肺炎链球菌pbp1a基因重组原核表达质粒的构建与鉴定

Construction and identification of prokaryotic expression vector for pbp1a gene of Streptococcus pneumoniae

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 and identify a prokaryotic expression vector for pbpla gene of Streptococcus pneumoniae. Methods The pbpla gene＇fragment of S. pneumoniae synthesized chemically, containing STMK region, was amplified by PCR and linked with vector pUC19. The constructed recombinant plasmid pUC19-pbpla was transformed to S. pneumoniae with penicillin- resistant gene mutation. The minimum inhibitory concentration （MIC） of penicillin to the transformed S. pneumoniae was determined to identify the activity of PBPla. The expression of recombinant PBPla protein was analyzed by SDS-PAGE and Western blot. Results The length of amplified pbpla gene was about 330 bp, which was consistent with that expected. Sequencing result showed that the full-length of recombinant plasmid was 360 bp, in which no mutation such as base deletion or insertion was found. The MIC of penicillin to transformed S. pneumoniae decreased from 8 to 2 μg/ml. The expressed recombinant PBPla protein, with a relative molecular mass of about 11 000, showed specific binding to rabbit sera against penicillin-sensitive S. pneumoniae strain. Conlusion The recombinant prokaryotic expression vector for pbpla gene of S. pneumoniae was constructed successfully, and competent PBPla protein was expressed in S. pneumoniae, which provided an experimental material for further study on the role of PBPla in drug- resistant variation of bacteria, and laid a foundation of further investigation on measure for elimination of the variation.