论文部分内容阅读
目的原核表达并纯化霍乱毒素A亚单位(cholera toxin subunitA,CTA)基因,建立分泌CTA单克隆抗体的杂交瘤细胞系,并制备单克隆抗体。方法构建原核表达质粒pET30a-CTA,转化大肠埃希菌(E.coli)BL21(DE3),IPTG诱导表达6 h,表达产物经12%SDS-PAGE分析后,用Ni2+亲和层析柱纯化;将纯化的重组CTA蛋白免疫C57BL/6小鼠,运用杂交瘤细胞技术制备抗CTA的单抗,采用Western blot法及体外阻断实验检测抗CTA单抗的特异性及对CT毒性作用的体外阻断。结果原核表达质粒pET30a-CTA经PCR及双酶切鉴定,证明构建正确;表达的重组CTA蛋白在相对分子质量25 000~35 000之间可见明显的目的蛋白条带,纯化后的重组CTA蛋白纯度达90%以上;获得1株可分泌抗CTA单抗的杂交瘤细胞株G6C3-D10,可与CTA蛋白特异性结合,在相对分子质量25 000~35 000之间可见明显的特异条带;杂交瘤培养上清中CTA特异性抗体滴度为54,与CT混合后,可明显抑制CT所诱导的细胞内环腺苷酸(cyclic adenosine monophosphate,cAMP)水平的升高(P<0.01)。结论原核表达并纯化了CTA基因,获得了能够分泌具有阻断CT毒性作用的单抗的细胞株,为CTA毒性作用和佐剂机制的进一步研究提供了参考。
Objective To express and purify cholera toxin subunit A (CTA) gene in prokaryotic cells and establish a hybridoma cell line secreting CTA monoclonal antibody and to prepare monoclonal antibody. Methods The prokaryotic expression plasmid pET30a-CTA was constructed and transformed into E. coli BL21 (DE3) and induced by IPTG for 6 h. The expressed product was analyzed by 12% SDS-PAGE and purified by Ni2 + affinity chromatography. C57BL / 6 mice were immunized with the purified recombinant CTA protein and monoclonal antibodies against CTA were prepared by hybridoma technology. Western blot and in vitro blocking assay were used to detect the specificity of anti-CTA mAb and the in vitro resistance to CT toxicity Off. Results The prokaryotic expression plasmid pET30a-CTA was identified by PCR and double enzyme digestion, which was proved to be correct. The expressed recombinant CTA protein showed obvious banding between 25 000 and 35 000 in molecular weight. Purified recombinant CTA protein purity Up to 90%. A hybridoma cell line G6C3-D10 secreting anti-CTA McAb was obtained, which could specifically bind with CTA protein and showed obvious specific bands between 25 000 ~ 35 000 in molecular weight. The CTA-specific antibody titer in tumor culture supernatants was 54, which was significantly inhibited by CT-induced increase of intracellular cAMP level (P <0.01). Conclusions Prokaryotic expression and purification of CTA gene and the ability to secrete monoclonal antibodies that block CT toxicity have provided a reference for the further study of CTA toxicity and adjuvant mechanism.