Preparation Of Bacterial Ghosts And Neisseria Gonorrhoeae Vaccine And Priming Study On Their Immunogenicity

Bacterial ghosts (BGs) are empty bacterial shells of Gram-negative bacteria devoid of nucleic acid, ribosomes or other constituents, and produced following expression of the cloned bacteriophage PhiX174lysis gene E. BGs share cellular morphology and surface structures with the natural cell, containing intrinsic adjuvant such as lipopolysaccharide, peptidoglycan, and that the periplasmic space and the cytoplasmic space of BGs can be filled with a lot of exogenous substances such as DNA and drugs. BGs have been investigated as vaccine and vector deliving vaccine and drugs.Gonorrhea is a sexually transmitted disease (STD) caused by Neisseria gonorrhoeae. The treatment of gonorrhea with antibiotics is sometimes inefficiency due to the high frequency of acquisition of resistance to antibiotics. Developing a new vaccine will play an important role in preventing and controlling gonorrhea.We plan to develop bacterial ghosts loading Gonorrhea DNA vaccine, the loading effency of BGs needs to be further improved. In this study, E.coli ghost, Salmonella ghost and Gonorrhea DNA vaccines delived by attenuated Salmonella were developed and their immunogenicity were investigated.1. Preparation of E.coli ghostThe lysis gene E and Em were amplified by PCR using bacteriophage PhiX174as the template and cloned into pBV220and pBV220m containing the XpL/pR-c1857regulator system. Four recombinant plasmids were constructed and designated as pBV220-E, pBV220m-E, pBV220-Em and pBV220m-Em, and transformed into E.coli. The recombinant bacteria were cultured at42℃for the generation of E.coli ghosts, which were in intact forms and their contents were released to extracellular region under scanning electron microscope and transmission electron microscope. The lysis efficency of E.coli(pBV220-Em) was99%, which was higher than that of the others. The mouse phage cell were transfected with the bacterial ghost loading plasmid pEGFP-N1, the results showed that the efficiency of transfection needs to be improved.2. Preparation of Salmonella ghost and their immunogencityThe temperature-controlled lysis cassette (EBOX) were amplified by PCR using the lysis plasmid as the template. The EBOX1and EBOX2were isolated from PCR product and subcloned in the Xho I-Spe I sites of the pBBR1MCS2vector. Then the recombinant plasmids were transformed by electroporation into Salmonella strain HA2. The expression of the lysis gene E was induced at42℃. The lysis rate was98.67%. The empty cell envelopes devoid of content could be observed significantly under scanning electron microscope and transmission electron microscope.6-8week-old BALB/c mice were devided into three groups and immunized with HA2-BG (orally and intranasally), inative HA2(intramuscularly) and PBS,3times at two weeks interval. Serum IgG antibody and fence IgA antibody was detected by indirect ELISA.The results showed that the serum IgG titers in HA2-BG group and inative HA2group were significant higher than that in PBS group, and the fence IgA titers in HA2-BG group was greater than those in the other2groups after the second and third immunization, indicating that HA2-BG can induced both systemic and local mucosal immunity.3. Construction and its immunogenicity of gonorrhoeae DNA vaccine delivered by attenuated SalmonellaThe NspA gene and PorB gene from gonorrhoeae strain WHO-A were amplified by PCR and the recombinant plasmids pVAX1-NspA和pVAXl-PorB were contructed. The mouse macrophage cell was transfected with recombinant plasmid. The transcription and expression NspA gene and PorB gene were successfully detected by RT-PCR and indirect immunofluorescent assay. The recombinant plasmids were transformed by electroporation into attenuated Salmonella.6-8week-old BALB/c mice were respectively inoculated orally and intranasally at the dosage of1×109CFU with SL7207(pVAXl-NspA), SL7207(pVAXl-PorB), SL7207(pVAXl-NspA)+SL7207(pVAX1-PorB), SL7207(pVAXl) and PBS, at2-week intervals for a total of three times. The antibody detected in serum and fence showed that specific antibodies against gonorrhoeae could be detected at day14post-priming, and the antibody levels continued to increase after priming, significantly higher than that of the negative control group after the third immunization (P<0.01).