Introduction

According to our experiment result, we have proved the secretion and expression possibility of TD1-antigen, TD1-adjuvant, also the antigenicity of TD1-antigen after transdermal process. So in the following experiments, we decided to utilize Bacillus Subtillis WB800N as engineering bacteria, plus shuttle vector pHT43 as secretion vector to build the Bacillus Subtillis 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 market to check the universal property of TD1-antigen. Besides, reporters that are essential during reality application have been found to make the final circuit come true.

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 Bacillus Subtillis, we inserted signal peptide between promoter and the TD1-Antigen/adjuvant sequence to secrete our recombinant protein. GFP has been chosen to check whether 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 one TD1-LTB protein, 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 Bacillus subtilis WB800N against neomycin As WB800N is a novel secretory expression system, most of its 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 Bacillus subtilis WB800N against neomycin

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