甲醇诱导条件对登革病毒四型联合重组包膜蛋白Ⅲ区表达的影响
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摘要
目的探讨甲醇诱导浓度和诱导时间对登革病毒(dengue virus,DENV)四型联合重组包膜蛋白Ⅲ区(envelope domainⅢ,EDⅢ)表达的影响,以期实现该蛋白在毕赤酵母中的高效表达。方法采用不同浓度甲醇(0. 05%、0. 1%、0. 25%、0. 5%、0. 75%、1%、1. 5%、2%、2. 5%、3%、5%、7. 5%)诱导表达带His标签的DENV四型联合重组EDⅢ蛋白,分别诱导18、24、30、48、72 h,样品经Western blot检测。最佳条件下诱导表达的蛋白液经Ni-Agarose His亲和层析纯化后,进行10%SDS-PAGE检测。结果最佳甲醇诱导浓度为2. 5%,最佳甲醇诱导时间为72 h。纯化产物经10%SDS-PAGE检测,于相对分子质量约41 000处可见单一目的蛋白条带,纯度为99%。结论当甲醇浓度为2. 5%,诱导时间为72 h时,可实现DENV四型联合重组EDⅢ蛋白在毕赤酵母中的高效表达。
Objective To investigate the effect of concentration and time for induction by methanol on expression of tetravalent recombinant envelope domain Ⅲ(EDⅢ)protein of dengue virus(DENV)so as to highly express the protein in Pichia pastoris. Methods The expression of His-tagged recombinant EDIII protein was induced with methanol at various concentrations(0. 05%,0. 1%,0. 25%,0. 5%,0. 75%,1%,1. 5%,2%,2. 5%,3%,5% and 7. 5%)for various(18,24,30,48 and 72) hours,and identified by Western blot. The protein expressed under the optimal condition was purified by Ni-Agarose His affinity chromatography and identified by 10% SDS-PAGE. Results The optimal methanol concentration and time for induction were 2. 5% and 72 h respectively. The purified protein showed a single band with a relative molecular mass of about 41 000 on 10% SDS-PAGE profile,of which the purity was 99%.Conclusion Tetravalent recombinant ED Ⅲ protein of DENV was highly expressed in P. pastoris after induction with2. 5% methanol for 72 h.
Objective To investigate the effect of concentration and time for induction by methanol on expression of tetravalent recombinant envelope domain Ⅲ(EDⅢ)protein of dengue virus(DENV)so as to highly express the protein in Pichia pastoris. Methods The expression of His-tagged recombinant EDIII protein was induced with methanol at various concentrations(0. 05%,0. 1%,0. 25%,0. 5%,0. 75%,1%,1. 5%,2%,2. 5%,3%,5% and 7. 5%)for various(18,24,30,48 and 72) hours,and identified by Western blot. The protein expressed under the optimal condition was purified by Ni-Agarose His affinity chromatography and identified by 10% SDS-PAGE. Results The optimal methanol concentration and time for induction were 2. 5% and 72 h respectively. The purified protein showed a single band with a relative molecular mass of about 41 000 on 10% SDS-PAGE profile,of which the purity was 99%.Conclusion Tetravalent recombinant ED Ⅲ protein of DENV was highly expressed in P. pastoris after induction with2. 5% methanol for 72 h.
引文
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[13] WU D,CHU J,WANG Y H,et al. Influence of methanol concentration on purification recovery of consensus interferon-αproduced by Pichia pastoris[J]. Chin J Biotech,2011,27(12):1789-1796.(in Chinese)吴丹,储炬,王永红,等.甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响[J].生物工程学报,2011,27(12):1789-1796.
[2] LI G H,NING Z J,LIU Y M,et al. Neurological manifestations of dengue infection[J]. Front Cell Infect Microbiol,2017,7:449. doi:10.3389/fcimb.2017.00449.
[3] NGONO A E,SHRESTA S. Immune response to Dengue and Zika[J]. Annu Rev Immunol,2018,36(4):279-308.
[4] KATZELNICK L C,GRESH L,HALLORAN M E,et al. Antibody-dependent enhancement of severe dengue disease in humans[J]. Science,2017,358(6365):929-932.
[5] FAHIMI H,MOHAMMADIPOUR M,HADDAD KASHANI H,et al. Dengue viruses and promising envelope protein domain III-based vaccines[J]. Appl Microbiol Biotechnol,2018,102(7):2977-2996.
[6] ETEMAD B,BATRA G,RAUT R,et al. An envelope domain III-based chimeric antigen produced in Pichia pastoris elicits neutralizing antibodies against all four dengue virus serotypes[J]. Am J Trop Med Hyg,2008,79(3):353-363.
[7] JI G H,DENG Y Q,YU X J,et al. Characterization of a novel dengue serotype 4 virus-specific neutralizing epitope on the envelope protein domainⅢ[J]. PLoS One,2015,10(10):e0139741. doi:10.1371/journal.pone.0139741.
[8] CRILL W D,ROEHRIG J T. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells[J]. J Virol,2001,75(16):7769-7773.
[9] QIU L J,ZHAO Y J,LI D,et al. Expression and immuⅢnological analysis of recombinant tetravalent envelope domainⅢprotein of dengue virus[J]. China Biotechnol,2016,36(1):1-6.(in Chinese)邱丽娟,赵玉娇,李多,等.登革病毒四型联合重组包膜蛋白Ⅲ区的表达和免疫原性鉴定[J].中国生物工程杂志,2016,36(1):1-6.
[10] WANG M, JIANG S, WANG Y. Recent advances in the production of recombinant subunit vaccines in Pichia pastoris[J]. Bioengineered,2016,7(3):155-165.
[11]王洪海,高卜渝,陈佩丽,等.酵母表达基因工程产物不均一性分析及其对策[J].中国科学(B辑),1994,24(12):1270-1274.
[12] LOOSER V,BRUHLMANN B,BUMBAK F,et al. Cultivation strategies to enhance productivity of Pichia pastoris:A review[J]. Biotechnol Adv,2015,33(6 Pt 2):1177-1193.
[13] WU D,CHU J,WANG Y H,et al. Influence of methanol concentration on purification recovery of consensus interferon-αproduced by Pichia pastoris[J]. Chin J Biotech,2011,27(12):1789-1796.(in Chinese)吴丹,储炬,王永红,等.甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响[J].生物工程学报,2011,27(12):1789-1796.