CAJ | 학술논문

目的克隆表达大鼠细胞因子信号转导抑制因子-1基因(SOCS-1).方法将大鼠SOCS-1全长编码基因的PCR产物,克隆到原核表达质粒pET-28a(+)中,构建重组质粒pET-28a(+)-ratSOCS-1.转化大肠埃希菌BL-21/DE3,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,应用镍离子金属螯合剂亲和层析柱从表达产物中纯化重组蛋白,通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对表达产物和重组蛋白进行鉴定.应用纯化的重组蛋白免疫新西兰大白兔,抗体滴度达到1:10000以上后进行末次免疫,2周后心脏取血、测定滴度,分离制备免疫血清.用红细胞裂解法制备大鼠外周血白细胞裂解全蛋白.将阴性血清(未用重组蛋白免疫的新西兰大白兔的血清)、免疫血清及兔抗组氨酸标签抗体转入各蛋白条带,用Western免疫印迹检测重组蛋白的免疫学活性.结果 PCR、双酶切及DNA测序分析均表明重组质粒pET-28a(+)-ratSOCS-1构建成功.SDS-PAGE结果可见转化了重组质粒的大肠埃希菌全菌和经超声裂解后的菌体沉淀的样品均在相对分子质量约26 000处有一明显的蛋白条带,经亲和层析获得的纯化重组蛋白有特异的单一条带与上述条带一致,而在超声裂解后的菌体上清中相同位置却未见有蛋白表达条带.Western免疫印迹表明重组蛋白、大鼠外周血白细胞裂解蛋白中相对分子质量约24000的蛋白条带、兔抗组氨酸标签抗体均可被免疫血清识别,分别出现清晰的相对分子质量26000、24 000、26000的反应条带,表明其具有免疫活性.结论 SOCS-1基因可以在大肠埃希菌BL-21/DE3中获得表达,其纯化重组蛋白具有良好的抗原性和免疫活性.
목적 극륭표체대서세포인자신호전도억제인자-1기인(SOCS-1).방법 장대서SOCS-1전장편마기인적PCR산물,극륭도원핵표체질립pET-28a(+)중,구건중조질립pET-28a(+)-ratSOCS-1.전화대장애희균BL-21/DE3,용이병기-β-D-류대반유당감(IPTG)유도표체,응용얼리자금속오합제친화층석주종표체산물중순화중조단백,통과십이완기광산납-취병희선알응효전영(SDS-PAGE)대표체산물화중조단백진행감정.응용순화적중조단백면역신서란대백토,항체적도체도1:10000이상후진행말차면역,2주후심장취혈、측정적도,분리제비면역혈청.용홍세포렬해법제비대서외주혈백세포렬해전단백.장음성혈청(미용중조단백면역적신서란대백토적혈청)、면역혈청급토항조안산표첨항체전입각단백조대,용Western면역인적검측중조단백적면역학활성.결과 PCR、쌍매절급DNA측서분석균표명중조질립pET-28a(+)-ratSOCS-1구건성공.SDS-PAGE결과가견전화료중조질립적대장애희균전균화경초성렬해후적균체침정적양품균재상대분자질량약26 000처유일명현적단백조대,경친화층석획득적순화중조단백유특이적단일조대여상술조대일치,이재초성렬해후적균체상청중상동위치각미견유단백표체조대.Western면역인적표명중조단백、대서외주혈백세포렬해단백중상대분자질량약24000적단백조대、토항조안산표첨항체균가피면역혈청식별,분별출현청석적상대분자질량26000、24 000、26000적반응조대,표명기구유면역활성.결론 SOCS-1기인가이재대장애희균BL-21/DE3중획득표체,기순화중조단백구유량호적항원성화면역활성.
Objective To clone and express the suppressor of cytokine signaling-1 gene (SOCS-1)in rats. Methods PCR product of full-length coding sequence of rat SOCS- 1 was cloned into a prokaryotic expression plasmid [pET-28a(+)] to construct the recombinant expression plasmid [pET-28a(+)-ratSOCS-1].Escherichia coli BL-21/DE3 was tranformed into the recombinant plasmid and expressed with isopropyl-β-D-thiogal (IPTG) induction. Recombinant proteins in the expression products were purified by Ni-IDA affinity chromatography. SDS-PAGE was used to verify the expression products and the recombinant protein. The purified recombinant protein was used to sensitized New Zealand rabbits until the last immunization at an samples which were then determined for antibody titers and separated for sera. Total proteins of peripheral blood WBC were prepared with RBC lysis method. Negative serum (from New Zealand rabbits not immunized with recombinant protein), immunized serum and rabbit anti-histidine labeled antibody were transferred to separate bands and the immunologic activities of recombinant proteins were determined with Western blotting. Results PCR, double enzyme digestion and DNA sequencing demonstrated successful construction of pET-28a(+)-ratSOCS-1 plasmid. The results of SDS-PAGE showed formation of an obvious protein band about 26 000 in relative molecular weight by both Escherichia coli transformed with recombinant plasmid and samples of precipitated bacterial debris from ultrasonic degradation. While characteristic single band from purified recombinant proteins obtained by affinity chromatography was shown to be consistent with this band, no protein expression in supernatant of bacterial debris from ultrasonic degradation was observed at the same location. In addition, Western blotting demonstrated the competency of immunized serum as it clearly recognized the recombinant protein, the 24 000 band of lysed protein from rat peripheral blood leukocytes, and rabbit anti-histidine labeled antibody, generating three distinct bands with relative molecule weights of 26 000, 24 000 and 26 000 respectively. Conclusions Rat SOCS- 1 can be successfully expressed in Escherichia coli (BL-21/DE3). The purified recombinant protein of SOCS-1 may have strong immunogenicity and immunocompetence.