Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris

A technology of growth hormone and Pichia pastoris, which is applied in the field of production and purification of recombinant protein to achieve the effects of improving fermentation efficiency, high purity and convenient separation

Pending Publication Date: 2016-06-22
CHANGSHU INSTITUTE OF TECHNOLOGY
View PDF6 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the Pichia pastoris expression system is a eukaryotic expression system that has developed rapidly and is widely used in recent years. At present, there is no report on the expression of chicken growth hormone recombinant protein in Pichia pastoris

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris
  • Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris
  • Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of the recombinant expression vector and transgenic recombinant yeast strain of chicken growth hormone recombinant protein

[0026] 1. Obtain cGH gene fragment from chicken pituitary

[0027] Total RNA was extracted from the pituitary gland and its cDNA was obtained by reverse transcription PCR. Primers were designed for the chicken growth hormone mRNA sequence (NM_204359) published by Genbank, and the cDNA obtained above was used as a template to perform PCR to obtain the full-length CDS fragment of the cGH gene. Insert the above cGH gene fragment into the pMD19-T vector, named pMD19-T-cGH, and sequence to confirm the correct acquisition of the target gene.

[0028] 2. Construction of pPIC9K-cGH recombinant expression vector

[0029] Design primers according to the gene sequence of chicken growth hormone mature polypeptide, wherein the upstream primer P1 has an NdeI restriction site, and the downstream primer P2 has an XhoI restriction site: d...

Embodiment 2

[0047] Example 2: Induced expression, purification and activity identification of chicken growth hormone recombinant protein

[0048] 1. Induced expression of chicken growth hormone recombinant protein with methanol

[0049] Inoculate a single colony of the transgenic recombinant yeast strain GS115-pPIC9K-cGH in 3mLYPD medium, and culture it on a shaking table at 28°C for 18 hours to activate it; OD 600 2-6, 2500×g, 5min centrifugation to collect the bacterial cells, change to BMMY medium to induce expression, add methanol to 1% every 24h, continuously induce for 96h, collect supernatant at 24h, 48h, 72h, 96h respectively, SDS- PAGE analysis protein ( image 3 ), as time increases, the better the expression effect is, and the 96h expression effect is the best, and then the high-expression strains are screened out.

[0050] 2. Purification of chicken growth hormone recombinant protein

[0051] Take 100mL of GS115-pPIC9K-cGH induction supernatant for 96h, dialyze in 2L nicke...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris. The expression and purification method includes the steps of firstly, extracting total RNA (ribonucleic acid) from chicken pituitary, performing reverse transcription PCR (polymerase chain reaction) to obtain cDNA (complementary deoxyribonucleic acid) and conducting PCR by taking the cDNA as a template so as to obtain chicken growth hormone gene fragments; secondly, using the Pichia pastoris for constructing a recombinant expression vector pPIC9K-cGH; thirdly, subjecting Pichia pastoris GS115 to electrotransformation by the recombinant expression vector pPIC9K-cGH and screening to obtain a transgenic recombinant yeast strain GS115-pPIC9K-cGH; fourthly, obtaining the chicken growth hormone recombinant protein through methanol induced expression and purification, and identifying activity of the chicken growth hormone recombinant protein through cell activity test. In the expression and purification method of the chicken growth hormone recombinant protein in the Pichia pastoris, high-efficiency expression of the cGH (chicken growth hormone) recombinant protein in the Pichia pastoris is achieved and a purification system is established firstly, so that the high-purity chicken growth hormone recombinant protein with activity can be obtained. The expression and purification method of the chicken growth hormone recombinant protein in the Pichia pastoris is simple and convenient to operate, high in yield and low in cost and provides a basis for further studies on structures, functions and production application of chicken growth hormone.

Description

technical field [0001] The invention relates to the production and purification method of recombinant protein in the field of genetic engineering, in particular to the high-efficiency expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris. Background technique [0002] Chicken growth hormone (cGH) not only plays a necessary role in regulating the normal growth and development of chickens, but also has a significant impact on various production traits of chickens, such as body weight, feed conversion rate, fat accumulation, egg production, resistance disease etc. In addition, by injecting exogenous GH, the weight of chicken embryos can be significantly increased during the embryonic period, and the development of long bones and cartilage tissues can be promoted, thereby increasing the market weight of broiler chickens. Compared with human and mammalian growth hormone research, chicken growth hormone research started late, but with...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C07K14/61C07K1/16C12R1/84
CPCC07K14/61C12N15/815C12N2800/102
Inventor 郁建锋顾志良何赛张艳萍金泽楷周欢庆姚丽娟
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com