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目的构建肺炎支原体(Mp)双蛋白多特异抗原表位表达载体,提高重组蛋白抗原的敏感性。方法应用生物信息学方法筛选Mp P116粘附蛋白抗原表位序列,PCR点突变技术获取P116蛋白基因片段,与pMD-T载体重组,转入大肠埃希菌JM109,通过限制性酶切图谱和基因序列分析鉴定重组质粒。酶切回收P116基因片段与pGEX 6P-1-P1 DNA重组,转入大肠埃希菌JM109菌株。用Glutathione Sepharose 4B纯化重组蛋白,SDS-PAGE分析表达产物的相对分子量,用Mp免疫血清进行免疫印迹试验,鉴定重组蛋白的免疫原性。结果 PCR点突变扩增Mp黏附蛋白P116的基因片段为597 bp,该基因片段与已知的基因库序列分析比较,除两个突变位点由UAG突变为UGG外,其余核苷酸序列同源性为100%。SDS-PAGE分析多表位重组蛋白相对分子质量(Mr)为77.8 kDa。免疫印迹结果显示,Mp兔多价血清能与纯化的78KDa的重组蛋白发生免疫反应。结论本研究成功构建了Mp双蛋白多表位的表达载体。该表达载体表达的重组蛋白具有Mp特异的免疫反应性。重组蛋白的敏感性有待进一步鉴定。
Objective To construct a Mp multi-epitope expression vector to improve the sensitivity of recombinant protein antigens. Methods The sequence of the Mp P116 adhesion protein epitope was screened by bioinformatics method. The P116 protein gene fragment was obtained by PCR point mutation technology. The fragment was recombined with pMD-T vector and transformed into Escherichia coli JM109. The restriction fragment and gene Sequence analysis identified the recombinant plasmid. The P116 gene fragment was digested and recombined with pGEX 6P-1-P1 DNA and transformed into Escherichia coli JM109 strain. The recombinant protein was purified with Glutathione Sepharose 4B, the relative molecular weight of the expressed product was analyzed by SDS-PAGE, and the immunogenicity of the recombinant protein was identified by immunoblotting with Mp immune serum. Results PCR amplification of Mp adhesion protein P116 gene fragment was 597 bp. Compared with the known gene library sequence analysis, except for the mutation of two mutations to UGG by UAG, the nucleotide sequence homology Sex is 100%. SDS-PAGE analysis of the molecular weight of the multi-epitope recombinant protein (Mr) was 77.8 kDa. Immunoblotting results showed that Mp rabbit polyvalent serum could immunoreact with the purified 78KDa recombinant protein. Conclusion This study successfully constructed a multi-epitope Mp protein expression vector. The recombinant protein expressed by the expression vector has Mp specific immunoreactivity. The sensitivity of recombinant proteins needs to be further identified.