Study On Oral Vaccine Of H5 Subtype Al And SARS Carried By Attenuated Salmonella Typhmurium

There are many human oral vaccine using on clinical practice since chicken cholera vaccine was developed by Pastuer such as well knowed rotula,which made big contribution to the poliomyelitis. Attenuated Salmonella typhimurium oral live vaccine is the other example which controled human typhoid fever. Human learned more about oral viccine from these two examples. Oral vaccine can stimulate local immunity of animal gastrointestinal tract by oral administration; oral vaccine can efficiently mimic infection route of pathogen.Firstly,it induce B lymphocyte and T lymphocyte respond in gastrointestinal tract and then obtain immunity of all of the body.oral vaccine is the new way to administrate vaccines.It can serve susceptible population immdietaly,and play a immportant role to the diease of H5 subtype Avian Influenza and SARS.Generally, the construction of gene vaccine is only acquired main protect antigen gene or epitopes. To avoid the danger which is brought by antibiotic resistance gene, we can reform gene vaccine vector in gene levels. Replace antibiotic resistance gene by nutrition screening marker, and deliver gene vaccine by nutrition vicious attenuate strain. Such vaccine design brings new hope to people for facing the unexpected important plague.In this experiment, we cloned HA gene of H5 subtype AI and S合gene of SARS-CoV. Then we reconstituted gene vaccine expression vector pVAX1.The recombined plasmid asd-pVAX1was constructed by this way which is complementary to the salmonella typhmurium X4550. We named this system as asd-balance lethal system. First of all, we expressed HA gene and S合gene in 293-T cells. Then the recombined plasmids pASD-HA,pASD-S合were transformed into mouse attenuated salmonella typhmurium nutrition deficiency strain X4550 respectively. We examined oral viaccine's safety, stability and make sure safe immunizing dose.At last we immunized oral vaccines to mouse, and then the specific humoral and cellular immunity were detected.According to the HA nucleotide sequence of H5 subtype AI in Genebank, we designed primers and abstained HA gene by PCR method. Total length of HA gene is 1704 base pairs, which encodes 567 amino acids. The sequence of splitting site is consistence with strong pathogenicity strain's feature (ERRRKR). S protein is the main protection antigen. Considering its length (3768 bp) is too long to express, we selected main epitopes of S gene and then synthesis it by artificial. The conformation was not changed by calculator assistance analyze.We destroyed kanamycine resistance gene by restriction enzyme PvuⅡ,vector fragment was abstained and processed by bovine alkaline phosphatase.pYA3342 vector was cut by restriction enzyme BglⅡand XbaⅠ,the asd gene was abtained and processed by Klenow fragment. The reconstructed vector pVAX1 and asd gene was ligated and transformed to E coli X6097 which is asd deficiency. The positive clone was selected by PCR and sequenced by TaKaRa company. The result showed us there are 1128 base pairs are complete identity to asd gene of mouse salmonella typhmurium. Although asd gene was incomplete (the complete ORF length of asd is 1175 bp), its function was not influent,and EGFP gene can be corrected expressed in vitro cells in this reconstructed vector.We constructed recombination plasmid pVAX-HA,pVAX-S合,pASD-HA and pASD-S合,then transfected these plasmids into 293-T cells by means of liposome respectively. In transcription level, mRNA of the HA gene and S合gene were detected by RT-PCR method. In protein level, protein of HA and S合were detected by ELISA assay and immunol fluorescence.Recombinant plasmids of pASD-HA and pASD-S合were transformed into attenuated X4550 Salmonella typhimurium by electroporation. Oral vaccine X4550(pASD-HA)and X4550(pASD-S合)were constructed and tested, and the results showed that they were stable,safety and could normally grow. It also shows that the recombinant strain which below 1×1010 cfu were safe when orally administered into BALB/c mouse.After recombinant orally administered of oral vaccine 109cfu which is a safe dose were respectively into BALB/c mice for 3 times every two weeks, the level of serum specific antibodies, cytotoxicity activity of specific CTL, the numbers of spleen T lymphocytes subgroups, and cytokine level of Th1 were examined. The results showed that the DNA vaccine could increase the percentage of the T cell subgroups, elicit serum specific antibody and specific cytotoxicity activity of CTL to HAAg and S合Ag.The immunized mice could produce Th1 and Th2 cytokine.This research offers technique storage for the vaccine.