目的原核表达、纯化肺炎链球菌表面蛋白A(pneumococcal surface protein A,Psp A),并制备多克隆抗体。方法应用ANTHEWIN、DNAstar等分子生物学软件,对Psp A氨基酸序列进行分析,筛选出抗原表位富集区(第33~109个氨基酸),选用原核生物偏爱的密码子优化基因序列,化学合成全新的基因序列pspa,插入质粒p GEX-4T-2和p ET28a(+)中,构建重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa,转化大肠埃希菌BL21(DE3),IPTG诱导表达。分别纯化带有GST标签和His标签的Psp A重组蛋白GST-Psp A和His-Psp A,以His-Psp A作为免疫原,经背部多点免疫新西兰大耳白兔,间接ELISA法检测血清抗体效价,Western blot法检测血清抗体特异性。结果两种重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa经双酶切鉴定构建正确;表达的两种重组蛋白GST-Psp A和His-Psp A相对分子质量分别约为33 000和18 000,均为可溶性表达,纯化后目的蛋白条带均无降解,纯度约为95%,蛋白浓度分别为2和0.2 mg/ml;制备的兔抗血清效价较高,可达1∶200 000,且特异性较好。结论原核表达并纯化了肺炎链球菌Psp A融合蛋白,并制备了特异性良好的高效价兔抗血清,为下一步建立肺炎链球菌快速检测技术奠定了基础。 Objective To prokaryotic express and purify pneumococcal surface protein A (Psp A), and to prepare polyclonal antibodies. Methods Amino acid sequences of Psp A were analyzed by molecular biology software ANTHEWIN, DNAstar and so on, and the antigenic epitope-rich region (33- 109 amino acids) was screened out. The preferred codon-optimized gene sequences of prokaryotes were selected and chemically synthesized The recombinant plasmid pGEX-4T-2-pspa and pET28a (+) - pspa were constructed and inserted into pGEX-4T-2 and pET28a (+) plasmids. The recombinant plasmids were transformed into Escherichia coli BL21 ), IPTG induced expression. Psp A recombinant proteins GST-Psp A and His-Psp A with GST tag and His-tag were purified respectively. His-Psp A was used as immunogen to immunize rabbits with New Zealand white rabbits. Indirect ELISA was used to detect serum antibody Titer, Western blot detection of serum antibody specificity. Results The two recombinant plasmids pGEX-4T-2-pspa and pET28a (+) - pspa were identified by double enzyme digestion. The relative molecular weights of the two recombinant proteins GST-Psp A and His-Psp A were approximately Were 33 000 and 18 000, respectively, and were soluble. The purified protein showed no degradation after purification. The purity was about 95% and the protein concentration was 2 and 0.2 mg / ml respectively. The titer of rabbit antiserum was high, Up to 1: 200 000, and the specificity is better. Conclusion Prokaryotic expression and purification of Pneumococcal Psp A fusion protein, and the preparation of a good specificity of high titer rabbit antiserum for the next step to establish rapid detection of Streptococcus pneumoniae technology laid the foundation.