Application of utilizing bacillus subtilis spore coat protein CotX as molecular carrier adopting spore surface to display exogenous proteins

A Bacillus subtilis, surface display technology, applied in the field of spore surface display of exogenous proteins, Bacillus subtilis spore capsid protein CotX, can solve the problems of different biological activities of recombinant spores, lack of application value of exogenous protein carriers, etc.

Inactive Publication Date: 2010-04-14
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, due to the differences in the molecular structure of different spore capsid proteins and the composition in spores, different spore capsid proteins are used as molecular carriers, and the biological activities of recombinant spores displaying foreign proteins on the surface are also different; Among the more than 20 kinds of Bacillus subtilis spore capsid proteins, some proteins may play a role in regulating or transporting other capsid proteins, and do not have the application value as carriers of exogenous proteins displayed on the surface of spores, except CotB, CotC, CotG which have been reported As a carrier molecule for displaying foreign proteins on the surface of Bacillus subtilis spores, there are no reports or patents of other capsid proteins as carriers

Method used

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  • Application of utilizing bacillus subtilis spore coat protein CotX as molecular carrier adopting spore surface to display exogenous proteins
  • Application of utilizing bacillus subtilis spore coat protein CotX as molecular carrier adopting spore surface to display exogenous proteins
  • Application of utilizing bacillus subtilis spore coat protein CotX as molecular carrier adopting spore surface to display exogenous proteins

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Embodiment 1

[0028] Preparation of Recombinant Spores of Bacillus subtilis Using CotX as Carrier Protein to Display Green Fluorescent Protein (GFP)

[0029] 1. Molecular Biology Operations

[0030] 1.2 Molecular biology techniques

[0031] Construction of all plasmids was carried out using standard molecular biology techniques as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989) for all recovered DNA of the present invention. Fragments were isolated and purified using gel extraction kit from Shanghai Sangon Bioengineering Co., Ltd. All cloned fragments of PCR products were sequenced by Shanghai Sangon Bioengineering Co., Ltd.

[0032] 1.3PCR amplification

[0033] The primers used for gene amplification were synthesized by Shanghai Sangon Biotechnology Co., Ltd. Restriction endonuclease recognition sites were added to the 5' end of the primers to facilitate the cloning of amplified pro...

Embodiment 2

[0056] Preparation of recombinant spores of Bacillus subtilis displaying Vp28 on the surface using CotX as carrier protein

[0057] 1. Molecular Biology Operations

[0058] 1.1 Extraction of Bacillus subtilis chromosome

[0059] Concrete operation method is with the extraction of the embodiment of the present invention 1,1.2 Bacillus subtilis chromosome

[0060] 1.2 Molecular biology techniques

[0061] Concrete operation method is with the embodiment of the present invention 1,1.3 molecular biology technology

[0062] 1.3PCR amplification

[0063] Concrete operation method is with the embodiment of the present invention 1,1.4 molecular biology technology

[0064] 2. Plasmid construction

[0065] 2.1 Basic plasmid construction

[0066] Concrete operation method is the same as embodiment 1, the construction of 2.1 basic plasmid

[0067] 2.2 Construction of integrated recombinant plasmid for fusion expression of CotX-Vp28 recombinant protein

[0068] Shrimp white spot sy...

Embodiment 3

[0076] Preparation of recombinant spores of Bacillus subtilis displaying Vp19 on the surface using CotX as carrier protein

[0077] The WSSV envelope protein gene vp19 used in this embodiment is cloned, sequenced and identified by the inventor and preserved: the following are WSSV vp19 clones, sequenced and identified:

[0078] The prawn samples that died after white spot syndrome virus infection were collected in Wanning City, Hainan Province, and 0.1 g gill tissues of the dead prawns were taken, and 5 times of TN buffer solution (0.02M Tris-HCl, 0.4M NaCl, pH 7.4) was added, Grind with a glass homogenizer, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant virus liquid. Add sodium dodecylsulfonate to a final concentration of 0.5%, proteinase K to a final concentration of 100mg / ml, treat at 37°C for 2 hours, extract with a phenol / chloroform mixture, add 1 / 4 volume of 5mol / ml L of sodium chloride and twice the volume of ethanol were precipitated at -20°C for 16...

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Abstract

The invention relates to an application of bacillus subtilis spore coat proteins CotX, in particular to the application of utilizing bacillus subtilis spore coat proteins CotX as a protein carrier adopting a spore surface to display exogenous proteins, which builds recombinant spores with surfaces displaying exogenous proteins through the spore surface displaying technique. The application is characterized by utilizing coding gene cotX of proteins of the carrier to perform gene recombination with coding sequences of exogenous proteins genes, building integrity recombinant plasmid fusing and expressing recombinant proteins, screening recombination strain after transforming bacillus subtilis, inducing the recombination strain to generate spores, and then obtaining the recombinant spores with surfaces displaying exogenous recombinant proteins.

Description

Technical field: [0001] The invention relates to a spore capsid protein displaying foreign protein on the surface of spores, in particular CotX, a spore capsid protein of Bacillus subtilis, as a molecular carrier, and the surface of the spores displays the foreign protein. Background technique [0002] Bacillus subtilis is an environmentally friendly, spore-forming, non-pathogenic Gram-positive bacterium. Under the induction of adverse environmental conditions such as nutrient deficiency, vegetative cells differentiate to form dormant bodies—spores. The spore consists of spore medulla, cortex, spore capsid and spore exine. The outer spore capsid is mainly composed of hydrophobic capsid protein, which has anti-enzyme and anti-drug functions. The formation of spores is a process in which a series of regulatory genes and structural genes are regulated and expressed in a certain sequence. These genes include more than 20 spore capsid protein genes and regulatory genes, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12R1/125
Inventor 宁德刚李倩徐卫东
Owner JIANGSU UNIV
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