Abstract： Objective To construct two oral rBCG vaccines expressing Der p2 and Omp D (in form of series connection and parallel connection) antigens on its cell wall.Methods The genes of Der p2 and Omp D were first amplified by PCR,and then were ligated into pProEX HTb vector respectively,and gained Der p2-Omp D fused gene.①Expressing in form of series connection:The Der p2-Omp D fused gene was subcloned into the shuttle plasmid pCW.The positive recombinant plasmid was named pCW Der p2-Omp D,and was transformed into BCG competent cells to construct rBCG oral vaccine,which could express the Der p2-Omp D fused protein on its cell wall in the form of series connection.②Expressing in form of parallel connection:The pCW-Der p2 and pCW-Omp D plasmids were transformed into BCG competent cells together to construct the another rBCG oral vaccine,which could express Der p2 and Omp D proteins on its cell wall in the form of parallel connection.The two rBCG oral vaccines were selected by hygromycine,and the positive rBCG were identified by PCR,spot immunifaction and indirect immunofluorescence.The PCR result showed that there was specific amplification of the target gene.Spot immunifaction showed that specific banding of rBCG expressing product with Omp D pAb and Der p2 pAb was observed,indicating that rBCG possesses immunological function.Indirect immunofluorescence showed that specifc green immunofluorescences were observed,indicating that rBCG could express the goal protein on its surface.Results The two rBCG oral vaccines expressing Der p2 and Omp D proteins on its cell wall in the forms of series connection and parallel connection were preparated by genetic engineering,and then identified by PCR,spot immunifaction and indirect immunofluorescence successfully.Conclusions Two rBCG oral vaccines were constructed successfully,which has provided foundation for in vitro experimental and clinical application.