美洲商陆抗病毒蛋白基因在毕赤酵母中的表达
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摘要
植物病毒病是农作物的主要病害之一，它危害严重，给农业生产带来的巨大经济损失。长期以来，人们一直在探索防治病毒病的方法，但到目前为止还未研制出有效防治试剂。许多研究发现美洲商陆抗病毒蛋白(pokeweed antiviral protein，PAP)对植物病毒具有良好的控制效果。然而，自然界天然存在的PAP资源有限，很难大规模利用。用生物反应器，低成本大量生产PAP，是解决这一问题的有效方法之一。目前已经有人在这方面做出了尝试，但蛋白表达产量还不高。Francis等在巴斯德毕赤酵母(Pichia pastoris)中表达了信号肽和毒性区缺失的PAP基因，使其表达量达到了10-12mg/L。中国农科院植保所植物病毒实验室陈定虎博士成功的筛选到P.pastoris PAP利用甲醇缓慢型重组子(Mut~s)，希望能利用微生物发酵的方法来大量生产PAP，将其应用于植物病毒病的防治。然而，Mut~s菌株由于发酵周期长，甲醇率利用低，并不适用于实际的大规模生产。
本研究将含有N末端信号肽和C末端毒性区缺失的PAP基因的表达载体pPIC9K-P用Sal I酶切线性化后，通过电击转化整合P.pastorisGS115菌株细胞中。PCR筛选出利用甲醇快速型(Mut~+)的重组子后用双膜免疫杂交法筛选出表达量较高的重组菌株。
在相同的培养条件下比较了Mut~+和Mut~s重组菌株表达PAP的异同。结果表明，诱导培养48小时后，Mut~+重组菌株表达产物在SDS-PAGE胶上显现出清晰的目的蛋白带，而Mut~s重组菌株培养72小时才能显示微弱的目的带；Western-blotting杂交信号强度表明，同样培养6天的Mut~+和Mut~s重组菌株表达产物在表达量上没有明显差别。抗病毒活性实验也表明，相同浓度的Mut~+重组子和Mut~s重组子的表达产物均对TMV病毒侵染有明显的抑制作用，且它们的抗病毒活性没有显著的差异。
通过摇瓶培养初步摸索了诱导时间、甲醇浓度、培养基pH值等培养条件对Mut~+重组菌株表达PAP的影响。SDS-PAGE分析结果表明，Mut~+组菌株在甲醇诱导第四天后PAP在培养液中积累量达到最高水平，延长培养时间会导致产量下降；在10g/L的甲醇浓度诱导下，PAP的表达量达到最高；培养基pH值在偏酸性条件下(6.0-6.4)PAP的表达量都维持在较高的水平。
Plant virus disease is one primary crop disease. It distributes wildly and does great harm to economy of agriculture yield. People search after ways to prevention and cure virus diseases for long time but not develop effective reagent yet. Many studies revealed that pokeweed antiviral protein (PAP) can control plant virus effectively. But PAP in nature is limited which could not apply cosmically. Using bioreacter to produce PAP is one efficacious way to get a mass of PAP. Someone tried this way but the yield is low. Francis expressed truncated mutant pokeweed antiviral protein gene in Pichia pastoris. The yield reached 10-12mg/L. Dr. Chen ding hu in plant virus group of Institute of plant protection of the CAAS (Chinese academy of agriculture sciences) selected PAP Muts recombinants and wanted to produce PAP through fermentation to prevention and cure plant virus disease. But as to Muts recombinants, the periods of fermentation is too long and the efficiency of using methanol is low. So using Muts recombinants to produce PAP in factory is not felicitousness.
Linearized the expression vector pPIC9K-P including the truncated mutant pokeweed antiviral protein (PAP) gene by restriction endonuclease Sal I and transformed it electrically into Pichia pastoris GS115.Mut+recombinants were selected by PCR and high yield Mut+ recombinant was picked out by double film immunoblotting.
To find out the difference between Mut+ and Muts recombinants we compared their expression of pokeweed antiviral protein in the same conditions. SDS-PAGE results showed that there was a clear target protein band in Mut+ recombinant supernatant after 48 hours of culturing, while a faint band only in Muts recombinant after 72 hours. Western-blotting result showed that there was no remarkable difference of yield between Mut+ and Muts recombinants after 6 days induced. Anti-virus activity tests revealed that culture supernatants of Mut+ and Muts recombinants could inhibit TMV infection with high efficiency in the same concentration and there was no significant difference between them.
