Study On The Transformation Of The Mycobacterium Tuberculosis ESAT-6 Gene Into Papaya

Oral vaccines produced by transgenic plants would change the traditional means of production and inoculation of vaccines, and the cost of vaccine production would be reduced greatly. The epidemic of tuberculosis made it necessary that the new and more effective vaccines for immunoprophylaxis of anti-tuberculosis would be developed. Consequently, it is very significant to develop transgenic plant vaccines.In our work, the ESAT-6 gene was modified by PCR amplification, the Kozak sequence was placed before the start codon of ESAT-6 gene and the KDEL sequence was added to the end of the gene. The modified gene was integrated into a binary vector pBI121. Then the ESAT-6 with 35S promoter and NOS terminator was excised from pBI121/E-6 to reconstruct plant expression vector with pCAMBIA1300. The reconstructed plant expression vector pCAMBIA1300/E-6 which contains hygromycin photransferase gene (HYT) was transferred to Agrobacerium Tumefaciens EHA105.The papaya leaves were infected with the Agrobacerium Tumefaciens pCAMBIA1300/E-6/EHA105, and callas was selected on solidified medium containing hygromycin. 13 regenerated plant with resistance were obtained through somatic embryogenesis from the embryogenic calli formed on the selection media. Some regenerated plants was checked by PCR, and it was confirmed that the ESAT-6 gene had been introduced into the plant genome of transgenic papaya by Southern blot analysis. Expression of ESAT-6 gene was further confirmed by Northern blot analysis. These transgenic plants will provide new material for the development of edible oral vaccines and the production of new effective vaccine of anti-tuberculosis.