Objective To construct the PUCD-dnaK recombinate luminescence bacteria vector containing a complete luxCDABE gene box to investigate the impaired mechanism of pollutant protein damage. Methods The gene of dnaK was amplified by PCR from E Coli W3110. The homology of sequencing result was tested by using BLAST and compared with danK in GenBank. The PCR products and PUCD615 vector were all digested with BamH I and EcoR I, then linked and imported into JM109 with electro transformation. Several clones were selected and identified by PCR and sequencing. Results The PCR amplification product of dnaK gene obtained 206 bp fragment, and the ratio of BLAST to PCR was 100% after sequencing, indicating that the amplification sequence was correct. PUCD-dnaK sequencing showed that the sequence contained the dnaK gene and the gene on the PUCD615 vector, and the corresponding cleavage site appeared correctly. Conclusion Construction of PUCD-dnaK vector is successful. PUCD615 containing the full luxCDABE gene box has the advantage over only contained luxAB, for the later set out bioluminescent only in the presence of substrate (fatty aldehyde). By optimizing the condition of ligation and transformation, the large fragment of PUCD615 and the short inserted sequence can be ligated successfully.%目的 构建含完整luxCDABE基因盒的PUCD-dnaK重组发光载体,用于对蛋白损伤污染进行毒性评价.方法 用PCR法从大肠杆菌W3110中扩增dnaK基因,将PCR产物测序后与GenBank中dnaK序列进行BLAST比对.将dnaK片段及PUCD615载体均用BamH I、EcoR I双酶切,连接后电转化导入宿主菌JM109.挑取克隆,提取质粒用PCR鉴定,阳性克隆再进行测序.结果 dnaK基因PCR扩增产物得到206 bp片段,PCR产物测序后BLAST比对同源性为100%,表明扩增序列正确.PUCD-dnaK测序结果表明序列含有dnaK基因及PUCD615载体上的基因,且与载体连接处正确地出现对应的酶切位点.结论 PUCD-dnaK载体构建成功.含luxCDABE全基因盒作为报告基因的载体PUCD615可克服luxAB必须外加底物(脂肪醛)才能实现生物发光的缺点,通过优化连接及转化条件,可将大片段的PUCD615载体与短片段的插入序列连接成功,构成重组载体.
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