Different cultivation condition such as induced time, methanol concentration and pH of culture medium was studied at shake flask cultivation. SDS-PAGE results showed that as to Mut+ recombinant highest yield was obtained after 4 days inducing and with the culture
time prolonged it reduced. Pokeweed antiviral protein gene expressed well when methanol concentration reached 10g/L. Pokeweed antiviral protein obtained high yield in thin acidic culture medium (pH6.0-6.4) and its quantity in total mass of secrete protein exceeded 30%.
本研究将含有N末端信号肽和C末端毒性区缺失的PAP基因的表达载体pPIC9K-P用Sal I酶切线性化后，通过电击转化整合P.pastorisGS115菌株细胞中。PCR筛选出利用甲醇快速型(Mut~+)的重组子后用双膜免疫杂交法筛选出表达量较高的重组菌株。
在相同的培养条件下比较了Mut~+和Mut~s重组菌株表达PAP的异同。结果表明，诱导培养48小时后，Mut~+重组菌株表达产物在SDS-PAGE胶上显现出清晰的目的蛋白带，而Mut~s重组菌株培养72小时才能显示微弱的目的带；Western-blotting杂交信号强度表明，同样培养6天的Mut~+和Mut~s重组菌株表达产物在表达量上没有明显差别。抗病毒活性实验也表明，相同浓度的Mut~+重组子和Mut~s重组子的表达产物均对TMV病毒侵染有明显的抑制作用，且它们的抗病毒活性没有显著的差异。
通过摇瓶培养初步摸索了诱导时间、甲醇浓度、培养基pH值等培养条件对Mut~+重组菌株表达PAP的影响。SDS-PAGE分析结果表明，Mut~+组菌株在甲醇诱导第四天后PAP在培养液中积累量达到最高水平，延长培养时间会导致产量下降；在10g/L的甲醇浓度诱导下，PAP的表达量达到最高；培养基pH值在偏酸性条件下(6.0-6.4)PAP的表达量都维持在较高的水平。
Plant virus disease is one primary crop disease. It distributes wildly and does great harm to economy of agriculture yield. People search after ways to prevention and cure virus diseases for long time but not develop effective reagent yet. Many studies revealed that pokeweed antiviral protein (PAP) can control plant virus effectively. But PAP in nature is limited which could not apply cosmically. Using bioreacter to produce PAP is one efficacious way to get a mass of PAP. Someone tried this way but the yield is low. Francis expressed truncated mutant pokeweed antiviral protein gene in Pichia pastoris. The yield reached 10-12mg/L. Dr. Chen ding hu in plant virus group of Institute of plant protection of the CAAS (Chinese academy of agriculture sciences) selected PAP Muts recombinants and wanted to produce PAP through fermentation to prevention and cure plant virus disease. But as to Muts recombinants, the periods of fermentation is too long and the efficiency of using methanol is low. So using Muts recombinants to produce PAP in factory is not felicitousness.
Linearized the expression vector pPIC9K-P including the truncated mutant pokeweed antiviral protein (PAP) gene by restriction endonuclease Sal I and transformed it electrically into Pichia pastoris GS115.Mut+recombinants were selected by PCR and high yield Mut+ recombinant was picked out by double film immunoblotting.
To find out the difference between Mut+ and Muts recombinants we compared their expression of pokeweed antiviral protein in the same conditions. SDS-PAGE results showed that there was a clear target protein band in Mut+ recombinant supernatant after 48 hours of culturing, while a faint band only in Muts recombinant after 72 hours. Western-blotting result showed that there was no remarkable difference of yield between Mut+ and Muts recombinants after 6 days induced. Anti-virus activity tests revealed that culture supernatants of Mut+ and Muts recombinants could inhibit TMV infection with high efficiency in the same concentration and there was no significant difference between them.
Different cultivation condition such as induced time, methanol concentration and pH of culture medium was studied at shake flask cultivation. SDS-PAGE results showed that as to Mut+ recombinant highest yield was obtained after 4 days inducing and with the culture
time prolonged it reduced. Pokeweed antiviral protein gene expressed well when methanol concentration reached 10g/L. Pokeweed antiviral protein obtained high yield in thin acidic culture medium (pH6.0-6.4) and its quantity in total mass of secrete protein exceeded 30%.
引文
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