CAJ | 학술논문

目的克隆猪囊尾蚴期特异性抗原cC1基因,并进行高效可溶性原核表达.方法利用PT-PCR从猪囊尾蚴中获得cC1编码基因,T-A克隆后,将cC1插入原核表达载体pET28a中,经酶切、测序鉴定后将其转入宿主菌E.coli BL21(DE3)中,通过IPTG诱导表达并优化表达条件,表达的融合蛋白Ni~+-亲和层析柱纯化后经快速液相蛋白层析分离系统检测其纯度.采用SDS-PAGE和Western-blotting对目的蛋白进行分析和鉴定.结果 RT-PCR扩增出的片段长度为1056 bp,构建的重组表达质粒经PCR、酶切验证,和预期的结果一致,测序鉴定与Genbank中cC1基因相比于423位存在1个同义突变.转化有重组质粒的E.coliBL21(DE3)经过0.05 mmol/L IPTG 37℃诱导6h高效表达了占菌体蛋白总量60%的可为囊虫病患者血清识别的相对分子质量约为40000的可溶性融合蛋白,经Ni+-亲和层析柱纯化后纯度达94%.结论成功克隆并高效可溶性原核表达了猪囊尾蚴期特异性抗原cC1,为其进一步研究和应用奠定了基础.
목적 극륭저낭미유기특이성항원cC1기인,병진행고효가용성원핵표체.방법 이용PT-PCR종저낭미유중획득cC1편마기인,T-A극륭후,장cC1삽입원핵표체재체pET28a중,경매절、측서감정후장기전입숙주균E.coli BL21(DE3)중,통과IPTG유도표체병우화표체조건,표체적융합단백Ni~+-친화층석주순화후경쾌속액상단백층석분리계통검측기순도.채용SDS-PAGE화Western-blotting대목적 단백진행분석화감정.결과 RT-PCR확증출적편단장도위1056 bp,구건적중조표체질립경PCR、매절험증,화예기적결과일치,측서감정여Genbank중cC1기인상비우423위존재1개동의돌변.전화유중조질립적E.coliBL21(DE3)경과0.05 mmol/L IPTG 37℃유도6h고효표체료점균체단백총량60%적가위낭충병환자혈청식별적상대분자질량약위40000적가용성융합단백,경Ni+-친화층석주순화후순도체94%.결론성공극륭병고효가용성원핵표체료저낭미유기특이성항원cC1,위기진일보연구화응용전정료기출.
Objective To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli. Methods The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD 18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the expression conditions were optimized. The expressed product was purified by Ni~+-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting. Results The fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 ℃, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography. Conclusion The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.