Method for preparing alanine aminotransferase and application

Alanine aminotransferase, alanine aminotransferase encoding technology, applied in the field of genetic engineering and protein expression, can solve the problems of few domestic products, lack of experience and strength of diagnostic reagents, elevated alanine aminotransferase, etc., and achieve high yield of finished products , simple operation and low preparation cost

Inactive Publication Date: 2013-04-03
肖飞
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 3. Alanine aminotransferase will also increase in heavy or long-term drinkers
Almost none of the major domestic manufacturers of diagnostic reagents has the experience and strength to produce corresponding quality control products, and there are very few domestically produced products recognized in the market

Method used

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  • Method for preparing alanine aminotransferase and application
  • Method for preparing alanine aminotransferase and application
  • Method for preparing alanine aminotransferase and application

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Embodiment Construction

[0058] The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0059] The invention establishes a method for preparing alanine transaminase with simple operation, rapid operation and low cost. The principle of this method mainly lies in: inserting the optimized alanine aminotransferase coding gene into the constructed expression vector, and inducing its high-level expression in Escherichia coli, the method is specifically as follows:

[0060] 1. Construction of alanine aminotransferase expression vector:

[0061] Transform the coding gene sequence of huma...

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Abstract

The invention discloses a method for preparing alanine aminotransferase, which includes the following steps: modifying a whole-genome-synthesized humanized alanine aminotransferase encoding gene sequence, and removing rare codons of escherichia coli; inserting the modified alanine aminotransferase encoding gene sequence into an expression vector, and adopting bacterial strains of escherichia coli for transformation and expression; inoculating the transformed and expressed bacterial strains with an LB culture medium and performing IPTG (isopropyl-Beta-d-thiogalactoside) induction; and breaking the obtained thalli, performing centrifugal separation, and passing by the Ni-NTA columns for purification. The operation for preparing alanine aminotransferase is very simple, the preparation cost is low, the product yield is high, the purity is high, and high biological activity is obtained; the prepared alanine aminotransferase serving as antigenic substances can be used for animal immunization, related antibodies can be screened; and meanwhile, the method lays the foundation of quick establishment for a calibrator preparing platform imperative to the domestic clinical chemistry diagnosis as well as development and preparation for diagnostic reagent standard substances and references.

Description

technical field [0001] The invention belongs to the field of genetic engineering and protein expression, and relates to a method for efficiently preparing alanine aminotransferase by using bioengineering technology. The product can be used as an antigenic substance for animal immunization, to screen related antibodies, and at the same time as a calibrator and standard for clinical chemical diagnosis. Lays the groundwork for the preparation and development of substances and reference materials. Background technique [0002] Alanine aminotransferase (alanine aminotransferase, ALT, EC2.6.1.2) is a transaminase between glutamic acid and pyruvate, also known as alanine aminotransferase, referred to as GPT and ALT. Elevated alanine aminotransferase is a common clinical phenomenon. The liver is the largest detoxification organ in the human body. Whether this organ is normal or not is very important to the human body. Elevated GPT is an important indicator of liver function proble...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/70C12N9/10C07K16/40G01N33/53
Inventor 肖飞黄薇张传宝王大光
Owner 肖飞
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