Expression, Purification and Preparation of Polyclonal Antibody of Bombyx mori Serpin-6

2013 ◽  
Vol 796 ◽  
pp. 72-76
Author(s):  
Ming Hui Wang ◽  
Kai-Zun Xu ◽  
Guo Sheng Li ◽  
Yan Yun Wang ◽  
Wei De Shen ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Protease Inhibitor ◽  
Bombyx Mori ◽  
Serine Protease ◽  
Polyclonal Antibody ◽  
Antibody Test ◽  
Serine Protease Inhibitor ◽  
Sds Page ◽  
Practical Tool

Serpin (Serine Protease Inhibitor) widely distributes in animals, plants, protozoan, prokaryotes and viruses. Serpin-6 belongs to the superfamily of serine protease inhibitor. In this study, we constructed a prokaryotic vector - pET28a-serpin-6, and used IPTG (isopropyl thiogalactoside) to induce serpin-6 protein expression, then applied the Ni-affinity chromatography to purify the collected recombinant protein. The assay of SDS-PAGE and Anti-his Polyclonal antibody Test showed that the recombinant serpin-6 protein have a molecular weight of 45 kDa. The highly purified sample of the recombinant protein was obtained. The protein has been used as antigen to immunizeKunMing miceusing the 4 times immunization method. We successfully attained the polyclonal antibody of serpin-6.The titer of the antibody is as high as 1:20000, with a good specificity. The polyclonal antibody can provide a practical tool to further study the distribution of protein expression and functions of serpin-6 in different states and breeds ofBombyx mori.

scholarly journals Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e31168 ◽  
Author(s):  
Ping Zhao ◽  
Zhaoming Dong ◽  
Jun Duan ◽  
Genhong Wang ◽  
Lingyan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Protease Inhibitor ◽  
Bombyx Mori ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Response Analysis ◽  
Genome Wide ◽  
Inhibitor Genes ◽  
Silkworm Bombyx Mori

scholarly journals Proteinase activity in latex of three plants of the family Euphorbiaceae

2014 ◽  
Vol 50 (3) ◽  
pp. 559-565 ◽  
Author(s):  
Andréa Michel Sobottka ◽  
Fabiana Tonial ◽  
Sonja Sytwala ◽  
Matthias Melzig
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Serine Protease Inhibitor ◽  
Polyacrylamide Gels ◽  
Protein Activity ◽  
Sds Page ◽  
The Family ◽  
Molecular Masses

In the family of Euphorbiaceae,the genera Euphorbia and Sapium are known to contain essentially latex-bearing species. In the present study, the latex of Euphorbia selloi(Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil., and Sapium glandulosum (L.) Morong, plants native from Brazil, were examined concerning proteolytic activity. All studied species have proteins with significant proteolytic activity and E. papillosa has the greatest specific activity. Aiming to verify the type of protease present, an assay with different inhibitors was performed. In the three tested plants, the proteolytic activity was significantly inhibited by a serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). Using techniques of electrophoresis with polyacrylamide gels (SDS-PAGE), the subunits of proteins were separated according to their molecular masses, and the protein activity was visually detected by zymography.

scholarly journals Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice

2021 ◽  
Author(s):  
Stefan Wörner ◽  
Bernhard N. Bohnert ◽  
Matthias Wörn ◽  
Mengyun Xiao ◽  
Andrea Janessa ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Kidney Injury ◽  
Serine Protease Inhibitor ◽  
Tubular Function ◽  
Proteolytic Activation ◽  
Salt Diet ◽  
Low Salt ◽  
Proximal Tubular ◽  
Proximal Tubular Function

AbstractTreatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.

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