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

Purification method of pBpp protein

A purification method and protein technology, applied in the field of purification of pBpp protein, can solve the problems of difficulty in large-scale preparation of pBpp protein and the like

Inactive Publication Date: 2017-07-21
NANCHANG UNIV
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides a purification method for pBpp protein, so as to solve the technical problem that pBpp protein is difficult to prepare on a large scale in the prior art

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Construction of pBpp protein recombinant expression system

[0039] Using zebrafish fecal flora DNA as a template and using the sequences of SEQ ID No.1 and SEQ ID No.2 as primers, the pBpp fragment shown in SEQ ID No.3 was obtained by PCR amplification, which was named pBpp.

[0040] The obtained pBpp fragment and plasmid pET28c were double-digested with NcoI and XhoI. After purification, the digested product was ligated into the pET28c (SEQ ID No.4) plasmid using T4 ligase. The recombinant plasmid was named pET28c-pBpp, and transformed into Enter E.coli BL21, and the obtained recombinant expression strain is named pET28c-pBpp-BL21.

[0041] 2. Expression and purification of pBpp protein

[0042] (1) pBpp protein expression: prepare 200ml LB medium, add 200ul ampicillin mother solution after cooling, so that the final concentration is 100ug / ml; select pET28c-pBpp-BL21 single colony for overnight shake flask culture; inoculate with 1% inoculum Put it into fresh LB me...

Embodiment 2

[0056] A purification method for pBpp protein, comprising the following steps:

[0057] 1) Using the total DNA of the zebrafish fecal flora as a template, the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No.2 are respectively used as upstream and downstream primers to perform PCR amplification to obtain a sequence such as SEQ ID No. The DNA fragment indicated by ID No.3 is the pBpp protein expression gene;

[0058] 2) Take the pBpp protein expression gene obtained in step 1) and connect it to the pET28c vector to obtain the recombinant plasmid pET28c-pBpp;

[0059] 3) Take the recombinant plasmid pET28c-pBpp obtained in step 2), and transfer it into Escherichia coli E.coli Top10 to obtain a recombinant strain;

[0060] 4) Cultivate the recombinant strain obtained in step 3), extract the recombinant plasmid pET28c-pBpp from it, and transform it into Escherichia coli E.coli BL21 to obtain the recombinant expression strain pET28c-pBpp-BL21;

[0061] 5) Cultiva...

Embodiment 3

[0073] A purification method for pBpp protein, comprising the following steps:

[0074] 1) Using the total DNA of the zebrafish fecal flora as a template, the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No.2 are respectively used as upstream and downstream primers to perform PCR amplification to obtain a sequence such as SEQ ID No. The DNA fragment indicated by ID No.3 is the pBpp protein expression gene;

[0075] 2) Take the pBpp protein expression gene obtained in step 1) and connect it to the pET28c vector to obtain the recombinant plasmid pET28c-pBpp;

[0076] 3) Take the recombinant plasmid pET28c-pBpp obtained in step 2), and transfer it into Escherichia coli E.coli Top10 to obtain a recombinant strain;

[0077] 4) Cultivate the recombinant strain obtained in step 3), extract the recombinant plasmid pET28c-pBpp from it, and transform it into Escherichia coli E.coli BL21 to obtain the recombinant expression strain pET28c-pBpp-BL21;

[0078] 5) Cultiva...

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 provides a purification method of pBpp protein. According to the purification method, zebra fish feces bacteria colony DNA is taken as a template, pBpp expression gene is obtained via amplification; pET28c plasmid is taken as a carrier, plasmid amplification is carried out in Escherichia coli TOP10, and then protein expression is carried out in Escherichia coli BL21. According to the production method, appropriate protein expression and induction conditions are designed based on the characteristics of recombination strains; effective purification of pBpp protein obtained via intracellular expression, bacteria is subjected to ultrasonication, combination of free protein is realized via nickel beads, protein elution is carried out, and finally dialysis is carried out with PBS and glycerin so as to realize effective purification of pBpp protein. It is found in experiments that the pBpp protein is capable of inducing propagation of beta cells in pancreas, so that possesses treatment effect on diabetes caused by islet beta cell damage.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and protein purification, in particular to a purification method for pBpp protein. Background technique [0002] Diabetes is caused by genetic factors, immune dysfunction, microbial infection and its toxins, free radical toxins, mental factors and other pathogenic factors acting on the body to cause hypofunction of pancreatic islets and insulin resistance. and electrolytes and a series of metabolic disorder syndromes, clinically characterized by hyperglycemia. [0003] At present, the prevalence rate of type Ⅱ diabetes in my country is increasing rapidly. According to the survey in 1979 among the 300,000 population in my country, the prevalence rate of diabetes was 0.6%, and it was 2.02% in 1989, with an average annual growth rate of about 0.1%. In 1994, my country According to a census of 200,000 people, the prevalence rate has risen to 2.5%. At present, the prevalence rate of diabete...

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): C07K14/195C12N15/70C07K1/14C07K1/22
CPCC07K14/195C12N15/70
Inventor 陈廷涛辛洪波王鑫
Owner NANCHANG UNIV
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