BCG_3174基因敲除卡介苗菌株的构建及鉴定

Construction and identification of BCG_3174 knockout BCG strain

目的为了证实BCG_3174是BCG基因组中存在的潜在抑制凋亡功能基因,拟构建BCG_3174敲除菌株并进行鉴定。方法以BCG中国株的基因组DNA为模板,PCR扩增BCG_3174基因,扩增产物用Van91Ⅰ酶切并回收产物。载体p0004S转化E.coli DH5α培养,用Van91I酶切载体p0004S。将酶切BCG_3174基因片段的左、右臂以及酶切载体p0004S的3.6 kb和1.6 kb两个片段连接,转化入DH5α中培养,筛选阳性克隆并测序鉴定,构建重组质粒p0004S-ΔBCG_3174。将载体phAE159转化E.coli HB101培养,提取phAE159后以PacI酶切,同时以PacI酶切重组质粒p0004S-ΔBCG_3174,以T4连接酶连接。加入E.coli HB101感受态细胞并培养,提取质粒后用PacI酶切鉴定,阳性克隆命名为phAE159-ΔBCG_3174,电击转化耻垢分枝杆菌mc2155感受态细胞。取BCG菌液与噬菌体裂解液混合共培养,提取敲除菌株的基因组DNA,鉴定正确的菌株命名为ΔBCG_3174。结果凝胶成像显示扩增产物与预期目标大小一致,左臂大小为887 bp,右臂大小为931 bp。阳性克隆测序确证左、右臂正确连入p0004S且序列无突变,p0004S-ΔBCG_3174序列与预期一致。PacⅠ酶切phAE159载体产生约42 kb和4 kb两条片段,酶切p0004S-ΔBCG_3174质粒产生7.1 kb片段,该片段亚克隆入PacⅠ酶切的phAE159载体后产生重组质粒phAE159-ΔBCG_3174,酶切鉴定证实phAE159-ΔBCG_3174构建正确。BCG_3174基因敲除的BCG基因组PCR扩增产物大小约为5.6 kb,与预期大小相符,BCG_3174基因敲除BCG菌株ΔBCG_3174构建成功。结论成功制备BCG_3174基因敲除的BCG菌株ΔBCG_3174,为BCG_3174基因的功能研究奠定了基础。

Objective To prepare and identify the BCG 3174 gene in a BCG knockout strain in order to confirm that BCG_3174 is a potential anti-apoptosis gene in the BCG vaccine.Methods Flanked by its left and right arms,the BCG_3174 gene was amplified with PCR using the genomic DNA of the BCG Chinese strain as a template and specific primers.The products of amplification were collected after Van91I enzyme digestion.The expression vector p0004 S was amplified in E.coli DH5α,extracted,and subjected to Van91 I digestion.Two of four segments 3.6 kb and 1.6 kb in length were ligated with the left and right flanks of BCG_3174,designated p0004 s-ΔBCG_3174,and transformed into E.coli DH5αfor replication.The positive clone,designated p0004 s-ΔBCG_3174,was selected using hygromycin and confirmed using sequencing.The vector phae159 was transformed into E.coli HB101 and cultured.After extraction of phAE159,the recombinant plasmid p004 s-ΔBCG_3174 was digested with PacI,ligated to fragments of phAE159 pretreated with PacI enzyme using T4 ligase,and transformed into E.coli HB101 competent cells.The plasmids were extracted and identified using PacI digestion.The positive clone was designated phAE159-ΔBCG_3174 and transformed into Mycobacterium smegmatis mc2155 competent cells via electroporation.The BCG culture suspension was co-incubated with a bacteriophage solution,and the genomic DNA of the surviving bacterium was extracted,sequenced,and designatedΔBCG_3174.Results Agarose gels indicated that the left and right flanks of the BCG_3174 gene in p0004 S-ΔBCG_3174 were 887 bp and 931 bp in length,and sequencing confirmed that they were inserted in the right direction in the vector without a mutation.After digestion with PacI,the two fragments 42 kb and 4 kb in length from phAE159 and the 7.1-kb fragment from p0004 S-ΔBCG_3174 were recombined as phAE159-ΔBCG_3174 without any genetic errors according to sequencing.The PCR products from the BCG genome with BCG_3174 knocked out were approximately 5.6 kb in length,which was consistent with expectations and which indicated that the BCGΔBCG_3174 strain was successfully constructed.Conclusion A BCG_3174 knockout BCG strain was constructed and identified.This finding lays the foundation for subsequent study of the functions of the BCG_3174 gene.