Expression of DNA vaccine against Trichinella spiralis in Chinese hamster ovary cells.
OBJECTIVE
To observe the in vitro expression of DNA vaccine (recombinant eukaryotic expression plasmid pcDNA3-TspE1) encoding a Mr 31,000 antigen of Trichinella spiralis in Chinese hamster ovary (CHO) cells and analyze the antigenicity of the products expressed.


METHODS
The recombinant plasmid pcDNA3-TspE1 was transfected into CHO cells by using cationic lipids (Lipofectamine 2000). The positive cell clones were screened by the selective antibiotic G418. The expressed products were identified by RT-PCR, IFAT, SDS-PAGE and Western blotting.


RESULTS
The results of RT-PCR amplification showed that there was one band with 876 bp in CHO cells transfected with pcDNA3-TspE1 and no any bands in CHO cells transfected with the empty plasmid pcDNA3. The IFAT demonstrated that the pcDNA3-TspE1 transfected CHO cells reacted with sera from mice immunized with the recombinant fusion protein and from mice infected with T. spiralis, the bright yellow green fluorescence staining appeared in the transfected CHO cells. The pcDNA3 transfected and un-transfected CHO cells exhibited as orange color. The results of SDS-PAGE showed that there was one band with Mr 31,000 in culture supernatant of CHO cells transfected with pcDNA3-TspE1. Western blotting confirmed that the band with Mr 31,000 could be recognized by sera from mice immunized with the recombinant fusion protein, from rabbits immunized with T. spiralis muscle larval soluble antigens, from mice infected with T. spiralis and from patients with trichinellosis. Conclusion The mammalian CHO cells were transfected by the recombinant plasmid pcDNA3-TspE1, and the TspE1 gene of T. spiralis was expressed in the transfected CHO cells. The proteins expressed are secreted into cell culture supernatants and show the antigenicity of T. spiralis.
Cui
Jing
J
Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou 450052, China.
Wang
Zhong-quan
ZQ
Han
Hua-Min
HM
Wei
Hai-Yan
HY
Zhang
Hong-wei
HW
Li
Yong-long
YL
D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000235:genetics / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D014162:Transfection; D017160:Trichinella spiralis / Q000276:immunology*; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000276:immunology
D000947:Antigens, Helminth; D004274:DNA, Recombinant; D019444:Vaccines, DNA