CAJ | 학술논문

目的：通过基因工程手段在大肠杆菌中表达并纯化融合蛋白 HSP65-PEAⅠ，建立小鼠实验模型，检测其抗体效价水平，初步评价免疫效果。方法扩增 HSP65-PEAⅠ片段插入表达载体 pET-28a 中，转化 E．coli BL21（DE3），IPTG 诱导表达，优化纯化条件，以 HSP65-PEAⅠ为免疫原对小鼠进行免疫接种。结果双酶切和测序鉴定结果证实 HSP65-PEAⅠ成功克隆入 pET-28a 载体中，表达的重组蛋白相对分子量约为97000，主要以包涵体形式表达，纯化后的重组蛋白纯度达90％，浓度为0．498 mg／ml。免疫小鼠均在首免1周后采集的血清中检测到特异性抗体，抗体效价水平持续增长且在42天均达到了210。结论成功在 E．coli 中表达并经过纯化得到重组蛋白 HSP65-PEAⅠ，确定了动物免疫程序，为制备抗烧烫伤多器官衰竭疫苗奠定了基础。
목적：통과기인공정수단재대장간균중표체병순화융합단백 HSP65-PEAⅠ，건립소서실험모형，검측기항체효개수평，초보평개면역효과。방법확증 HSP65-PEAⅠ편단삽입표체재체 pET-28a 중，전화 E．coli BL21（DE3），IPTG 유도표체，우화순화조건，이 HSP65-PEAⅠ위면역원대소서진행면역접충。결과쌍매절화측서감정결과증실 HSP65-PEAⅠ성공극륭입 pET-28a 재체중，표체적중조단백상대분자량약위97000，주요이포함체형식표체，순화후적중조단백순도체90％，농도위0．498 mg／ml。면역소서균재수면1주후채집적혈청중검측도특이성항체，항체효개수평지속증장차재42천균체도료210。결론성공재 E．coli 중표체병경과순화득도중조단백 HSP65-PEAⅠ，학정료동물면역정서，위제비항소탕상다기관쇠갈역묘전정료기출。
Objective Express protein HSP65-PEAⅠ in E.coli and obtain purified protein,establish mice model, preliminary evaluate the immune efficacy by detecting antibody titer levels.Methods Amplify HSP65-PEAⅠand then inserted into expression vector pET-28a,transform E.coli BL21(DE3),express with the induction of IPTG,optimize the purification condition,then the protein was inoculated in mice as immunogen.Results Double digestion and sequen-cing results proved that the HSP65-PEAⅠ gene was cloned into vector pET-28a,the relative molecular mass of ex-pressed recombinant protein was about 97000,mainly expressed as inclusion body.After purification,purity of recombi-nant protein was more than 90%,concentration was 0.498 mg/ml.The specific antibody was detected in the serum of mice in the first week after immunization,the titer level continued growing and rise to 2 10 after 42 days.Conclusion Re-combinant protein HSP65-PEAⅠwas successfully expressed in E.coli and purified.Animal immunization program was further established which laid a foundation for preparation of vaccine for multiple organ failure by burns.