Active lipoprotein-associated phospholipase A2 of natural enzymes and procedure for preparing active lipoprotein-associated phospholipase A2
A technology of preparation process and enzymatic activity, applied in the direction of anti-enzyme immunoglobulin, serum immunoglobulin, immunoglobulin, etc. PLA2 enzyme activity inhibitor and other problems, to achieve the effect of wide application, strong specificity and high detection sensitivity
Inactive Publication Date: 2016-01-06
CHANGCHUN HENGXIAO BIOTECH CO LTD
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Problems solved by technology
[0011] The double-antibody sandwich ELISA is used to determine the content of LP-PLA2 in the sample, but cannot measure the activity of LP-PLA2 enzyme, and cannot be used to screen inhibitors of LP-PLA2 enzyme activity
The method of directly measuring LP-PLA2 enzyme activity in a sample with an enzyme substrate can be used to screen plant extracts for LP-PLA2 enzyme activity inhibitors, but it has great defects, mainly because plant extracts may reduce the detection results of LP-PLA2 enzyme activity. It cannot be determined whether these substances interfere with the enzyme substrate, or affect the function of LP-PLA2 enzyme, and cannot be determined to be LP-PLA2 specific inhibitors
Method used
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example 1
[0357] Example 1: 100ng / mlFlag-LP-PLA2 enzyme activity (IU)=K(OD 4min –OD 3mim / 4min-3mnin) / 0.02ml
[0358] 100ng / ml Flag-LP-PLA2 enzyme activity (IU)=85.1nmol (0.294-0.218 / 4-1min) / 0.02ml=323nmol / min / ml.
[0359] 10. Production of LP-PLA2 Antibody
[0360] 1. (Day0) Collect 2ml NewZealand rabbit blood
[0361] 2. (Day1) 1ml Flag-Lp-PLA2 (0.4mg / ml) was mixed with 1ml Freund's complete adjuvant (CFA), and subcutaneously injected into NewZealand rabbits at 10 points (0.2ml / each point)
[0362] 3. (Day14) 0.5ml Flag-Lp-PLA2 (0.4mg / ml) was mixed with 0.5ml Freund's incomplete adjuvant (IFA), and subcutaneously injected into NewZealand rabbits at 5 points (0.2ml / each point)
[0363] 4. (Day28) Mix 0.5ml Flag-Lp-PLA2 (0.4mg / ml) with 0.5ml Freund's incomplete adjuvant (IFA), and inject subcutaneously into NewZealand rabbits at 5 points (0.2ml / point)
[0364] 5. (Day35) Collect 25ml NewZealand rabbit blood
[0365] 6. (Day42) Mix 0.5ml Flag-Lp-PLA2 (0.4mg / ml) with 0.5ml Freund’s ...
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The invention discloses active lipoprotein-associated phospholipase A2 of natural enzymes and a procedure for preparing the active lipoprotein-associated phospholipase A2, and belongs to the field of biotechnologies. By the aid of the active lipoprotein-associated phospholipase A2 of the natural enzymes and the procedure, LP-PLA2-N end antibodies can be specific to LP-PLA2-N ends without interfering the enzyme activity of enzyme catalysis structural regions of LP-PLA2 C ends and are suitable for antibody LP-PLA2 capturing enzyme activity determination tests, so that LP-PLA2 or LP-PLA2-micromolecule compounds in blood can be captured, and the enzyme activity of the LP-PLA2 can be specifically determined. The procedure includes steps of constructing pVL1392-Flag-LP-PLA2 vectors; carrying out co-transfection on the pVL1392-Flag-LP-PLA2 vectors and linear AcNPV (autographa califomica nuclear polyhedrosisvirus) DNA (deoxyribonucleic acid) to generate recombinant Flag-LP-PLA2 AcNPV; expressing the eight short peptides LP-PLA2 with amino acid residues; purifying the short peptides to obtain Flag-LP-PLA2 proteins with the natural enzyme activity and the antigenicity; constructing pVL1392-His-LP-PLA2-N vectors; preparing His-LP-PLA2-N end protein affinity chromatography columns; obtaining anti-LP-PLA2-N end antibodies by means of separation and purification. The active lipoprotein-associated phospholipase A2 and the procedure have the advantage that a method for determining the enzyme activity of the LP-PLA2 is high in specificity and detection sensitivity and wide in application.
Description
technical field [0001] The invention belongs to the field of biotechnology. Background technique [0002] Lipoprotein-associated phospholipase A2 (Lipoprotein-associated phospholipase A2, LP-PLA2) is a kind of PLA2 in the phospholipase family, a secreted protein (45KDa) with 441 amino acid residues, which circulates in the blood in the form of enzyme activity. LP-PLA2 is a serine-dependent phospholipase, and its catalytic activity does not require calcium ions. The enzyme active region of LP-PLA2 is located at the C-terminus of the protein, and the amino acid residues S273, D296, and H351 constitute the core site of enzyme catalytic activity; amino acid residues 189-239 form a sheet structure, which is involved in controlling the specificity of the enzyme substrate, and is low Density lipoprotein (LDL) binding domain; 367-370 amino acid residues involved in high-density lipoprotein (HDL) binding. [0003] There are two sources of LP-PLA2 in the blood, one is produced and s...
Claims
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Login to View More IPC IPC(8): C12N9/18C12N15/866C07K16/40C07K16/06C07K1/22
CPCC12N9/18C07K16/065C07K16/40C12Y301/01004
Inventor 关大伟李宝民刘木清于顺蔡燕宁高大松关恒赵雪
Owner CHANGCHUN HENGXIAO BIOTECH CO LTD