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.

① Expression of antigen/adjuvant in Bacillus Subtillis

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.

Fig4. SDS PAGE shows the expression of LTB in WB800N