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Preparation method of recombinant pBpp protein based on escherichia coli expression system

A technology of Escherichia coli and expression system, which is applied in the field of preparation of recombinant pBpp protein based on Escherichia coli expression system, which can solve the problems of difficult large-scale preparation of pBpp protein

Inactive Publication Date: 2017-06-27
NANCHANG UNIV
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  • 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 method for preparing recombinant pBpp protein based on an Escherichia coli expression system, so as to solve the technical problem that pBpp protein is difficult to produce on a large scale in the prior art

Method used

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Experimental program
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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 method for preparing recombinant pBpp protein based on Escherichia coli expression system, 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 first double digest with NcoI and XhoI, and then use T4 ligase to connect the digested product to pET28c after purification 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-p...

Embodiment 3

[0073] A method for preparing recombinant pBpp protein based on Escherichia coli expression system, 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 first double digest with NcoI and XhoI, and then use T4 ligase to connect the digested product to pET28c after purification 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-p...

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Abstract

The invention provides a preparation method of recombinant pBpp protein based on an escherichia coli expression system. Through the technical scheme, DNAs in zebra fish faecal bacterium populations are used as templates, pBpp expressed genes are obtained through amplification, then pET28c plasmids are used as carriers, the plasmids are amplified in Escherichia coli TOP10, and then the plasmids are converted into escherichia coli BL21 for protein expression. On this basis, appropriate protein expression and induction conductions are designed for the characteristics of recombination strains, for the intracellular expression pBpp protein, after ultrasonication is carried out on the thallus, nickel beads are firstly used for combining dissociative protein, then protein elution is carried out, and finally dialysis is carried out by using PBS and glycerinum respectively, so that effective purification of the pBpp protein is realized. Experiments find that the pBpp protein prepared in the invention can definitely induce multiplication of beta cells in pancreas, so that the pBpp protein plays a therapeutic effect on diabetes caused by damage of pancreatic Beta cells.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and protein purification, in particular to a method for preparing recombinant pBpp protein based on an Escherichia coli expression system. 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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C07K14/46
CPCC12N15/70C07K14/461
Inventor 陈廷涛辛洪波王鑫
Owner NANCHANG UNIV
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