[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.
Fang
Qiang
Q
Department of Microbiology and Parasitology, Bengbu Medical College, Bengbu 233030, China. fq333@sohu.com
Luo
Jiang-kun
JK
Cui
Zhuo
Z
Qi
Wen-juan
WJ
Hu
Yuan-sheng
YS
Shen
Ji-long
JL
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