[Cloning and efficient prokaryotic expression of soluble stage-specific antigen cC1 from Cysticercus cellulosae].
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 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.


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 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.


CONCLUSIONS
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.
Department of Microbiology and Parasitology, Bengbu Medical College, Bengbu 233030, China. fq333@sohu.com
Qiang
Q
Fang
Jiang-kun
JK
Luo
Zhuo
Z
Cui
Wen-juan
WJ
Qi
Yuan-sheng
YS
Hu
Ji-long
JL
Shen
D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D003552:Cysticercus / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D012995:Solubility; D013552:Swine; D041201:Taenia solium / Q000276:immunology
D000947:Antigens, Helminth; D011993:Recombinant Fusion Proteins