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目的:制备金黄色葡萄球菌蛋白A(Sp A)Z结构域的原核表达蛋白及其多克隆抗体,并对其进行特异性鉴定。方法:3段Sp A-Z结构域经柔性肽(GS)串联连接构成三串联体(Sp A-3Z),经原核密码子优化后全基因合成,克隆至p ET21a(+)载体,构建p ET21a(+)/Sp A-3Z重组质粒,测序鉴定;将重组质粒转化大肠杆菌BL21(DE3),经异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达重组蛋白,用Ni-NTA亲和层析法纯化并经SDS-PAGE及Western Blot法分析鉴定;纯化的Sp A-3Z重组蛋白免疫日本大耳白兔,并分别于免疫前和免疫后采集血清,用ELISA法检测其多克隆抗体的反应和效价,并用Western Blot法分析多克隆抗体结合天然Sp A的特异性。结果:成功构建了p ET21a(+)/Sp A-3Z重组质粒;该重组质粒在原核表达系统表达并获得纯化的Sp A-3Z重组蛋白,SDS-PAGE电泳显示该蛋白的分子质量约为21 k Da,与理论分子量大小相符;日本大耳白兔经该蛋白免疫后可产生特异性的多克隆血清Ig G抗体,效价可达1:40 000;Western Blot法检测结果显示,在分子质量约42 k Da(天然Sp A蛋白)处出现单一条带,提示兔多克隆血清抗体可识别天然Sp A。结论:Sp A-3Z原核蛋白有较好的免疫原性和抗原性,制备的Sp A-3Z兔多克隆血清抗体Ig G效价高、特异性好,可进一步用于Sp A-Z相关的生物学和免疫学等功能研究。
Objective: To prepare the prokaryotic expression protein and its polyclonal antibody against Staphylococcus aureus protein A (Sp A) Z domain and to identify it. METHODS: Three Sp AZ domains were connected in series by flexible peptide (GS) to form a triple tandem body (Sp A-3Z), which was synthesized by pronucleus codon optimization and cloned into p ET21a (+) vector to construct pET21a +) / Sp A-3Z recombinant plasmid was constructed and sequenced. The recombinant plasmid was transformed into E.coli BL21 (DE3) and recombinant protein was induced by isopropyl thio-β-D-thiogalactopyranoside (IPTG) Purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western Blot. Purified Sp A-3Z recombinant protein was immunized with Japanese white rabbits and serum was collected before immunization and after immunization. Method was used to detect the reaction and titer of the polyclonal antibody. The specificity of the polyclonal antibody bound to native Sp A was analyzed by Western Blot. Results: The recombinant plasmid pET21a (+) / Sp A-3Z was successfully constructed. The recombinant plasmid was expressed in prokaryotic expression system and purified the recombinant protein Sp A-3Z. The molecular weight of the recombinant protein was about 21 k Da, consistent with the theoretical molecular size; Japanese white rabbits after immunization of the protein can produce specific polyclonal serum Ig G antibody titers up to 1:40 000; Western Blot test results show that the molecular mass A single band at about 42 kDa (native Sp A protein) suggested that rabbit polyclonal serum antibodies recognize native Sp A. CONCLUSION: Sp A-3Z prokaryotic protein has good immunogenicity and antigenicity. The prepared polyclonal antibody against Sp A-3Z rabbit Ig G has high titer and good specificity and can be further used in Sp AZ biology And immunology and other functional studies.