| Tuberculosis (TB) is one of the leading infectious disease in adults, causing over 8 million new cases and 3 million deaths annually. It is estimated that one third of the world,s population were infected with Mycobacterium tuberculosis.The human immuneodeficiency virus pandemic and the emergency of multi-drug resistant strains of the causative bacilli have led to an elevated incidence of TB. The World Health Organization has recently declared the current situation to be a global emergency and has made it a priority to develop more effective vaccines against TB.Bacille Calmette-Guerin(BCG),a live,attenuated strain of Mycobacterium bovis, is currently the only available vaccine for the prevention of TB,BCG has demonstrated protection against severe and fatal TB in children . However, BCG has shown itself to be of varible efficacy (ranging from 0-80%)for protecting adults from pulmonary TB,depending on the population tested . Thus,the generation of an improved vaccine to replace BCG and to prevent TB is urgently needed.Several new types of TB vaccine preparations,including subunit, live attenuated,recombinant BCG and DNA vaccines,are currently being investingated experimentally.BCG has shown itself to be of extensive safety(there are over 3 billion people who have inoculated BCG all around the world) , rebuild BCG is a strategy to develop TB vaccine.ERP gene is a very important virous gene in Mycobacterium tuberculosis.It exist not only in Mycobacterium tuberculosis, but also in BCG.The suffer whose immune function is defective was inoculated BCG,it maybe will arouse severe diffused tuberculosis.Objective In order to knockout erp gene in BCG,the sideward gene of ERP was amplified and gene targeting vector was constructed. constructing BCG mutation.Methods Culturing the BCG in vitro,extracting the genome DNA ,amplifying the targeted gene with PCR,constructing the gene targeting vector of the BCG and identifying it.and then electroporating it into the BCG.Results The fragment was amplified successfully.The replacement vector with deleted ERP gene was constructed. BCG mutation with deleted ERP gene was constructed.Conclusion Constructing the replacement vector which is used to the gene knockout in BCG. constructing the BCG mutation with deleted ERP gene. |
