High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization
High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization
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DOI：
10.1186/1472-6750-9-55
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发表时间：
2009-06-12
影响因子：
3.5
通讯作者：
Park, Enoch Y.
中科院分区：
文献类型：
--
作者：
Kato, Tatsuya;Manoha, Suganthi Lavender;Park, Enoch Y.
背景：芽生杆状病毒被用于疫苗、抗体的生产和跨膜蛋白的功能分析。在本研究中，我们试图制备和纯化重组家蚕核型多角体病毒(rBmNPV-hPRR)，在其自身表面显示与标志肽序列相连的人(PRO)肾素受体(HPRR)。这些颗粒被用来进一步分析hPRR与人Prorenin的结合。家蚕幼虫体内产生rBmNPV-hPRR，用体积排阻色谱(SEC)从家蚕血淋巴中分离纯化重组BmNPV-hPRR。结果：建立了一种快速检测血淋巴中BmNPV滴度的方法。终点稀释法与Q-PCR法测定BmNPV的相关系数为0.99。RBmNPV-hPRR杆状病毒在家蚕血淋巴中产生的重组杆状病毒空斑形成单位(PFU)为1.31×10(8)，是Bm5细胞转染液的2.8×10(4)倍。经Sephacryl S-1000SF柱层析纯化，注射后4d幼虫血淋巴的纯化倍数为264倍，注射后4.5d和5d分别为35倍和39倍。纯化后第4天和第5天的rBmNPV-hPRR的蛋白质图谱相同，而囊膜蛋白(76、45和35 kDa)与核衣壳蛋白VP39的比值在第5天有所增加。结论：rBmNPV-hPRR的成功纯化表明，利用家蚕幼虫生产杆状病毒，并用Sephacryl S-1000SF柱层析从血淋巴中纯化hPRR，为大规模生产获得高滴度的纯化BmNPV毒种提供了一条经济的途径。此外，它还可用于进一步的结合分析和hPRR抑制剂的筛选。
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..Conclusion: 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.