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目的:通过原核表达系统制备绿脓杆菌外毒素(PE)PE38KDEL重组蛋白,并制备特异性兔免疫血清多克隆抗体。方法:选择PE部分基因(PE38),并将C端609-613位的氨基酸REDLK突变为KDEL,通过原核密码子优化(http://www.jcat.de)后全基因合成,经Hind III和Xho I酶切位点克隆至p ET32a(+)原核表达载体,构建p ET32a(+)/PE38KDEL重组质粒,将测序正确的重组质粒转化到E.coli BL21(DE3)感受态细菌中,经异丙基硫代半乳糖苷(IPTG)诱导表达重组PE38KDEL蛋白,经Ni-NTA亲和层析法制备纯化蛋白,采用SDS-PAGE电泳和Western blot法进行鉴定。进一步将PE38KDEL纯化蛋白免疫日本大耳白兔制备PE38KDEL特异性多克隆抗体,并采集免疫前后血清,用ELISA法检测免疫后的抗体反应和效价。结果:成功构建了p ET32a(+)/PE38KDEL重组质粒。在原核表达系统中该质粒成功表达并获得纯化的PE38KDEL蛋白。SDSPAGE电泳显示蛋白分子质量约为57 k Da,与预计理论值大小相符合。Western blot法检测结果显示,在分子质量约57 k Da处出现单一条带。通过免疫大耳白兔成功获得PE38KDEL特异性多克隆抗体,抗体效价高达1:60 000。结论:PE38KDEL蛋白可诱导兔产生特异性多克隆血清抗体,且该抗体效价高、特异性强,为进一步研究基于PE38KDEL毒素的生物学和免疫学等功能奠定了基础。
Objective: To prepare the PE38KDEL recombinant protein of Pseudomonas aeruginosa exotoxin (PE) by prokaryotic expression system and to prepare polyclonal antibody against rabbit polyclonal antibody. Methods: The partial PE gene (PE38) was selected and the amino acid REDLK at position 609-613 of C terminus was mutated to KDEL. After complete gene synthesis by prokaryotic codon usage (http://www.jcat.de) Xho I restriction enzyme site was cloned into p ET32a (+) prokaryotic expression vector to construct p ET32a (+) / PE38KDEL recombinant plasmid, the correct recombinant plasmid was transformed into E. coli BL21 (DE3) competent bacteria, Recombinant PE38KDEL protein was induced by propylthiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blot. PE38KDEL purified protein was further immunized with Japanese white rabbits to prepare PE38KDEL specific polyclonal antibody, and serum before and after immunization was collected. The antibody response and titer after immunization were detected by ELISA. Results: The p ET32a (+) / PE38KDEL recombinant plasmid was successfully constructed. The plasmid was successfully expressed in prokaryotic expression system and obtained purified PE38KDEL protein. SDSPAGE electrophoresis showed that the molecular weight of the protein was about 57 kDa, which was consistent with the predicted theoretical value. Western blot results showed that a single band appeared at about 57 kDa molecular weight. PE38KDEL specific polyclonal antibody was successfully obtained by immunizing large white rabbits and the antibody titer was as high as 1:60 000. CONCLUSION: PE38KDEL protein can induce specific polyclonal antibody in rabbits. The antibody is highly potent and specific, which lays the foundation for further study on the biological and immunological functions of PE38KDEL toxin.