Expression Of Secreted Ag85A Of BCG In Tomato

Background Mycobacterium tuberculosis remains a significant public health problem in the world. The tuberculosis control program has been took out in developing countries, however the improvement of the epidemiological situation has been rather sluggish. There are about 4,510 thousand patients with active pulmonary tuberculosis, and 1,500 thousand smear-positive cases, and 1,960 thousand culture-positive cases in China. There was only a minimal decrease of 25% for active pulmonary tuberculosis cases in the last 10 years according to the estimated numbers of patients with active pulmonary tuberculosis. The prevalence of tuberculosis infection is based on the sum of 44.5%. In another word, there are a half population (about 550 millions people) infected by mycobacterium tuberculosis. The overall prevalence of tuberculosis infection in Years 0 and 14 is calculated at 5.5%. 130 thousand patients died from tuberculosis each year. The death from tuberculosis had 2 times more than from any other infectious diseases and parasites. The patients died at average 55.2 years old.The prevalence of HIV/AIDS resulted in the increase of tuberculosis in the last 20 years. Human is faced a severe threatening from tuberculosis and HIV/AIDS co-infection. Tuberculosis activated latent HIV infection. On the other hand, HIV increased the progress of latent mycobacterium tuberculosis infection. Tuberculosis and HIV cooperated with each other and deteriorated the other. In the first side,Immunity of patients with HIV can not hold back the growth of mycobacterium tuberculosis and dissemination. Tuberculosis is the most opportunistic infection and cause of patients with HIV. In the second side, patients with tuberculosis activated macrophage that induced TNF- a stimulating HTV reproductive, and developed in AIDS.Mycobacterium bovis BCG prevented people from tuberculosis almost 100 years. Its protection against tuberculosis was limited. BCG protected babies and children against incidence and mortality of tuberculosis. Especially tuberculosis meningitis milary cases decreased among children. However BCG has little protection against pulmonary and extra-pulmonary tuberculosis in adults. Pulmonary tuberculosis in adults was the most cause of death, and the only source of infection. It is the most important to develop new vaccine against tuberculosis.Antigen 85A from mycobacterium is the main extracellular protein including secreted and mature form. Mature Ag85A located in culture filtrate, while secreted form containing signal peptide and mature protein located in cell. Many researches showed that Ag85 A is potentially protective. Subunit protein vaccine, DNA vaccine, recombinant virus vaccine and recombinant BCG can induce immunity activities. And these vaccines protected mice against the challenge from tuberculosis.Transgenic plants expressing foreign genes are suitable systems for the production of relevant immunogens that can be used to develop a new generation of vaccines, and was regarded by researchers in recent years. Many pre-clinical animal trials and human trials indicated that antigen expressed in plants was assembled into suitable structures, and induced immune response, and was protective.Whole fbpA gene coding secreted Ag85A was transformed into 'Yellow Cherry' tomato to investigate the expression of fbpA gene in tomato.Materials and methods PCR amplification of the fbpA gene from BCG D2 genomic DNA was performed. The amplified products were purified, and theninserted into pUCm-T vectors. Recombinant pUCm-T-fbpA was transformed into E. coli DH5 a . Characterization of the fbpA gene cloned in pUCm-T vectors was carried out by PCR screening individual bacterial colonies, single digestion with restriction endonuclease HmdIII, double digestion with restriction endonucleases BamH I + Sac I, and DNA sequencing. After identification, the excised fbpA insert from recombinant pUCm-T-fbpA with endonucleases BamH I + Sac I was subcloned in vector pBI121 to obtain recombinant plant expression plasmid of pBI121-fbpA. Recombinant pBI121-fbpA cloned in E. coli DH5 a was identified by PCR screening individual bacterial colonies and double digestion with restriction endonucleases BamH I + Sac I . Then, Agrobacterium tumefaciens strain EHA105 was transformed with pBI121-fbpA. After that, colonies were selected by PCR and extracting recombinant pBI121-fbpA. Agrobacterium tumefaciens strain EHA105 carrying recombinant pBI121-fbpA was stored at -70 °C ready to use.Cotyledons and hypocotyls from tomato cultivars 'Yllow Cherry' and 'Red Cherry' were cultured on basic