Construction And Elvalution Of A Novel MntA And Nos Null Vaccine Candidate Of Bacillus Anthracis

Bacillus anthracis was confirmed as the first bacterial pathogens.It is gram positive bacteria.The cells are arranged corrugated shape without flagella.Bacillus anthracis is easy to form spore in the comfortable temperature,oxygen,plenty of external environment or artificial medium.It has the ability to resist heat,drying,radiation,chemical drugs.So the spores were used as the main form of biological weapons of Bacillus anthracis.Anthrax is the disease caused by Bacillus anthracis infection.It is an acute infectious diseases and recognized as one of the five zoonosis in the world.Therefore,the prevention and treatment of anthrax is particularly important.inh A gene and nos gene are important virulence genes of Bacillus anthracis.inh A plays an important role in regulating protein secretion of bacteria and the host.nos gene plays a significant role to remove the host within the antibacterial substance of host and restrain oxidative reaction.firstly,upstream homologous fragment and downstream homologous fragment of inh A gene were amplified by PCR to construct the targeting vector.We introduced the recombinant into B.anthracis AP422 by electroporation and screened the mutant by using Cre-Lox P system and establish the optimization scheme of gene knockout.The recombinant strain was identified by PCR and Western blotting.Its biological character was also analyzed.The recombinant plasmid was constructed,and the inh A deletion mutant was got,and the resistance selection marker was deleted.Compared to the AP422,the ability of growth and spore formation of the mutant strain decreased significantly,and the expression of other secreted proteins was affected.We constructed inh A gene deletion mutant of Bacillus anthracis AP422.This research will be helpful to study the functions of inh A gene and the forming mechanism of Bacillus anthracis spore.Then,we constructed the lack of nos gene of accine candidate strains based on B.anthracis AP430.Upstream homologous fragment and downstream homologous fragment of nos gene were amplified by PCR to construct the targeting vector.Weintroduced the recombinant into B.anthracis AP430 by electroporation and screened the mutant by using Cre-Lox P system.The recombinant strain was identified by PCR and the biological character was also analyzed.The recombinant plasmid was constructed,and the inh A deletion mutant was got,and the resistance selection marker was deleted.Compared to the AP430,the ability of growth and the sensitivity of hydrogen peroxide of the mutant strain decreased significantly.Spore formation of the mutant strain is no affected.We constructed nos and mnt A gene deletion mutant of Bacillus anthracis.This research will be helpful to study the functions of nos gene and provide an potential vaccine candidate strains for anthrax.Finally,to judg the immunity and the safety of vaccine candidate strains,the spores of vaccine candidate strains were giving to mice and guinea pigs by ntramuscular injection with a dose of 2?107CFU each animal.Though ELISA test,the antisera titers of mice against PA caused by A16 R,vaccine candidate,and blank in mice were 0.56,0.57,and 0.06;the antisera titers against spore caused by A16 R,vaccine candidate,and blank in mice were 0.64,0.65,and 0.02;the antisera titers of guinea pig against PA caused by A16 R,vaccine candidate,and blank in mice were 0.63,0.61,and 0.14.The results show that compared with the blank,immune response caused by A16 R and vaccine candidate strains increased significantly;but compared with the A16 R,immune response caused by vaccine candidate strains did not change significantly.The spores of vaccine candidate strains were giving to mice by ntramuscular and subcutaneous injection with a dose of 2?108CFU each animal.The number of deaths for mice given A16 R by ntramuscular was 7 and the number given vaccine candidate strains was 0;the number of deaths for mice given A16 R by subcutaneous injection was 6 and the number given vaccine candidate strains was 0.The results show that compared with the A16 R,the toxicity of vaccine candidate strains has been decreased greatly.This has layed a good foundation for the study of anthrax vaccine.