Objective To constract the Rv2647-deleted strain and Rv2647-complemented strain of Mycobacterium tuberculosis (M. tb) and Rv2647-overexpressing Mycobacterium smegmatis (Ms::Rv2647) by phage recombination technique and to evaluate the effect of M. tb Rv2647 protein on lung injury in model mice. Methods The affinal exchange sites was constructed and integrated into the phage genomes of M. tb, then the phagemids were obtained and introduced into Mycobacterium smegmatis (Ms). Ultimately, the recombinant phages with the same AES were constructed. Obtaining high titer recombinant phages via amplification in vitro and transfecting them into M. tb, which was cultured for 28 days at 37℃. The single clone was selected and verified by PCR and the Rv2647-deleted strain (H37RvΔRv2647) was obtained. The Rv2647 gene was amplified by PCR and was integrated into the multiple clone sites of vector pMV361 and pMV261 through seamless cloning to obtain the positive plasmids, which were transfected into H37RvΔRv2647 and Ms to obtain the Rv2647-complemented strain (H37RvΔRv2647::Rv2647) and Rv2647-overexpressing Ms (Ms::Rv2647), respectively. The suspension of H37Rv, H37RvΔRv2647, H37RvΔRv2647::Rv2647, Ms, and Ms::Rv2647 were used to infect C57BL/6 mice via tracheal injection. After infection for 30 days and 7 days, the survival, lung bacterial load, and lung injury of model mice were evaluated. Results (1) The PCR showed that Rv2647 gene was missing in the H37RvΔRv2647, while it was present in the H37RvΔRv2647::Rv2647 and Ms::Rv2647; (2) The 30-day survival of model mice infected with H37RvΔRv2647, H37Rv, and H37RvΔRv2647::Rv2647 were 100.00%, 83.33%, and 83.33%, respectively; The 7-day survival of model mice infected with Ms and Ms::Rv2647 were 100.00% and 83.33%; The lung bacterial load (Log10 CFU) of model mice in H37RvΔRv2647 group was 3.40±0.18, which was significantly lower than those of H37Rv group (3.86±0.15, P<0.001) and H37RvΔRv2647::Rv2647 group (3.80±0.16, P<0.01); The inflammation area (%) in lung tissues of model mice in the H37RvΔRv2647 group was 4.37±3.06, which was significantly lower than those in the H37Rv group (62.76±14.24, P<0.001) and H37RvΔRv2647::Rv2647 group (67.37±0.45, P<0.001). The lung bacterial load (Log10 CFU) of model mice in Ms group was 2.53±0.16, which was significantly lower than that of Ms::Rv2647 group (2.81±0.13, P<0.01); The inflammation area (%) in lung tissue of model mice in the Ms group was 5.71±1.29, which was significantly lower than that in the Ms::Rv2647 group (33.13±13.84, P<0.05). Conclusion The Rv2647-deleted strain (H37RvΔRv2647) and Rv2647-complementary strain (H37RvΔRv2647::Rv2647) of M. tb and Rv2647-overexpressing Ms (Ms::Rv2647) were successfully constructed. Rv2647 protein may aggravate lung injury via inhibiting host clearance of M. tb, of which mechanism is worthy of further investigation.