Preparation And Identification Of Salmonella Omp D-mediated Rbcg Oral Vaccine

Live recombinant vaccines are based on the use of a live microorganism (virus or bacteria) that acts as a vector for the expression of genes from another organism. The new recombinant microorganism can be used as a vaccine for both organisms. Recombinant Bacillus Calmette-Guerin (BCG) is a typical vaccine of live recombinant vaccines, that insets foreign genes into BCG to construct multivalent vaccines by using molecular biology techniques, then induces long-term cellular and humoral immune responses. Recombinant BCG (rBCG) can prevent and cure many kinds of diseases by expressing associated antigens in vivo. We have constructed rBCG by transferring Dermatophagoides pteronyssinus antigen Der p2 into BCG in our lab. The recombinant Der p2-BCG could stimulate Th1 predominant immune response in mice when injected intraperitoneally or subcutaneously. The results indicated that rBCG vaccines could be used to treat some diseases which were hypersensitive to some specific antigens.However, if rBCG vaccines were injected repeatedly in the short term, serious local delayed-type hypersensitivity could be induced. Then, we considered that oral vaccines were administered to mice, and results showed that it could also induce antigen specific Th1 predominant immune response. Nevertheless, BCG doesn't inhabit in the intestinal tract because of its low affinity to intestinal epithelia. Furthermore, a large dosis of BCG could cause dysbacteria and oropharynx adenitis, thus the immune efficacy is limited. Therefore, in order to improve the affinity of rBCG to intestinal epithelia, the protein Omp D, one of the major adhesion molecules, was used to prepare the rBCG oral vaccine.AIM: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 AND RESULTS:1. Expression and purification of Omp D proteinThe Omp D gene amplification was encoded by PCR from genomic DNA of Salmonella Typhimurium, and cloned into vector pMD-18T. The DNA sequence of Omp D was identical with that of Genbank reported. After confirmed by sequencing, the target gene was inserted into expression plasmid pET-28a (+) to construct recombinant plasmid pET28a (+)-Omp D, which was identified by PCR and restrictive enzyme digestion. The recombinant plasmid was transformed into E. coli BL21(DE3) which then expressed Omp D under IPTG induction. The analysis of SDS-PAGE showed that there was a specific protein expression at 40 kDa molecular marker, and the protein was further identified by Western-blot using anti-6×His mAb. Then, it was purified by Ni+-NTA purification system under denaturing condition and its purity was about ninety percent. 2. Preparation of Omp D polyclonal antibody in rabbitA rabbit was immunized with Omp D protein according to the following procedure. The reannealed Omp D protein dissolved in PBS was emulsified with an equal volume of Freund's complete adjuvant. On days 0, 14 and 28, 2 ml of the prepared mixture was intracutaneously injected into the rabbit. On days 42,2 ml of the Omp D protein dissolved in PBS was injected into the rabbit. On days 49, antibody titers of Omp D protein was measured by ELISA and attained to 1: 10 000. The specificity and sensitivity of Omp D antibody were also analyzed by Western-blot.3. Preparation and identification of rBCG oral vaccineThe 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. CONCLUSIONS:1. Omp D protein was purified successfully in E.coli DH5α. Omp D polyclonal antibody in rabbit was obtained, whose antibody titer attained to 1: 10 000 detected by ELISA. Its high specificity and sensitivity were analyzed by Western-blot.2. 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.