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pBpp protein, and function verification method thereof

A protein, pet28c-pbpp technology, applied in the direction of peptide preparation methods, peptide/protein components, chemical instruments and methods, etc., can solve the problems of difficult large-scale preparation of pBpp proteins

Inactive Publication Date: 2017-07-21
NANCHANG UNIV
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  • 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 pBpp protein and its function verification method, so as to solve the technical problem that the pBpp protein is difficult to produce on a large scale in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Construction of pBpp protein recombinant expression system

[0042] 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.

[0043] 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.

[0044] 2. Expression and purification of pBpp protein

[0045] (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 ...

Embodiment 2

[0059] A pBpp protein, the pBpp protein is prepared by the following method:

[0060] 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 5) Cultivate...

Embodiment 3

[0081] A pBpp protein, the pBpp protein is prepared by the following method:

[0082] 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;

[0083] 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;

[0084] 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;

[0085] 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;

[0086]5) Cultivate ...

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Abstract

The invention provides a pBpp protein, and a function verification method thereof. According to a production 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 realized effective purification of the 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 fields of genetic engineering and protein purification, in particular to a pBpp protein and a method for verifying its function. 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 prevalenc...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/70C07K1/14C07K1/22A61K38/16A61P3/10
CPCA61K38/00C07K14/195C12N15/70
Inventor 陈廷涛辛洪波王鑫
Owner NANCHANG UNIV
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