[Cloning and expression of the F0-ATP synthase B chain of Clonorchis sinensis and immunogenicity identification of the recombinant protein].
OBJECTIVE
To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein.


METHODS
The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity.


RESULTS
The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein.


CONCLUSIONS
The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.
Zhou
Hong-juan
HJ
Department of Parasitology, Zhongshan Medical College of Sun Yat-sen University, Guangzhou 510080, China.
Hu
Xu-chu
XC
Hu
Feng-yu
FY
Xu
Jing
J
Ma
Chang-Ling
CL
Zheng
Xiao-ling
XL
Chen
Xiao-xiang
XX
Yu
Xin-bing
XB
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D015153:Blotting, Western; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D015801:Helminth Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000909:Antibodies, Helminth; D018076:DNA, Complementary; D015801:Helminth Proteins; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases