The Effect Of BCG With Knockout Of The Heat Shock Protein 16.3 Gene On The Mouse Macrophage RAW 264.7

Objectives:To construct and identify BCG strain and Mycobacterium tuberculosis(MTB)H37Rv strain with heat shock protein(Hsp16.3)gene mutations, further to study the autophagy function of macrophages RAW 264.7 after infected with those mutation strains, which will provide certain theoretical basis for explore the mechanisms of latent tuberculosis infection( LTBI).Methods:The BCG strain and H37 Rv strain were cultured in 7H9 medium at 37 ℃ for 4weeks,and the genome of those strains were extracted by commercial Kit. We designed different primers to amplify Hsp16.3 gene with 435 bp, 3’ and 5’ side sequence of Hsp16.3 gene by polymerase chain reaction(PCR), respectively. All DNA sequences were confirmed by sequencing. The gene of 3’ and 5’ side sequence of Hsp16.3 was inserted into the corresponding multiple cloning sites region of vector p KO. Positive plasmid was screened by PCR, restriction enzyme cutting and sequencing, named p KO-Hsp16.3. This p KO-Hsp16.3 plasmid was transfected into competent BCG/H37 Rv cell by electroporation, and the transformants were screened on 7H10 agar plates containing kanamycin and saccharose respectively. We further describe the relative curve of OD value and mutation bacterial CFU and confirmed the bacterium dosage infection mouse macrophage. The effect of BCG with Hsp16.3 gene mutation on autophagy function of mouse macrophage RAW 264.7 was assayed by Western Blot and Immunocytochemistry.Results:We have successfully amplified a serial of genes from MTB genome, including Hsp16.3 gene with 435 bp, its 5’ gene with 663 bp and 3’ gene with 684 bp by PCR. The recombinant plasmid p KO-Hsp16.3 was constructed by insert the 5’ gene and 3’DNA fragments into p KO vector. After the electrictransformation, the positive BCG strain/H37 Rv strain with Hsp16.3 gene mutation was screened on the 7H10 agar or sauton medium with kanamycin sulfate and sucrose. Hsp16.3 gene mutation was further confirmed by PCR analysis. Through detection of LC3 expression, We found BCG strain with gene Hsp16.3 mutation infection mouse macrophage RAW264.7 could promote the autophagy formation in macrophage.Conclusion:Successfully construct the recombinant plasmid p KO-Hsp16.3 and the BCG and H37 Rv strain with Hsp16.3 gene mutation. Autophagy could be induced after BCG strain with Hsp16.3 gene mutation infection mouse macrophage.