Fang Qiang, Luo Jiang-kun, Cui Zhuo, Qi Wen-juan, Hu Yuan-sheng, Shen Ji-long
Department of Microbiology and Parasitology, Bengbu Medical College, Bengbu 233030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Feb;30(2):206-9.
To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli.
The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-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.
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 degrees celsius;, 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.
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.
克隆猪囊尾蚴阶段特异性抗原cC1的编码基因，并在大肠杆菌中高效表达可溶性cC1。
采用RT-PCR从猪囊尾蚴中扩增cC1基因，克隆至pMD18-T载体，随后亚克隆至原核表达质粒pET28a。将重组质粒转化至大肠杆菌BL21(DE3)中并优化表达条件。表达产物经镍离子亲和层析纯化，通过高效液相色谱（HPLC）分析，并用SDS-PAGE和Western印迹法进行鉴定。
RT-PCR扩增产物片段长度为1056 bp。将扩增的基因序列与Genbank中的cC1基因比较，发现在克隆至表达质粒的基因中423位存在同义点突变。在37℃用0.05 mmol/L IPTG诱导6小时后，40 kd可溶性融合蛋白的表达量超过细菌总蛋白的60%，且该融合蛋白能被囊尾蚴感染的人血清识别。经亲和层析纯化后，融合蛋白纯度约为94%。
成功克隆了猪囊尾蚴阶段特异性抗原cC1，并在大肠杆菌中高效表达了可溶性蛋白，为其进一步研究和应用奠定了基础。
