Method for guiding expression of major capsid proteins L1 of HPV16 (human papilloma virus 16) in pichia pastoris by using formaldehyde dehydrogenase promoter

A technology of formaldehyde dehydrogenase and Pichia pastoris, which is applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of limited protein expression types, single regulatory pathways, and relatively demanding growth conditions. advanced questions

Inactive Publication Date: 2015-05-06
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

[0004] Although the constitutive promoter simplifies the production steps, if a higher protein yield is obtained, the growth of t...

Method used

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  • Method for guiding expression of major capsid proteins L1 of HPV16 (human papilloma virus 16) in pichia pastoris by using formaldehyde dehydrogenase promoter
  • Method for guiding expression of major capsid proteins L1 of HPV16 (human papilloma virus 16) in pichia pastoris by using formaldehyde dehydrogenase promoter
  • Method for guiding expression of major capsid proteins L1 of HPV16 (human papilloma virus 16) in pichia pastoris by using formaldehyde dehydrogenase promoter

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Embodiment

[0071] Example: Under the control of formaldehyde dehydrogenase promoter control, induced expressions of human papilloma virus type 16 major shell protein L1

[0072] The clone and construct of formaldehyde dehydrogenase promoter are completed through the following steps.Search for formaldehyde dehydrogenase promoter gene sequences from the GeneBank database (Genebank serial number: AF066054), and design the promoter nucleotide segment amplification primer according to the gene sequence.

[0073] Use 20 micro -rise reaction liquid to perform formaldehyde dehydrinase promoter nucleotide fragments amplification, which include 2 microcontrolls 10 × extaq enzyme buffer, 1.6 micro -rise DNTP mixture, 50 microcomol concentration up and downstream primers0.25 slightly ascended, 0.5 micro -rising EXTAQ enzymes, 2 micro -rising red yeast cells GS115 bacteria liquid, double steamed water 13.4 micro -rise.The PCR amplification reaction condition is 95 ° C, prestigious 3 minutes; 72 ° C, 2 mi...

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Abstract

The invention relates to a method for guiding the expression of major capsid proteins L1 of HPV16 (human papilloma virus 16) in pichia pastoris by using a formaldehyde dehydrogenase promoter, wherein the expression of major capsid proteins L1 of HPV16 is controlled by a pichia pastoris formaldehyde dehydrogenase promoter. The method disclosed by the invention mainly comprises the following steps: i) building a pichia pastoris expression vector guided by a pichia pastoris formaldehyde dehydrogenase promoter; and ii) under the guidance of the formaldehyde dehydrogenase promoter, expressing the major capsid proteins L1 of HPV16 in different carbon/nitrogen source combined culture media. According to the invention, based on a pichia pastoris expression system, and starting with the regulating characteristics of the formaldehyde dehydrogenase promoter, the optimized expression of major capsid proteins L1 of HPV16 is implemented in the different carbon/nitrogen source combined culture media according to the characteristics of the promoter, so that the efficient expression of the major capsid proteins L1 of HPV16 is realized, thereby obtaining a low-cost virus-like particle preparation technology and implementing the low-cost preparation of human papilloma virus vaccines.

Description

Technical field [0001] The present invention belongs to the field of biological engineering technology, which involves the use of the use of Bichic yeast formaldehyde dehydrogenase (FLD) in the use of the chinensis host cells in the hyaluronic types of the hypolitic yeast host cells.) Expressive method. Background technique [0002] Bi Chi yeast expression system can effectively secrete heterogeneous proteins and perform post -translation modification. The operation is easy to achieve high -density fermentation, and does not produce internal toxins and mammalian nucleic acid components. Therefore, it is a widely used protein.Expression system.Transcription is one of the most critical links of gene expression regulation. The promoter can significantly affect the beginning, level, and persistence of heterogeneous transcription, thereby directly affecting the expression efficiency of heterogeneous protein.The most commonly used and classic promoter in Bi Chi yeast is type of mosquit...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/37C12N15/66C12P21/02C12R1/84
Inventor 马雁冰金晓媚姚宇峰杨旭黄惟巍刘存宝孙文佳龙琼李杨褚晓杰
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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