Construction of human paraoxonase 3 gene expression vector

A paraoxonase and gene technology, applied in the field of biotechnology and pharmacy, can solve the problems of long-term multiple medication, slow onset of action, short action time, etc. Effect

Inactive Publication Date: 2008-08-13
NANJING UNIV
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Problems solved by technology

However, most of them have shortcomings such as short action time, slow onset, and long-term multiple medications. Using gene therapy to introduce liver-pr

Method used

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  • Construction of human paraoxonase 3 gene expression vector
  • Construction of human paraoxonase 3 gene expression vector
  • Construction of human paraoxonase 3 gene expression vector

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Embodiment Construction

[0039] 1. The pMD18T-hPON3 plasmid was constructed and preserved by our laboratory. Using pMD18T-hPON3 as a template, primers 1 and 2 were used to amplify the full-length gene of hPON3 with a total of 1065 bp by PCR, and inserted into the corresponding multiple cloning site of the pCI plasmid after double digestion with EcoR I and SalI. The hPON3 gene is under the control of the CMV promoter. Sequencing proved that the result was completely consistent with the designed sequence (see Figure 1 for the process flow). Transform pCI-hPON3 with CaCl 2 The competent TOP10 Escherichia coli was prepared by the method, and the Escherichia coli was amplified according to the operation process of the third edition of "Molecular Cloning", and a large number of plasmids were extracted by the PEG method. The purity and yield of the extracted plasmids were determined by UV spectrophotometry, and the plasmid concentration was adjusted to 2 μg / μl with PBS. All biochemical reagents were purch...

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Abstract

The invention belongs to biotechnology pharmacy technology field. The recombination plasmid Pci-Hpon3 expressed in zooblast is constructed by used human mintacol enzyme 3 gene (Hpon3), Hpon3 is expressed efficiently and secreted into blood in small murine skeletal muscles organizing in electric transferring method to improve PON3 in blood serum and enforce the ability of small mouse antioxidation pressure so that it can control liver damage efficiently. The activity of Hpon3 transgene expression small mouse blood serum hydrolytic decomposition melilotol is improved by 1.4 times and can maintain 24 days. After small mouse acute or subacute liver damage mould is built by injecting carbon tetrachloride into abdominal cavities of transgene group and normal small mouse, the blood serum ALT and AST level of transgene small mouse are lower greatly, malondialdehyde (MDA) level of liver homogenate is lower, reducing glutathione (GSH) and total antioxidant ability (T-AOC) are higher than contrasting group, and transgene group small mouse liver cell damage extent indicated in nosology slicer result is lower than CC1(4) control group's indicate that recombination human PON3 plasmid transgene expression can improve efficiently blood serum PON3 activity and reduce liver oxygenizing pressure so that it can be used for preventing and treating liver damage.

Description

1. Technical field [0001] The invention belongs to the technical field of biotechnology and pharmacy. 2. Background technology [0002] Paraoxonase 3 (PON3) is the latest discovered and least studied member of the paraoxonase gene family. The full length of human PON3 gene cDNA is 1075bp, and the coding region is 1065bp in length, encoding 354 amino acids. The gene expression product PON3 is a glycoprotein with a molecular weight of about 40kDa. It is a calcium ion-dependent esterase with aromatic esterase activity, lactonase activity and antioxidant function [Lu H Q, et al. Biochem Pharmaco, 2005, 70: 1019-1025]. Human PON3 is mainly synthesized and secreted into the blood by hepatocytes, and 98% of it is combined with HDL in serum, but the content of PON3 in serum is only 1 / 50 of that of PON1. Experiments have proved that PON3 not only inhibits the oxidative modification of HDL and LDL, but also has stronger antioxidant capacity. The ability of purified rabbit serum PON...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/55C12N15/89C12N9/16A61K48/00A61P1/16
Inventor 秦浚川彭薇吕海芹臧宇辉朱洁张驰姜晓玲
Owner NANJING UNIV
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