ABSTRACT
Objective CARD9 can activate several pathways involved in immunity, such as NF-ΚB, MAPK, etc. However the mechanism of this process has not yet been elucidated. For conducting relevant experiments in vitro, a prokaryotic expression vector of CARD9-MBP fusion protein has to been construct, and the fusion protein need to be expressed and purified. Methods The coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-30a(+) vector. The recombinant plasmid was transformed into E.coli DH5α competent cells and proceeded PCR identification, restriction analysis and gene sequencing. The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The target protein was induced to express by IPTG under different conditions. Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis. The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column, and identificated by MALDI-TOF mass spectrometry after MBP-tag to be removed by HRV3C enzyme. Results The CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis. The result of gene sequencing was consistent with the target sequence. The SDS-PAGE electrophoresis showed that the target protein with molecular mass (MR) about 105 000 was successfully induced to express in E.coli BL21 (DE3). A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column. Identification by MALDI-TOF mass spectrometry demonstrated that the target protein after MBP-tag to be removed by HRV3C enzyme is CARD9 protein. In the later stage, gel filtration chromatography column was used to obtain further pure CARD9-MBP fusion protein. Conclusion The prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed. The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.
ABSTRACT
Objective Erythroderma is a very serious disease that affects nearly the entire cutaneous surface and are highly subjected to secondary hypoalbuminemia, infection, cardiovascular diseases, complex causes and high death rates. The article aimed to explore the etiology, comorbidities and complicated infection of erythroderma.Methods Retrospective analysis was conducted on clinical data of 95 cases of erythroderma in our department from January 2009 to August 2016. Observations were made on the patients' clinical characteristics, etiology and inducement, lab examination, complications and complicated infection.Results There were 73 first-episode and 22 recurrent patients, among which 14 cases are psoriasis as the basic disease. As to etiological factors, there were 57 cases secondary to other skin diseases (60%) and 25 cases by drug reactions (26%). As to inducing factors, there were 6 cases by upper respiratory tract infection, 38 cases by irrational application of glucocorticoids, and 7 cases by external stimulants (traditional Chinese medicine scrubbing and external medicinal liquor). The main complications were 38 cases of cardiovascular diseases (40%). The complicated infection rates of plasma albumin in patients <35g/L and ≥35g/L were 65.78% and 12.28%(P<0.01). The complicated infection rates of the patients with hypoalbuminemia and electrolyte disturbance were 44.2% and 25% respectively (P<0.05).Conclusion The erythroderma is mainly secondary to previous skin diseases, mostly psoriasis, with cardiovascular diseases as the main comorbidities. In clinical practice, importance should be attached to monitoring decreased plasma albumin level and electrolyte disturbances in order to reduce the risk of infection.