Mycobacterium tuberculosis Rv2450c gene cloning and prokaryotic expression of.doc

 PAGE \* MERGEFORMAT 11 Mycobacterium tuberculosis Rv2450c gene cloning and prokaryotic expression of Authors: LI Li-qing, SU Ming-quan, Li-Wen Li, Hao Ke [Keywords:] Mycobacterium tuberculosis Cloning and prokaryotic expression of Mycobacterium tuberculosis Rv2450c gene [Abstract] AIM: To explore whether the Mycobacterium tuberculosis Rv2450c gene can promote its own resuscitation and growth. METHODS: Mycobacterium tuberculosis was cultured and genome DNA was extracted. Rv2450c gene was obtained by PCR using specific primers, cloned into BamH Ⅰ and EcoR Ⅰ site of pGEX4T2 expression vector and confirmed by sequencing. After the recombinant expression vector being transformed into E.coli BL21 (DE3), the recombinant bacteria were induced at 30 ℃ for 5 h and the fusion protein GSTRv2450c was analyzed by SDSPAGE. RESULTS: DNA sequencing results showed that the Rv2450c gene amplified was exactly consistent with the sequence reported in GenBank and the SDSPAGE analysis demonstrated that the Rv2450c was expressed in E.coli BL21 (DE3). The protein band amounted to 22.9% of total bacteria protein. CONCLUSION: Mycobacterium tuberculosis Rv2450c gene is successfully cloned and expressed in E.coli BL21 (DE3). [Keywords] Mycobacterium tuberculosis; resuscitationpromoting factor; cloning, molecular; gene expression [Abstract] Objective: To study whether the Mycobacterium tuberculosis Rv2450c gene can promote its revival and growth. Methods: M. tuberculosis, extract its genome, PCR amplified Rv2450c gene was cloned into the pSP73 vector and sequenced. The purpose of pGEX4T2 gene was subcloned into the expression vector, sequencing confirmed that is correct transformed into E. coli BL21 (DE3), 30 ℃ with IPTG induction 5 h and then SDSPAGE analysis. Results: The sequencing results confirm that won the Rv2450c gene, which sequence is fully consistent with the reported in GenBank . SDSPAGE analysis showed that, Rv2450c gene in E. coli BL21 (DE3) expressed recombinant pr