百日咳毒素S1亚单位截短片段的表达、纯化及初步应用

目的表达并纯化百日咳毒素(PT)S1亚单位截短片段S146,以其制备抗体,初步建立检测PT的双抗体夹心ELISA法。方法PCR扩增S146基因,亚克隆至载体pUC18,鉴定正确后,插入表达载体pET16b,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot鉴定后,用镍离子亲和层析柱纯化,复性后的蛋白免疫家兔,制备抗血清,纯化后作为包被抗体,建立检测PT的双抗体夹心ELISA法,并检测其特异性。结果表达的重组蛋白相对分子质量约为19000,主要存在于破菌沉淀中,表达量占菌体总蛋白的44%;纯化后蛋白纯度为94.5%;家兔抗血清可特异性识别天然PTSI;建立的双抗体夹心ELISA法特异性良好。结论已成功表达、纯化了PTS1亚单位截短片段S146,并以纯化的抗S146抗体作为包被抗体,初步建立了检测PT的双抗体夹心ELISA法,为研制特异性检测试剂和提供一种疫苗的质量控制方法奠定了基础。

Expression and Purification of Truncated Fragment of Pertussis Toxin S1 Sub unit and Preliminary Application of Expressed Product

Objective To express the truncated fragment S146 of pertussis toxin(PT)S1 subunit in E.coli and prepare anti-body with the purified expressed product to develop a double antibody sandwich ELISA for determination of PT.Methods S146 gene fragment was amplified by PCR and subcloned to vector pUC18.The constructed recombinant plasmid pUC18-S146 was identi-fied by sequencing,from which the target gene was extracted and inserted into expression vector pET16b.The constructed recombi-nant plasmid pET16b-S146 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by nickel ion affinity chromatography and renaturalized.Rabbits were immunized with the renaturalized protein,and the antisera were collected,purified and used as coating antibody to develop a double antibody sandwich ELISA for determination of PT.The developed ELISA method was evaluated for specificity.Results The ex-pressed product,with a relative molecular mass of about 19 000,mainly existed in the ultrasonic precipitate of recombinant E.coli,contained 44%of total somatic protein and reached a purity of 94.5%after purification.The prepared rabbit antisera recognized nat-ural PT specifically,and the developed double antibody sandwich ELISA showed good specificity.Conclusion The truncated frag-ment S146 of PT S1 subunit was successfully expressed and purified,and a double antibody sandwich ELISA was preliminarily devel-oped using the expressed product as coating antibody,which laid a foundation of development of specific detection kit for quality con-trol of vaccine.