Mycobacterium Tuberculosis The RpfE And RipB Cloning,Expression And Biological Activity Performance

Tuberculosis (TB) is respiratory disease caused by infection with mycobacterium Tuberculosis, which is the second biggest infectious killer of human worldwide. Now the detection of MTB in sputum is the gold standard of diagnosis of tuberculosis. However, it takes long time to recover dormant MTB in the process of testing, which delays the diagnosis and treatment of TB.Resuscitation promoting factor (Rpf) can promote recovery and growth of gram-positive bacterium with high G+C content such as Micrococcus luteus, mycobacterium marinum and MTB. Resuscitation promoting factor interacting protein A, which can accelerate the recovery of dormant MTB cooperating with Rpf B, is a kind of peptidoglycan hydrolase. We found that Rip B shared high identity with the N-terminal of Rip A, and speculated Rip B could degrade the peptidoglycan of gram-positive bacterium and promote their recovery. In this research, we used Escherichia coli to express Rpf E and Rip B, analyzed their function of promoting bacteria multiplication and evaluated the feasibility of Rpf E and Rip B served as candidate components of rapid detecting MTBAccording to the sequence of rpfE and RipB, we designed primers, and amplified the gene of rpfE and RipB with the genome of Mycobacterium tuberculosis as templates. We constructed plasmid pGEX-4T-rpfE and pET-21a-RipB, and transformed them into the recombinant E. coli, respectively. According to the experiment of inducing express conditions optimization, we found the optimal expression conditions of RpfE was0.75mmol/L IPTG, inducing10h at28℃, and RipB was0.5mmol/L, inducing12h at15℃.We tested virous concentration of rpfE and RipB promoted the recovery and multiplication of Micrococcus luteus, and found that both rpfE and RipB accelerated the multiplication of Micrococcus luteus at100pmol/L.