Construction And Immunogenicity Study Of MVA-Ag85B-TB10.4and MVA-tPA-Ag85B-TB10.4

Tuberculosis as a human disease occurs at approximately70,000years ago in Africa, and Mycobacterium tuberculosis as the pathogen was also found more than a century. But now, TB remains a major diseases afflicting humankind. Based on World Health Organization statistics in2012, the number of new TB patients worldwide is8.6million and1.3million dead due to tuberculosis. Currently the only widely used Mycobacterium tuberculosis is vaccine BCG, but its protective effect for adult is not good. So the development of new and effective TB vaccine is imminent. There are numerous combination of bacteria secreted proteins, some of which have been shown to have good immunogenicity, and has been used in some vaccines. Ag85B is one kind of protein to be used in many new tuberculosis vaccine as antigen protein, which is belong to Fbp family. It can help M. tuberculosis adhere to the mucosal surface, thereby facilitating them entry into the host cell. TB10.4is a member of the ESAT‐6family, which is a iron and zinc intake route of Mycobacterium. It is necessary for the growth of Mycobacterium tuberculosis. Viral vectors in the current vaccine research is being taken seriously, and poxvirus is considered a good candidate vaccine vector vectors due to its good immunogenicity and excellent safety.In this study, we construct MVA‐Ag85B‐TB10.4, which expresses recombinant Ag85B‐TB10,4fusion protein with poxvirus vector. In order to enhance the expression of the protein, we also construct MVA‐tPA‐Ag85B‐TB10.4, that introduced tissue plasminogen activator signal peptide (tPA) on the N‐terminal of Ag85B‐TB10.4. Specific contents are as follows:Firstly, we obtain Ag85B‐TB10.4and Ag85B‐TB10.4with tPA or HA by PCR. We connect them into pcDNA3.1(‐) and then transform them into293T cell. The result of western blot show that, Ag85B‐TB10.4can express normally in cell, while tPA signal peptide for improved protein expression plays a significant role.Secondly, Ag85B‐TB10.4gene and tPA‐Ag85B‐TB10.4gene are recombined into MVA by pSC11M1. Result of western blot show that recombinant virus strains can express foreign protein normally.Thirdly, the two strains of recombinant viruses were amplified and purified, then immune Balb/c mouse. MVA‐Blank and PBS are also immured as control groups. The result show that with antigen protein and peptide stimulation, MVA‐Ag85B‐TB10.4induced IFN‐γ secretion weakly. By contrast, MVA‐Ag85B‐TB10.4induced much stronger IFN‐γ secretion. It prove that tPA can enhance the level of response after vaccination.In this study, the immune effects of MVA‐Ag85B‐TB10.4and MVA‐tPA‐Ag85B‐TB10.4were investigated initially, confirmed that tPA signal peptide can enhance the immune response effect. However, the specific mechanism of action and the protective effect of the vaccine require further study.