卡介苗Hsp16.3基因突变株对小鼠巨噬细胞RAW 264.7自噬的影响

The Effect of BCG with Knockout of the Heat Shock Protein 16. 3 Gene on the Mouse Macrophage RAW 264. 7

构建及鉴定卡介苗(BCG)Hsp16.3基因突变株,观察该突变株对小鼠巨噬细胞自噬功能的影响。通过聚合酶链反应(PCR)的方法扩增Hsp16.3基因两侧分子量分别为663 bp和684 bp的两个DNA片段,并将这两个目的片段分别插入p KO载体上相应的位点。通过硫酸卡那霉素筛选,利用双酶切以及测序的方法,获得阳性重组质粒p KO-Hsp16.3。将阳性重组质粒p KO-Hsp16.3电转入BCG感受态细胞中,经过硫酸卡那霉素和蔗糖两次筛选,得到BCG Hsp16.3基因突变株。将筛选培养的BCG Hsp16.3突变株用于小鼠巨噬细胞的感染实验,并通过Western Blot和细胞免疫荧光技术对自噬相关蛋白的表达水平的变化进行观察及分析。成功获得重组质粒p KO-Hsp16.3的阳性克隆;电转化该重组质粒,并经过硫酸卡那霉素筛选和蔗糖反筛选,成功得到BCG Hsp16.3基因突变株。通过Western Blot以及免疫荧光分析发现,BCG Hsp16.3基因的突变株促进了小鼠巨噬细胞自噬的发生。

To construct and ident i fy BCG strain wi th heat shock protein（ Hspl6. 3 ） gene mutat ions, the autophagy function of macrophages RAW 264. 7 after those mutation strains infection was further studied. Different primers were designed to ampl ify 3 ’ and 5 ’ side sequence of Hspl6. 3 gene by polymerase chain reaction （PCR） , respec-t ively. All DNA sequences were confirmed by sequencing. The gene of 3 , and 5 ’ side sequence of Hspl6 . 3 was inserted into the corresponding multiple cloning sites region of vector pKO. Positive plasmid was screened by re-strict ion enzyme cutting and sequencing, named pKO-Hspl6. 3. This pKO-Hspl6. 3 plasmid was transfected into comepetent BCG/H37Rv cell by electroporat ion, and the transformants were screened on 7H10 agar plates contai-ning kanamycin and saccharose respectively. The effect of BCG with Hspl6. 3 gene mutation on autophagy function of mouse macrophage RAW 264. 7 was assayed by Western Blot and Immunocytochemistry. The recombinant plamid pKO-Hspl6. 3 have been successfully gotten,which was constructed by insert ing the 5 gene and 3 DNA fragments into pKO vector. After the electrictransformat ion, the positive BCG strain with Hspl6. 3 gene mutation was screened on the 7H10 agar or sauton medium with kanamycin sulfate and sucrose. And the BCG Hspl6. 3 gene mu-tation was gotten. Through detection of LC3 expression, BCG strain with gene Hspl6. 3 mutation was found infec-ting mouse macrophage RAW264. 7 could promote the autophagy formation in macrophage.