| Brucellosis is a bacterial infectious disease caused by intracellular parasite Brucell osis,which can be infected by both human and animals.Brucellosis is caused by intr acellular parasite Brucellosis.Brucellosis occurs in most parts of the world.It has become a serious threat to human health and economic development.In recent years,the prevalence of brucellosis has become more and more serious,and China is no exception.It is particularly important to prevent and control brucellosis.Immunization is an important means to prevent brucellosis.At present,the most commonly used vaccines are live attenuated Brucella vaccines.These vaccines have good protective ability,but they also have some shortcomings,such as toxicity and susceptibility to infection,and interfere with serological testing,leading to the inability to distinguish between natura lly infected animals and immunized animals.Therefore,the study of new Brucella vaccine has become a new hot spot.In recent years,the research of genetic engineering vaccine is getting hotter and hotter.It is more and more important to study a better new vaccine against brucellosis.Lactococcus lactis expression vector is a safe and efficient expression vector,which is often used in the production of subunit vaccines.In this study,three dominant antigens of Brucella Omp10,Omp28 and L7/L12 were introduced into Lactococcus lactis in series to construct recombinant Lactococcus lactisimm unopreparations capable of expressing Brucella fusion protein Omp10-Omp28-L7/L12.Lactococcus lactis is a common probiotic bacteria in nature,which is recognized as a safe microorganism.Lactococcus lactis can colonize in the intestine for a long time and stimulate the mucosal immune response of the body,thus causing systemicim mune response.Therefore,food-grade Lactococcus lactis was selected as expression vector in this experiment.Firstly,the Omp10-Omp28-L7/L12 fusion gene synthesized by the company was amplified by PCR.Then,the Omp10-Omp28-L7/L12 fusion gene was linked with Lactococcus lactis expression vector pNZ8148 by DNA recombination technology,and then transferred into Lactococcus lactis NZ9000 competent cells by electrotransformation.After induction with Nisin,the expressed products were treated withmouse anti-His label antibody,Omp10 polyclonal antibody,Omp28 polyclonal antibody and L7/L12 polyclonal antibody respectively,and HRP-labeled sheep anti-mouse IgG as secondary antibody.The results showed that the specific bands of 55.4 kDa appeared,it indicated that the expression of Omp10-Omp28-L7/L12 fusion protein in Brucell a was successful.SPF BALB/c mice were immunized orally with recombinant Lactococcus lactis p NZ8148-Omp10-Omp28-L7/L12/NZ9000 by oral gavage for the first time in 0-4 days.The mice were immunized continuously for 4 days and strengthened on 10-14 days.Three blood samples and small intestinal mucosa were collected randomly from each group on the 0th,7th,14 th,21th,28 th and 35 th days after the first immunization.The specific antibodies of Brucella IgG,intestinal mucosa sIgA and serum I were detected.The levels of L-2,IL-4,IFN-gamma and the number of CD4+,CD8+T lymphocyt e in peripheral blood were studied.The results showed that recombinant Lactococcus pNZ8148-Omp10-Omp28-L7/L12/NZ9000 could induce the production of specific sIgA and IgG antibodies,increase the levels of IL-2,IL-4 and IFN-gamma,and increase the number of CD4+,CD8+T lymphocytes.In conclusion,recombinant Lactococcus lactis can not only express Brucella Omp10-Omp28-L7/L12 fusion protein,but also produce humoral and cellular immune responses in mice.It provides experimental basis for further development of new Brucellosis vaccine. |
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