phase enhancement scan combined with angiography (CTA)in the diagnosis of pulmonary mass.Methods Two hundred and sixty-five patients with pulmonary mass confirmed by pa-thology were checked,analyzed the CT sign of multi-phase enhancement scan and the blood supply of pulmonary mass displayed by CTA.Results Lung cancer was mainly supplied by bronchial arteries,some by body arteries,the feeding arteries display rate of lung cancer group was significantly higher than that of benign disease group(P 0.05).Enhancement dynamic curves of lung cancer group was differ-ent from benign lesion groups:Lung cancer without obvious enhancement in pulmonary artery phase,CT value increased rapidly in aorta phase,120 s reached peak,and declined slowly in delay phase;CT value of tuberculoma was increased slowly without obvious peak;CT value of inflammatory increased gradually in pulmonary artery phase,90 s reached the peak;hamartoma was no obvious enhancement;Hemangioma enhanced rapidly after strengthening in the pulmonary artery phase,reached the peak at about 15 s,and then decreased slowly.Conclusion 64-row helical CT multi-phase enhancement scan combined with angiography have important clinical value,which can differentiate malignant mass from benign ones.