Modified pig alpha-interferon genes, construction of expression vector and preparation of protein thereof

Abstract

The invention discloses construction of modified porcine alpha-interferon gene and an expression vector as well as a method for making protein of the modified porcine alpha-interferon gene. A sequence table of the modified porcine alpha-interferon gene is shown in SEQ ID NO.1. The invention discloses a method for making the protein of the modified porcine alpha-interferon gene at the same time, relating to the steps of reformation of the porcine alpha-interferon codon, clone of the gene and high expression in pichia, in particular to improvements on operation methods relevant to matching and reformation of yeast codon preferendum by porcine alpha-interferon codon; a culture method for effectively producing porcine alpha-interferon; measures for preventing pollution; the control of determining quantity and the time of adding methanol and the time of protein expression, namely protein harvest time; asepsis examination; the test of protein content and activity, and a method for enlarging culture condition. The method is easy to operate with low cost, thereby realizing the stable and effective expression of the porcine alpha-interferon in pichia, and improving as much as 300 times of an antiviral activity of the interferon.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the modification of porcine α-interferon gene, the construction of expression vector, its high-efficiency expression in Pichia pastoris and its protein preparation method. Background technique [0002] Interferon (IFN) is a kind of protein with a wide range of biological activities. It has various functions such as regulating the body's immune function, anti-virus and anti-tumor, and is an important part of the body's defense system. A large number of in vitro and in vivo tests have shown that porcine interferon has defensive and inhibitory effects on viral infectious diseases that are a major threat to production. Interferon has broad application prospects due to its wide range of effects and small residues. However, due to the high cost of interferon and the economic value of animals, its clinical application in veterinary medicine is mainly limited to t...

AI Technical Summary

Problems solved by technology

[0004] Although the use of yeast to express interferon has achieved some results, how to modify the interferon gene so that it matches the preference of yeast to increase the expression of interferon; how to cultivate during the expression process and how to ensure the protein Dissolved oxygen required for expression, how to avoid contamination, the amount and time of adding methanol to induce, the control of protein expression time, that is, the protein harvest time, the method of sterility test, the determination of protein content and activity, etc. are all quite different. Failure to form a stable and complete technical solution is not conducive to the development of industrialized production of porcine α-interferon, and cannot fundamentally solve the cost problem of porcine α-interferon, which affects its popularization and application

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