Expression of recombinant baculovirus carrying Schistosoma japonicum 26 ku GST in mammalian cells.
In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 x 10(8). Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.
Yu
Guangqing
G
Department of Parasitology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Song
Jianhua
J
Liu
Wenqi
W
Long
Xiaochun
X
Mo
Hongmei
H
Li
Yonglong
Y
Chen
Xinwen
X
D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012549:Schistosoma japonicum / Q000235:genetics / Q000276:immunology*; D018411:Spodoptera; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology
D000947:Antigens, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D005982:Glutathione Transferase