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目的构建肺炎链球菌(Streptococcus pneumoniae,S.pneumoniae)pbp1a基因重组的原核表达质粒,并进行鉴定。方法化学合成S.pneumoniae含STMK区的pbp1a基因片段,PCR扩增后,与pUC19载体连接,构建重组表达质粒pUC19-pbp1a,转化耐青霉素的pbp1a基因突变的S.pneumoniae,测定青霉素对转化菌的最低抑菌浓度(Minimum inhibitory concentration,MIC),以鉴定PBP1a的活性;SDS-PAGE和Western blot分析重组PBP1a蛋白的表达。结果 pbp1a基因扩增产物可见约330 bp的特异条带,大小与预期一致,测序表明重组质粒全长360 bp,无碱基的缺失、插入等突变;青霉素对转化菌的MIC从8μg/ml下降为2μg/ml;表达的重组PBP1a蛋白相对分子质量约为11 000,可与抗S.pneumoniae青霉素敏感株兔血清特异性结合。结论成功构建了S.pneumoniae pbp1a基因重组原核表达质粒,其能在S.pneumoniae中表达有活性的PBP1a蛋白,为进一步研究PBPs在细菌耐药变异中的作用提供了实验材料,也为进一步探讨消除细菌耐药性变异的措施奠定了基础。
Objective To construct a prokaryotic expression plasmid of pbp1a gene of Streptococcus pneumoniae (S. pneumoniae) and identify it. Methods The gene fragment of pbp1a in STMK region was chemically synthesized. After PCR amplification, it was ligated with pUC19 vector to construct recombinant plasmid pUC19-pbp1a and transformed into S.pneumoniae with pbp1a mutation of penicillin. Minimum inhibitory concentration (MIC) was used to identify the activity of PBP1a. The expression of recombinant PBP1a protein was analyzed by SDS-PAGE and Western blot. Results The specific amplified fragment of pbp1a was about 330 bp with the same size as expected. The sequencing showed that the full length of the recombinant plasmid was 360 bp with no base deletion and insertion. The MIC of penicillin to the transformed bacteria decreased from 8 μg / ml Was 2μg / ml. The expressed recombinant PBP1a protein had a relative molecular mass of about 11 000 and could specifically bind to S. pneumoniae penicillin-sensitive rabbit serum. Conclusion The prokaryotic expression plasmid pPp1a of S.pneumoniae was successfully constructed, which can express the active PBP1a protein in S.pneumoniae, which provided experimental materials for further study on the role of PBPs in bacterial resistance mutation. Bacterial resistance mutations laid the foundation for the measure.