结核分枝杆菌T7噬菌体展示基因组DNA文库的构建与鉴定

目的 利用T7噬菌体作为载体,构建结核分枝杆菌的基因组DNA文库。方法 提取MTB的基因组DNA,用EcoR I和Hind III进行双酶切,以对基因组DNA进行片段化,并在其末端加上定向的EcoR I和Hind III黏性末端,用DNA片段纯化分离柱除去小于200 bp的片段和多余接头。将DNA片段与T7 10-3b噬菌体连接,经包装蛋白进行包装后转入BLT5403宿主菌以构建T7噬菌体展示基因组DNA文库,并检测文库的滴度和随机性。结果 成功提取到较高质量的MTB基因组DNA;构建的T7噬菌体展示基因组DNA文库的滴度约为6×10⁶ pfu/cm³;用PCR检测结果表明,在随机挑取的16个噬菌斑中,其重组率为100%,且插入片段都介于250~2 000 bp左右。结论 成功构建了MTB T7噬菌体展示基因组DNA文库,为从基因组范围筛选MTB的优势抗原奠定了前期实验基础。

Construction and identification of T7 phage display genome DNA library of Mycobacterium tuberculosis

T7 phage displayed library of genomic DNA of Mycobacterium tuberculosis was constructed to further screen the immunodominant antigens from the whole genome of Mycobacterium tuberculosis. The total genomic DNA of Mycobacterium tuberculosis was extracted and digested with EcoR I and Hind III in order to fragment the genomic DNA and ligated with EcoR I / Hind III sticky ends. The DNA fragments that were longer than 200 bp were collected and ligated into the T7 10-3b phage vector. After packaged in vitro, the recombinant T7 phage vectors were transformed into BLT5403 to construct the T7 phage display library. The genomic DNA with high quality was successfully extracted. The titer of T7 phage display library was about 6×10⁶ pfu/cm³. The 16 phage plaques were randomly selected and amplified by PCR. The results of PCR showed that the recombination ratio was about 100%, and the inserts were long between 250 bp and 2 000 bp. And the results of BLAST analysis showed all sequences are originated from Mycobacterium tuberculosis. These results indicated that the T7 phage display genome DNA library of Mycobacterium tuberculosis was successfully constructed, which lay the foundation for the screening of the immunodominant antigens from the whole genome of Mycobacterium tuberculosis.