[Transfection and expression of recombinant human beta-defensin-2 gene in insect cells].
OBJECTIVE
This study was conducted to determine the feasibility and relevant technique itinerary for the production of hBD-2 with Baculovirus Expression Vector System (BEVS).


METHODS
The full hBD-2/His cDNA was amplified from rpcDNA3.1/hBD-2/myc-His by using PCR with a pair of primers (hBD2p10 and hBD2p11) and was inserted into the MCS of transfer vector: pAcGHLT-A. AcNPV DNA and rpAcGHLT-A/hBD-2/His were co-transfected into Sf21 cells. Recombination would take place within the Sf21 cells between the homologous regions in the transfer vector and AcNPV DNA. After 5 days of co-transfer, both supernatant of the experimental cells and positive control cells were collected. Sf21 cells were infected with virus rAcNPVhBD-2/His and then determined by end-point dilution assay. The expression of hBD-2/His in both cell lysate and supernatant was analyzed by western blot with specific 6 poly-histamines antibody.


RESULTS
Both enzyme cutting result and sequence analysis showed that recombinant hBD-2 with C terminal of bi-tags of myc and 6xHis had been inserted into the transfer vector of BEVS system correctly, and recombinant transfer vector rpAcGHLT-A/hBD-2/His had been constructed successfully. End-point dilution assay proved that recombinant virus rAcNPVhBD-2/His had been acquired. Western blot revealed that lysate of Sf21 cells transfected by rAcNPVhBD-2/His showed a band of relative moleculal mass about 47.5 x 10(3) which implied that a fuse peptide of hBD-2/His with up-stream of GST tag, 6xHistag, protein kinase A site and thrombin cleavage had expressed. The culture supernatant showed two bands of relative moleculal mass about 40 x 10(3) and 30 x 10(3), which were inferred to be the proceeded products of the fuse peptide during secretion process from cell into culture supernatant.


CONCLUSIONS
These results suggested that it may be feasible to use BEVS system as a high efficient biologic reactor for producing recombinant hBD-2.
Cai
Shao-hui
SH
School of Pharmacy of Jinan University, Guangzhou 510632, China.
Du
Jun
J
Huang
Ning
N
Wang
Bo-yao
BY
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000235:genetics* / Q000378:metabolism; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
C106641:DEFB4A protein, human; D011994:Recombinant Proteins; D023083:beta-Defensins