Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.
Cloning, Molecular ; Escherichia coli/genetics* ; Humans ; Recombinant Fusion Proteins/genetics* ; Recombinant Proteins ; Tissue Inhibitor of Metalloproteinase-2/genetics*

Objective:To explore the relationship between sleep status and the disease in children with recurrent vertigo（RVC） by analyzing the objective sleep condition of children with recurrent vertigo. Methods:According to the diagnostic criteria of RVC, 50 children with RVC and 20 normal controls without RVC were selected. According to the vertigo questionnaire score, the RVC group was divided into mild, moderate and severe groups according to severity. Continuous polysomnography（PSG） was performed for all participants, and SPSS 25.0 statistical software was used to analyze the monitoring results. Results:①There were significant differences in sleep time of each period, total sleep time and sleep efficiency between RVC group and control group（P<0.05）, but there was no significant difference in sleep latency（P>0.05）. The specific manifestations were that the proportion of sleep time in N1 and N2 phases increased, the proportion of sleep time in N3 and REM phases decreased, the total sleep time and sleep efficiency decreased in RVC group. ②The abnormal rate of sleep apnea hypopnea index, that is, the proportion of AHI≥5 times/h and the abnormal rate of lowest blood oxygen saturation in RVC group were higher than those in normal control group. There was significant difference between the two groups（P<0.05）. ③There were significant differences in the proportion of AHI≥5 times/h and lowest SpO2 among mild group, moderate group and severe group（P<0.05）. ④There was no significant correlation between the degree of vertigo and the abnormal rate of AHI in children with RVC, but there was a negative correlation between the degree of vertigo and the abnormal rate of lowest SpO2 in children with RVC. Conclusion:Children with RVC are often accompanied by sleep disorders, clinicians should pay attention to both the symptoms of vertigo and sleep condition in children. Polysomnography is non-invasive and operable, providing a new idea to the auxiliary examination of RVC in children. It is of certain clinical significance for the comprehensive treatment of children with RVC to actively improve vertigo symptoms and pay attention to improving sleep quality.
Humans ; Child ; Polysomnography ; Sleep Apnea, Obstructive/diagnosis* ; Sleep ; Dizziness ; Vertigo/diagnosis*