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Development and Characterization of in Vivo and in Vitro Surface Display Systems of Bacillus Using an Autolysin Anchor

Author: JiangMengTian
Tutor: LiLin
School: Huazhong Agricultural University
Course: Microbiology
Keywords: Of Bacillus thuringiensis Peptidoglycan hydrolase Surface Display Anchored in vitro
CLC: Q93
Type: Master's thesis
Year: 2011
Downloads: 20
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Abstract

Bacillus (Bacillus) cell surface display system is a heat-stable and can be used for a variety of exogenous protein shows biotechnology platform. Bacillus thuringiensis (B. thuringiensis) in Bacillus is second only mode species Bacillus subtilis (B. subtilis) bacterial species. Bacteria prominent feature is in the form spores at the same time, can produce a parasporal crystal having insecticidal activity, it has become important insecticidal bacterial surface display of the application side of the multifunctional reactive systems, while also exhibit good the potential development of bacterial surface display system excellent host bacteria and attention at home and abroad. In this study, using a Bacillus cell wall anchored proteins as a carrier protein, the development of a continuity from the body show vegetative cells and spores of Bacillus thuringiensis, in vitro show a variety of vegetative cells and spores of Bacillus multiple active display system and each of the green fluorescent protein, and a bacterial mutant laccase as a target protein for the show activity analysis. Multiple active bacterial surface display system at home and abroad, has not been reported. Bacillus thuringiensis m altar is presumed from the wild strain YBT-1520 genome analysis, encodes a strong cell wall anchor binding properties of autolysin. This study was measured using RT-PCR technology of the relative transcriptional activity in the protein-encoding gene mbg 7 consecutive days in the wild strain through the culture period, the results show that the the mbg gene transcription in the cell culture to about 80% Bacillus highest cleavage The activity value. By on Mbg protein structure prediction, inferred is a N-acetyl-glucosaminidase, on its C-terminal catalytic domain, N-terminal domain contains two LysM anchoring. To explore the white lysin Mbg N-terminal domain (Mbgn) as carrying protein surface display system activity, the study by three gene fragments were amplified Pcry3Aa-mbgn-gfp connected to get the plasmid vector pHT304 on available expression the recombinant plasmid pMB164 fusion protein Mbgn-GFP in the vegetative stage of starting. Bacillus thuringiensis transformed BMB171, screened recombinant strain MB164. Recombinant strain MB164 cells GFP-specific fluorescence activity assay, the immunofluorescence microscope observation and flow cytometry analysis of measurement results show that in vivo expression Mbgn-GFP fusion protein is not only a high level to anchor on the surface of the vegetative cells, and exogenous protein (GFP) is in its active form and displayed on the cell surface. Regular sampling of the various periods in the the recombinant bacteria the MB164 growth cycle in GFP-specific fluorescence activity monitoring, results showed that after 96h (microscope observation, this time about 70% of the spores are released), about 33% of the MB164 total fluorescence activity occurred control MB165 total fluorescence activity value occurred a reduction of about 34%, indicating that GFP can be displayed on the anchored by Mbgn, spore surface. In order to further validate and analyze, build the expression and purification of the fusion protein carrier pMB313 the fusion gene mbgn-gfp connection to pET22b () vector. Anchoring experiments performed in vitro with purified fusion protein Mbgn-GFP cells and spores of Bacillus thuringiensis Vegetative cell fluorescence intensity potential reached 180, and the vegetative spores and Bacillus subtilis, Bacillus can reach 140 and this in vitro anchored to realize the performance still other types of Gram-positive bacteria, proved Mbgn anchoring function extensiveness. For then with the laccase protein WlacD as functional protein alternative reporter protein GFP construct surface display system recombinant plasmid was named Mbgn-WlacD pMB316 into recombinant strain of Bacillus thuringiensis MB316. Measured MB316 vegetative activity up to 10.56 U / ml is much higher than the control strain (1.148 U / ml) BMB171 the laccase activity of the recombinant bacterial cells, Bacillus (4.648 U / ml) also has a certain activity. Subsequent determination of immune fluorescence microscopy and flow cytometry analysis (Cy5 fluorescence efficiency of 33.43%) results show that the in vivo expression of the fusion protein of Mbgn-WlacD not only a high level anchored on the surface of the vegetative cells, and WlacD its active form exhibited on the cell surface using the same purified fusion protein on cells and spores vitro anchor fusion protein purified vector construct Mbgn-WlacD to fusion gene mbgn-wlacD connected to pTrcHisC named pMB317. Was incubated via Mbgn-WlacD purified protein from Bacillus thuringiensis vegetative activity of 2.74 U / ml (non-incubated only 1.15 U / ml in), the Bacillus activity was 1.86 U / ml (0.744 U / ml in not incubated) , Bacillus subtilis vegetative (2.22 U / ml), and Bacillus (1.72 U / ml) was also detected on the surface of laccase activity, again confirmed Mbgn anchoring function as a carrier protein having extensive.

Development and Characterization of in Vivo and in Vitro Surface Display Systems of Bacillus Using an Autolysin Anchor

Abstract

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