Construction And Identification Of A Recombinant BCG Vaccine Secreting The Fusion Protein GM-CSF-Ag85B-Rv3872-CFP10-ESAT6

Tuberculosis (TB) is a zoonotic disease caused by the Mycobacterium tuberculosis complex group, which has a high rate of infection and death roll. The disease not only seriously affects the health of the human body, but also causes heavy economic losses to the livestock industry. Each year, there are about 9.4 million new cases of tuberculosis and 1.7 million people die from TB infection. What’s more alarming is that it is believed that one-third of the world’s population is latently infected with TB per year. As for the animal infection, about 50 million cattle are infected with Mycobacterium bovis annually, resulting in the loss of at least 3 billion each year.In order to prevent human tuberculosis, people takes two main measures:BCG immunize new-born infants and antibiotic treatment of TB patients. BCG can be protective on only children but not adults, and the effect of different parts in the world varies largely. TB antibiotic treatment lasts for more than six months; and drug side effects and the emergence of multi-drug-resistant and pan-drug-resistant strains render treatment ineffective. Therefore, it is an urgent need to develop new and efficient vaccines that increase the protection for children and provide protection for adults.Due to zoonotic characteristics of TB, the measure to prevent and control the animal tuberculosis is quarantine-culling, without immunization. Quarantine-culling policy has many defects, and is difficult to be carried out. Firstly, the cost of quarantine-culling is so high that the developing countries can not afford; Secondly, animal tuberculosis is caused by Mycobacterium bovis, whose hosts range widely, the disseminations between different hosts leads to poor effects of Quarantine-culling policy. In addition, wildlife is difficult to be detected and culled. Therefore, an effective vaccine is also needed to prevent and control the animal tuberculosis.At present, there are mainly two approaches in TB vaccine development:1) improving traditional BCG strains by introducting protective antigen into BCG,; 2) attenuating virulent Mycobacterium tuberculosis with genetic tools. Because BCG comes from Mycobacterium bovis but human Tuberculosis is caused by Mycobacterium tuberculosis, it is hoped to develop new vaccine from same species of bacteria caused TB. Since animal tuberculosis is mainly due to Mycobacterium bovis, the improvements of BCG would not only provide new vaccines against human tuberculosis, but also new means against animal tuberculosis.Compared with virulence Mycobacterium bovis, BCG genome lacks many genes within RD regions, and some of which have good immunogenicity and can induce protective immunity. Introduction of some genes into BCG can improve the immune protective effect of BCG against TB. Some of the above genes are:the TB10.4, Ag85A, Ag85B, CFP-10, ESAT-6, HSP65 and Mtb39A, and several of them have been introduced into the BCG and proved to enhance the immune protection. On the other hand, some cytokines acted as molecular adjuvants are also applied to increase effect of recombinant BCG on TB, such as GM-CSF, IL-2, IL-12, and IFN-γ.In my study, the protective antigens Ag85B, Rv3872, CFP10, ESAT6 and cytokine GM-CSF, were introduced into BCG together, aiming to construct an ideal candidate vaccine strains for human and animals. The main contents and results are described as follows:1. Cloning and identification of genesBy using MtbH37Rv genome as the template, Ag85B (A), Rv3872 (R), CFP10 (C) and ESAT6 (E) were amplified by PCR, forming ARCE in fusion, then cloned into pET32. The recombinant plasmid was verified to be correct by restriction enzyme digestion and sequencing, and transformed into E. coli BL21, then induced for expression, The results showed that the fused gene can be expressed; the recombinant protein was analyzed by Western blot with RCE polyclonal antibody, and confirmed to be of immunogenicity.GM-CSF (G) was synthesized from Sangon Biotech Co. and connected with gene Ag85B、Rv3872、CFP10 and ESAT6, forming GARCE, then cloned into the vector pET32. The constructed plasmid was verified to be correct by enzyme digestion and sequencing, and then transformed into E. coli BL21, then was induced for expression, The results showed that fused gene can be expressed; the recombinant protein was analyzed by Western blot with RCE polyclonal antibody, and confirmed to be of immunogenicity.2. Construction and identification of plasmidsGM-CSF (referred to as G), Ag85B (A), Rv3872 (R), CFP10 (C) and EAST6 (E) were connected in series, then cloned into the E. coli-M. Bacillus shuttle plasmid pMV261 and integration vector pTIC6a, to construct recombinant plasmids of antigen gene with GM-CSF and without GM-CSF. The resultant plasmids are named as pMV261-GARCE, pMV261-ARCE, pTIC6a-GARCE, pTIC6a-ARCE respectively. The recombinant plasmids were proved to be correct by restriction enzyme digestion, PCR amplification and sequencing.3. Construction and identification of recombinant BCGFour recombinant plasmids were transformed into BCG by electroporation, the expression and reactogenicity of the recombinant protein expressed by the recombinant BCG were analyzed by Western blot. The results showed that the foreign genes were successfully expressed in rBCG. Furthermore the results showed that rBCG and parental BCG have similar growth curves, colony morphology, and staining properties.4. The immunological test with the recombinant BCGsA total of 120 mice (BALB/C) were divided into six groups (n= 20), including four recombinant BCG immunization group, one PBS group as negative control, one BCG parental strain group as positive control. Dose of immuniziation was 106CFU, and subcutaneous inoculation was applied. Three mice of each group were sacrificed every three weeks to observe both humoral and cellular immune induced by BCG and recombinant protein in the bodies. We have detected the levels of antibodies in the mouse sera, the lymphocyte proliferation, and cytokine secretion levels and confirmed that immunized mice can produce specific humoral and cellular immune against the BCG exogenous protein.In summary, four recombinant BCGs that contain Ag85B, Rv3872, CFP10, ESAT6 and GM-CSF gene were constructed successfully and introduced protein could be expressed and induce immune response.