medium MS. Four groups of medium supplemented with different concentrations of hormones ZT and IAA were optimized to induce callus and shoots. The best explant (cotyledon of 'Yellow Cherry') was tested on medium supplanted with Km, Cb and Cef. Then, EHA105 suspending culture carrying recombinant pBI121-fbpA infected cotyledons after 2 days of pre-culture. The optical density of EHA105 suspending culture was about 0.3, and infection continued for 10 minutes. Then, co-culture of ERA 105 and cotyledons continued for 3 days in dark (in the duration of Agrobacterium tumefaciens strain EHA105 reproduction YEB medium was supplemented with 200 uM AS and 5% glucose, while in the duration of infection and co-culture MS medium was supplemented with 20 uM AS and 5% glucose). Cotyledons were selected in the medium with 10 mg/L Km, 500 mg/L Cb after co-culture. When there were shoots from callus, the shoots were cut and continuously selected in the root medium with 10 mg/L Km, 500 mg/LCef. When there were roots from the bottom of stem, the plants transformed by EHA105 carrying fbpA gene were trained and transplanted from medium to field.Collect small amounts of fresh and younger plant tissue in a microfuge tube and split. Supernatant was tested to amplify the target gene fbpA. Tomato plants total DNA was extracted to amplify fbpA gene and hybridize by Southern blotting in order to conform the integration of fbpA gene in tomato DNA. Then total protein was extracted from tomato fruits in order to characterize the expression of fbpA by Western blotting hybridiation.Results The DNA sequence fbpA gene contained a 1014-bp open reading frame. Upstream of this DNA region, the gene codes for a signal peptide required for the secretion of a 295-amino-acid-long mature protein. The DNA sequence coding for the signal peptide contains 129-base-pair, and the deduced amino acid sequence corresponds to a 43-residue protein. The DNA sequence coding for the mature protein contains 885-base-pair. The fbpA geng was identical to that from At. bovis BCG strain 1173P2 in homology between DNA sequencesCotyledons of 'Yellow Cherry' were the best explant than others in four groups of medium. Its shooting rate was 100%, and an average days of 11 were needed to induce shoots. The optimal medium was MS supplemented with 1 mg/L ZT and 0.8 mg/L IAA. cotyledon explants of 'Yellow Cherry' grew badly with increasing of kanamycin concentration. The percentage of callus and shoots significantly reduced. However, days of inducing callus and shoots prolonged. Regeneration of cotyledons were inhibited completely when the concentration of kanamycin was 10 mg/L. Therefore, 10 mg/L kanamycin was used to select transformed cotyledons. There were no effects of 0-500 mg/L carbenicillin on the regeneration of cotyledons. However, 500 mg/L carbenicillin inhibited the growth of roots. The same concentration of cefotaxin had no effects on roots. So, 500 mg/L carbenicillin was used during inducing shoots while 500 mg/L cefotaxin was used during inducingroots against Agrobacterium tumefaciens strain EHA105.After infection and co-culture, cotyledons were selected in MS medium with 10 mg/L Km and 500 mg/L Cb. The research showed that callus and shoots transformed by EHA105 carrying fbpA grew slowly and badly. There were a little induced callus and shoots among explants. And only one shoot grew in per explant at most. Moreover, shoots rate increased to 13.1% in the medium with supplement of AS and glucose, while it was 6.1% in the medium with no supplements. Then shoots were cut and transplanted into the medium with 10 mg/L Km and 500 mg/L Cef to induce roots. It was observed that there were a few roots and the roots grew slowly. These tomato plants were transplanted to field when roots grew strong.These tomato plants were examined and the results showed that fbpA gene was integrated in the tomato genomic DNA and antigen 85A expressed in tomato fruits were recognized by the serum of patients with tuberculosis.Conclusions 1. The fbpA geng from M. bovis BCG strain D2 was identical to that from 1173P2 in homology between DNA sequences.2. Cotyledons of 'Yellow Cherry' were suitable to accept exogenous gene.3. 10 mg/L kanamycin was suitable to select transformed cotyledons. 500 mg/L carbenicillin was used during inducing shoots while 500 mg/L cefotaxin was used during inducing roots against Agrobacterium tumefaciens strain EHA105.4. AS and glucose increased shooting rate after transformation.5. fbpA gene was integrated in the tomato genomic DNA and antigen 85A expressed in tomato fruits was recognized by the serum of patients with tuberculosis.