Construction Of Bacillus Subtilis Spore Surface Display Vectors And The Display Of S-layer Protein Of Lactobacillus Cripatus On Bacillus Subtilisspore

It has been identified that S-layer protein of Lacticacid bacteria plays a major role in the adhesion function of Lacticacid bacteria.Bacillus subtilis and its spore are beneficial to animal organism,but they stay in intestinal tract shortly.It can make the probiotic of Bacillussubtilis bigger by the display of S-layer protein on the spore of Bacillus subtilisbecause it can prolong the time of Bacillus subtilis in intestinal tract.The styles of the display of heterologous protein on the spore include plasmid vetor,single-cross homologous recombination and double-cross homologous recombination which is commonly used but the efficiency is low.In this study we constructed three styles of surface display vectors and tried to explore advantages and disadvantages of the surface display vectors We also tried to display the S-layer of Lactobacillus crispatus on Bacillus subtillus spore through the three ways above.1.To construct the shuttle expression vector pLLF-2 harboring promoter Pglv and multiple cloning site,the tk gene fragment was replaced by multiple cloning site fragment generated by primer annealing.The GFP protein and ?-galactosidase were expressed in Bacillus subtillusby the shuttle expression vector pLLF-2.This indicated that the shuttle expression vector pLLF-2 was constructed successfully and it could express the heterologous protein.2.The gfp gene was respectively linked to 3' end of cotB and cotZ gene fragment which contained connecting peptides.To construct plasmid type surface display vectors pEYGJ52 and pEYW27,the fusion gene fragment cotB-gfp and cot Z-gfp were inserted into the shuttle expression vector pLLF-2 through relevant restriction enzyme site.Recombination strains BSYW27 and BSYGJ52 were obtained after pEYW27 and pEYGJ52 were transformed into Bacillus subtilis PY79.That the green fluorescence was not detected on the spores of Recombination strains BSYW27 and BSYGJ52 under fluorescence microscope illustrated that plasmid type surface display vector using coat protein CotB and CotZ as anchoring proteion was not suitable for displaying heterologous protein on spore.3.Recombination strains BSYW25 and BSYW26 were getted from that single cross integration vectors pEYW25 and pEYW26 generated by inserting fusion gene fragment cotB-gfp and cotZ-fp into pLX-2 were transformed into Bacillus subtilis PY79.That the green fluorescence was only detected on the spore of strain BSYW26 indicated that single integration type could be used for displaying heterologous protein on spore and the failure of the recombination strain BSYW25 might be dued to the location of CotB protein on spore.4.We only constructed the double cross integration vector using CotZ protein as anchoring protein and GFP as reporter protein based on the result of single cross integration vector.To get double cross over integration vector pEYW68,fusion gene fragment was inserted into pLX-2AmyE through relevant restriction enzyme cutting site.BSYW68 was obtained by transforming pEYW68 into Bacillus subtilis PY79.That the green fluorescence could be detected on the spore of strain BSYW68 generated by Amylase ectopic integration illustrated that double cross integration type was also suitable for displaying heterologous protein on spore.5.The vectors harboring sip gene pEYW10,pEYW14,pEYW20,pEYW21 and pEYW72 were obtained after the encoding gene of S-layer protein of Lacticacidcrispatusreplaced the gfp gene fragment of p EYGJ52,pEYW27,pEYW25,pEYW26 and pEYW68.Transforming pEYW10,pEYW14,pEYW20,pEYW21 and pEYW72 into Bacillus subtilis PY79,the obtained recombination strains BSYW10,BSYW14,BSYW20,BSYW21 and BSYW72 were induced to spore.The coat protein of the recombinantion strains above was extracted.But the Sip protein could not be detected on any spore of the recombination strains BSYW10,BSYW14,BSYW20,BSYW21 and BSYW72 by Western blot.The result might depend on the character of S-layer protein of Lacticacidcrispatus.In conclusion,plasmid type surface display vector,single cross integration vector and double cross integration vector were constracted in this study.We also tried to display the S-layer protein of Lacticacidcrispatuson the spore of Bacillus subtilis PY79.That We preliminarily understood the merit and dismerit,the feasibility of three different surface display vectors contributed to the next work about Bacillus subtilis spore surface display.