Introduction

According to our experiment result, we have proved the secretion and expression possibility of TD1-antigen, TD1-adjuvant, and the antigenicity of TD1-antigen after transdermal process. So in the following experiments, we decided to utilize B.subtilis WB800N as engineered bacteria, plus shuttle vector pHT43 as secretion vector to build the B.subtilis secretory expression system. On top of this, we have taken advantage of different kinds of TD1-antigen, testing HBsAg, PA, Ag85b that have been applied into the market to check the universal property of TD1-antigen. Besides, reporters essential during real application have been found to realize the final circuit.

ONE: Expression of TD1-"X" in WB800N

1.Expression of TD1-GFP

Fig4. fluorescent microscope shows the expression of GFP in WB800N

2.Expression of TD1-antigen/adjuvant

In order to realize the secretory expression in B.subtilis, we inserted signal peptide between promoter and the TD1-Antigen/adjuvant sequence to secrete our recombinant protein. GFP was chosen to check the reliability of this circuit. After large amounts of experiments, GFP has finally been found via fluorescence microscope.

Then, three kinds of recombinant antigen, TD1-HBsAg, TD1-PA, TD1-Ag85b, and recombinant immunologic adjuvant were designed. The earliest successful protein TD1-LTB can not only be observed clear stripes in SDS-PAGE, but also be proved according to HPLC-MS.

Fig5. SDS PAGE shows the expression of LTB in WB800N

TWO: Neomycin resistance of WB800N

Selection on the of resistance of B.subtilis WB800N against neomycin

As WB800N is a novel secretory expression system, most of whose parameters remain unclear, among which the chief problem is that its unique resistance against neomycin has never been quantitatively analyzed, and our work made up for this blank.

Fig6. Selection on the of resistance of B.subtilis WB800N against neomycin

We have figured it out that the best concentration of neomycin for selection use is 128mg/ml.