Plasma SCF/c-kit Levels in Patients with Dipper and Non-Dipper Hypertension.

Objective The aim of this study was to investigate the relationship between peripheral plasma stem cell factor (SCF)/c-kit levels and the types of dipper and non-dipper hypertension in hypertensive patients. Methods This cross-sectional study included newly diagnosed hypertensive patients who underwent 24-hour ambulatory blood pressure monitor (ABPM) between January 2009 and 2012 in Jiangning city. Patients were divided into the dipper group and the non-dipper group according to ABPM measurements. The levels of SCF and its receptor c-kit, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in peripheral blood were measured via enzyme-linked immunosorbent assays. The serum levels of glucose and lipid were examined as well. The levels of SCF/c-kit were compared between the dippers and the non-dippers; and their correlation with 24-hour mean systolic blood pressure (MSBP), 24-hour mean diastolic blood pressure (MDBP), TNF-α and IL-6 were investigated using linear regression analyses statistically. Results A total of 247 patients with newly diagnosed hypertension were recruited into the study, including 116 non-dippers and 131 dippers. The levels of peripheral plasma SCF were higher in non-dipper group (907.1±52.7 ng/L vs. 778.7±44.6 ng/L; t=2.837, P<0.01), and the levels of c-kit were higher in non-dipper group too (13.2±1.7 µg/L vs 9.57±1.4 µg/L; t=2.831, P<0.01). Linear regression analysis revealed that SCF/c-kit levels were significantly positively correlated with MSBP, MDBP, plasma TNF-α, and IL-6 levels (all P<0.01). Conclusions Peripheral plasma SCF/c-kit levels are higher in patients with non-dipper hypertension than those with dipper one, and significantly correlate with 24-hour MSBP, 24-hour MDBP, serum TNF-α and IL-6 levels.

[Expression of alpha-toxin gene of Clostridium perfringens type A and its primary immunological protective function].

Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type A by polymerase chain reaction (PCR). PCR product was inserted into vector pGEM-T directly. The cloned recombinant plasmid pXCPA02 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases Nco I /EcoR I from plasmid pXCPA02, and then inserted into an expression vector pET-28c which cleaved with Nco I /EcoR I by blunt-end ligation. The recombinant expression plasmid pXETA02 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pXETA02 possessed a positive alpha-toxin gene sequence and reading frame. BL21 (DE3) (pXETA02) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies with ELISA and Western blot. The expression optimization result indicated that the alpha toxin gene expression optimized condition with IPTG induction is culture medium pH 7.5, culture temperature 37 degrees C, joining IPTG to final concentration 0.8 mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 34.28% of total cellular protein with IPTG induction by SDS-PAGE and thin-layer gel scanning analysis. The alpha toxin gene expression optimized condition with lactose induction is culture medium pH 7.5, culture temperature 37 degrees C, joining lactose to final concentration 0.1 g/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha-toxin proteins were about 23.82% of total cellular protein with lactose induction by SDS-PAGE and thin-layer gel scanning analysis. More importantly, Immunization in a mouse model with crude preparation containing the alpha-toxin protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type A.

[Fusion of betal-toxin gene and beta2-toxin gene from Clostridium perfringens type C].

Betal-toxin and beta2-toxin genes from chromosomal DNA of Clostridium perfringens type C were amplified by PCR, PCR products were cleaved with restriction endonucleases and recovered. The recombinant plasmid pETXB1-2 containing beta1-beta2 fusion genes was constructed by recombinant technique and then transformed into Escherichia coli BL21 (DE3). The beta1-beta2 fusion proteins were expressed in recombinant strain BL21 (DE3) (pETXB1-2), and the expression level of the beta1-beta2 fusion proteins was about 15.36% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, immunization in a mouse model with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain induced protection against at least 1 MLD of the toxin from Clostridium perfringens type C. Hence the fusion proteins possess a good immunogenicity. The constructed recombinant strain BL21 (DE3) (pETXB1-2) can be used as a candidate of vaccine strain.