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A kind of preparation method of biologically active cholera toxin b subunit protein

A cholera toxin and biological activity technology, which is applied in the fields of biotechnology, resource utilization and sustainable development, can solve the problems of unclear biological activity and low expression of proteins, and achieve the effects of high purity, high protein expression and convenient operation.

Active Publication Date: 2020-06-02
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there have been many reports in the literature on the expression of CTB in vitro, including prokaryotic and eukaryotic expression, the expression level is not high, and it is not clear whether the obtained protein has biological activity

Method used

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  • A kind of preparation method of biologically active cholera toxin b subunit protein
  • A kind of preparation method of biologically active cholera toxin b subunit protein
  • A kind of preparation method of biologically active cholera toxin b subunit protein

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Embodiment 1

[0046] A preparation method of biologically active cholera toxin B subunit protein, comprising the steps of:

[0047] 1. Construction of a prokaryotic expression vector: the CTB gene (nucleotide sequence shown in SEQ ID NO.1, corresponding amino acid sequence shown in SEQ ID NO.2) is cloned onto the pET28a-OH vector (restriction site BamH I and Xho I), get

[0048] CTB-pET28a-OH expression vector.

[0049] The pET28a-OH vector is obtained by transforming the pET-28a(+) vector. Specifically, the sequence from the N-terminal His-Tag to BamH I of pET-28a(+) is deleted by PCR. This transformation can make the cloned CTB -The N-terminus of the pET28a-OH expression vector only has His-Tag, which can increase the expression of CTB and reduce the restriction enzyme cutting procedure.

[0050] 2. Expression vector transformation: the CTB-pET28a-OH expression vector was transformed into Escherichia coli BL21(DE3).

[0051] 3. Monoclonal activation: Pick a single clone containing the ...

Embodiment 2

[0071] Activity detection of cholera toxin B subunit protein:

[0072] 1. During the expression of cholera toxin B subunit, in the culture medium, add 5-fluoro-tryptophan to mark the 88th tryptophan of CTB, the purpose is to use 19 F NMR directly observes the renaturation process of inclusion bodies. During the renaturation process, each time the buffer is changed, 450uL of the sample in the dialysis bag is added to 50uL D 2 O, mix well, pack into 5mm NMR tube, adopt fluorine spectrum on 600MHz NMR spectrometer, according to 19 The peak of F can clearly observe the denaturation-intermediate-renaturation process ( figure 1 ). At the same time, take 10uL of each spectrum sample and run SDS-Page, and you can clearly see the appearance of pentamer after CTB renaturation ( figure 2 ).

[0073] 2. The cholera toxin subunit B after refolding, in order to detect its biological activity, we use fluorescein isothiocyanate (FITC) to label the protein to obtain fluorescent FITC-CTB, ...

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Abstract

The present invention discloses a preparation method of cholera toxin B subunit protein having biological activity. According to the present invention, a lot of cholera toxin subunits are expressed in Escherichia coli by using a prokaryotic expression vector, and then in vitro efficient renaturation is performed to obtain a large number of the cholera toxin B subunit having biological activity; the operations can be performed in the general laboratory, and no pollution is generated; and the most important advantage is that the labeling with various fluorescence, isotopes and tracers can be performed according to the experimental needs.

Description

technical field [0001] The invention relates to the fields of biotechnology, resource utilization and sustainable development, in particular to a method for preparing a biologically active cholera toxin B subunit protein. Background technique [0002] Vibrio cholerae (Vibrio cholerae) causes a severe intestinal infectious disease, manifested as diarrhea, dehydration, shock and even death, which belongs to the international quarantine infectious disease. Statistics from the World Health Organization in 2016 showed that 1.3 to 4 million cholera cases broke out in the world every year, and cholera caused 21,000 to 143,000 deaths each year. Cholera is a serious threat to human health, and cholera toxin (CT) is a toxin secreted by Vibrio cholerae with ADP-ribosyltransferase activity, which is the main factor causing diarrhea. CT is a toxin with a typical AB5 structure. Five B subunits are non-covalently combined into a very compact and stable barrel-shaped pentamer; the A subuni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/28C07K1/34C12N15/70
CPCC07K14/28C12N15/70
Inventor 刘晓黎李从刚裴云山吴琼刘买利
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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