绿脓杆菌外毒素PE38KDEL重组蛋白原核表达及多克隆抗体的制备

Prokaryotic expression of recombinant PE38KDEL protein and preparation of its polyclonal antibody

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 PE38 KDEL recombinant protein from Pseudomonas exotoxin A in prokaryotic expression system and prepare the PE38 KDEL specific polyclonal antibody. Methods： The C-terminal 609-613 amine acid REDLK from Pseudomonas aeruginosa exotoxin（PE） genes（PE38） was mutanted to KDEL. After prokaryotic codon optimization（http：//www.jcat.de）, the full gene sequences were synthesized and then cloned into expression vector p ET32a（＋） prokaryotic expression vector inserted into Hind III and Xho I and constructed the recombinant plasmid p ET32a（＋）/PE38 KDEL. After sequenced, the plasmid was transformed into E.coli BL21（DE3） and induced by isopropyl beta-D-1-thiogalactoside（IPTG） to get the PE38 KDEL protein. The recombinant protein was purified by Ni-NTA affinity chromatography, SDS-PAGE and Western blot were used to identify it. Then, the rabbits were immunized by the PE38 KDEL purified protein. The sera were collected before and after immunized. The antibody reaction and titer were detected by ELISA. Results： The p ET32a（＋）/PE38 KDEL recombinant plasmid was successfully constructed and the protein was expressed and purified in the prokaryotic expression system. The protein molecular weight was about 57 k Da which was in accordance with the expected theoretical value. The PE38 KDEL polyclonal antibody was obtained by immunization of rabbits and the titer of it was as high as 1：60 000. Conclusion： The PE38 KDEL recombinant protein can induce the rabbit to produce serum polyclonal antibody which has high titer and strong specificity and can be used for further study which laid a solid foundation on the biology and immunology function.