Background[1-3]
Human paraoxonase (PON) ELISA KIT uses a double-antibody sandwich method to determine the level of human paraoxonase (PON) in specimens. Use purified human paraoxonase (PON) antibody to coat the microwell plate to make a solid-phase antibody. Add paraoxonase 1 (PON1) to the microwells coated with monoclonal antibody, and then mix it with HRP-labeled paraoxonase 1 (PON1). The phosphatase (PON) antibody is combined to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of color is positively correlated with the paraoxonase (PON) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm, and calculate the concentration of human paraoxonase (PON) in the sample through the standard curve.
The paraoxonase (PON) gene family has at least three members, including PON1, PON2 and PON3, which are located on chromosome 7q21.3-22.1. The PON1 gene product, also called serum paraoxonase, not only plays an important role in the detoxification of organophosphorus nerve agents, but is also closely related to the onset of atherosclerosis, coronary heart disease, type 2 diabetes and other diseases. Serum paraoxonase can catalyze the hydrolysis of phosphate bonds and degrade organophosphates, aromatic carboxylic acid esters and carbamates, which is the molecular basis of PON1’s protective effect on organophosphorus compound poisoning.
However, the affinity and conversion rate of PON1 for organophosphorus in mammals are very limited. Therefore, during acute organophosphorus poisoning, PON1 has no chance to play a protective role. Only at low levels and chronic poisoning, PON1 can There is time and opportunity to make a difference. Animal and in vivo experiments with serum paraoxonase suggest that this protein may have important pathophysiological and pharmacological effects. With the development of molecular biology technology, in vitro expression and gene therapy of PON1 gene products will become possible, allowing it to play an important role in the treatment of organophosphorus pesticide poisoning, cardiovascular disease, and diabetic vascular complications.
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Human paraoxonase (PON) ELISA KIT is used to study the expression and enzymatic properties of human paraoxonase 1 in Pichia pastoris.
Human Paraoxonase 1 (hPON1) is a non-specific esterase in human blood that can hydrolyze a variety of organophosphorus compounds. Studies have shown that PON1 has anti-inflammatory and antioxidant properties , anti-diabetic, anti-atherosclerosis, and rescue organophosphorus poisoning, and PON1 has better detoxification effects than existing organophosphorus detoxifiers. The price of Tosoh matting agent in Japan. However, purifying PON1 from natural sources is difficult to operate and has low efficiency; while using the E. coli expression system, although the expression level of foreign proteins is high, it is mostly expressed in the form of inclusion bodies, which requires renaturation, and there are often problems with protein folding. hPON1 was expressed intracellularly using the Pichia pastoris expression system. First, the hPON1 gene was cloned into the pPICZA expression vector after codon optimization, and the pPICZA-PON1 recombinant expression plasmid was constructed. After linearization, it was transformed into the Pichia pastoris X33 strain, and the positive recombinant strain was screened. The recombinant strain was fermented in shake flask for 120 h, and the enzyme activity was 0.15 U/mL. After collecting the fermentation broth and cell lysis, it was purified using Ni-NTA chelate affinity chromatography to obtain purified hPON1. The SDS-PAGE results showed that the size of the expression product was about 37 kDa, which was consistent with the expected protein molecular weight. Secondly, a strategy of effective combination of single factor experiment and Box-Behnken combinatorial design method was used to optimize the fermentation conditions expressed in Pichia pastoris to increase the expression of rhPON1. The best optimization conditions are: methanol concentration 1.42%, initial culture medium pH value 5.91, culture medium filling volume 9.39%, and the predicted maximum theoretical enzyme activity of rhPON1 is 0.737 U/mL. The rhPON1 enzyme activity obtained under the best optimized conditions was 0.735 U/mL, which was 1.78 times the enzyme activity under pre-optimized conditions. At the same time, the effect of the initial induction bacterial concentration on the expression of rhPON1 was also investigated. Appropriately increasing the initial induction bacterial concentration can promote the expression of rhPON1 during the induction process, providing a basis for further optimizing the expression of rhPON1 in Pichia pastoris.
