High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization.
BACKGROUND
Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).


RESULTS
A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 x 10(8) plaque forming unit (pfu) in hemolymph, which was 2.8 x 10(4) times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..


CONCLUSIONS
The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.
Kato
Tatsuya
T
Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. actkato@shizuoka.ac.jp
Manoha
Suganthi Lavender
SL
Tanaka
Shigeyasu
S
Park
Enoch Y
EY
D000818:Animals; D012831:Bombyx / Q000378:metabolism* / Q000821:virology; D002850:Chromatography, Gel; D006458:Hemolymph / Q000378:metabolism; D006801:Humans; D007814:Larva / Q000378:metabolism / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014461:Ultracentrifugation; D014776:Virus Cultivation; D000093342:Prorenin Receptor
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000093342:Prorenin Receptor