猪γ-干扰素基因在毕赤酵母中的表达与活性检测
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摘要
猪干扰素-γ(PoIFN-γ)是由激活的T细胞和NK细胞产生的一类细胞因子，它具有高效的抗病毒感染、抑制肿瘤细胞生长和调节机体免疫功能的作用，作为生物药剂和疫苗佐剂具有广阔的应用前景。
为了更好地利用基因工程技术来开发重组猪干扰素-γ制剂，本研究将去除信号肽的猪干扰素-γ基因置于毕赤酵母a因子分泌信号的DNA序列后，构建成能分泌表达PoIFN-γ蛋白的重组表达载体pPIC9K-PoIFN-γ，然后将其导入高效表达系统—毕赤酵母中。培养毕赤酵母，表达PoIFN-γ蛋白，测定其效价，为PoIFN-γ进一步的研究与开发打下基础。本研究的主要结果如下：
1、用Oligo.exe(3.3)软件设计一对引物，以携带猪IFN-γ全基因的重组质粒PMD-18T-PoIFN-γ为模板，通过PCR方法，扩增出长约441bp左右的特异性条带，胶回收该片段，用EcoRⅠ和NotⅠ分别双酶切克隆质粒PoIFN-γ成熟蛋白基因片段和pPIC9K质粒，回收并用连接试剂盒酶连接目的片段，转化大肠杆菌，质粒抽提、酶切及PCR鉴定。结果表明，表达载体pPIC9K-PoIFN-γ得以成功构建，DNA测序结果表明，PoIFN-γ成熟蛋白基因表达片段定向插入到表达载体启动子下并形成正确的开放阅读框(ORF)。
2、用salⅠ酶切pPIC9K-PoIFN-γ重组质粒，采用原生质体法转化毕赤酵母，通过MD平板和G418抗性筛选，得到重组转化子53个。用特异性引物，在提取质粒之后，对转化菌株进行PCR鉴定，结果显示扩增出了特异条带，表明外源基因整合进了宿主细胞。同时SDS-PAGE鉴定、效价测定等实验结果也表明重组菌株得以成功转化。
3、重组酵母菌株通过MGY培养基培养，对其上清液直接进行SDS-PAGE分析，没有发现17KD左右的蛋白质条带，但对其进行超声裂解之后，得到了预期的17KD目的蛋白带，并对其超声裂解上清液通过细胞病变抑制法测定蛋白活性。结果表明，重组猪干扰素-γ蛋白能够很好地保护WISH细胞免受VSV水泡性口炎病毒的感染。
Porcine interferon-gamma (PoIFN-γ),produced by activated cytotoxic T cells and natural killer cells, plays important roles on anti-infectious, Antitumor Activity and immunomoduLatory effects.PoIFN-γhave capacious applied foreground as biological fungicide and Vaccine Adjuvant.
In order to develop the application of recombinant PoIFN-γ, a recombinant expression vector was constructed in this study.The porcine interferon-gamma gene, in which the sequence encoding signal peptide was cutted then followed that of the a-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. in order to secrete express the PoIFN-γ.The recombinant plasmid pPIC9K-PoIFN-γwas constructed. The objective of this research is to transform the cloned PoIFN-γgene into Pichia pastoris. Then culture the Pichia pastori, expressing the PoIFN-γ, determining the activity. which provides a good foundation for the research and development of PoIFN-γ.Main resuLts of this research are as followed:
1、Expression fragment of mature protein of PoIFN-γabout 441bp was obtained from recombinant plasmid of PMD-18T-PoIFN-γcontaining complete PoIFN-γby PCR amplification with primers designed according to the sequence PoIFN-γ. Desired fragments obtained from double-digestion of plasmid PoIFN-γand pPIC9K with EcoRI and NotI were restored and linked together. thus a recombinant plasmid vector pPIC9K-PoIFN-γwas constructed. Sequencing resuLts of plasmid pPIC9K-PoIFN-γindicated that structural PoIFN-γgene was integrated into the correct reading frame.
2、salⅠ-linized recombinant plasmid pPIC9K-PoIFN-γwas transformed into Pichia pastoris by produce plasmatosome.53 positive transformants were screened on MD plates containing G418. Spcific Pichia clony PCR product showed that foreign PoIFN-γgene was integrated into the host cell. The experimental resuLts from SDS-PAGE and protien of PoIFN-γactivity assay showed recombined pichia was successfuLly transformed into by the PoIFN-γgene.
3、combined pichia cell was cuLtured in the MGY.The experimental resuLts of SDS-PAGE assay of cuLture medium showed protein was not successfuLly secreted expression. But the 17KD target proteins can be found in the uLtrasonic lysate. The specific activity of uLtrasonic lysate was assayed by the standard atuviral activity test on WISH cells challenged with VSV virus.resuLt showed.The recombinant PoIFN-γcan well protect WISH cells against viruLent vesicuLar stomatitis virus (VSV) infection in vitro.
为了更好地利用基因工程技术来开发重组猪干扰素-γ制剂，本研究将去除信号肽的猪干扰素-γ基因置于毕赤酵母a因子分泌信号的DNA序列后，构建成能分泌表达PoIFN-γ蛋白的重组表达载体pPIC9K-PoIFN-γ，然后将其导入高效表达系统—毕赤酵母中。培养毕赤酵母，表达PoIFN-γ蛋白，测定其效价，为PoIFN-γ进一步的研究与开发打下基础。本研究的主要结果如下：
1、用Oligo.exe(3.3)软件设计一对引物，以携带猪IFN-γ全基因的重组质粒PMD-18T-PoIFN-γ为模板，通过PCR方法，扩增出长约441bp左右的特异性条带，胶回收该片段，用EcoRⅠ和NotⅠ分别双酶切克隆质粒PoIFN-γ成熟蛋白基因片段和pPIC9K质粒，回收并用连接试剂盒酶连接目的片段，转化大肠杆菌，质粒抽提、酶切及PCR鉴定。结果表明，表达载体pPIC9K-PoIFN-γ得以成功构建，DNA测序结果表明，PoIFN-γ成熟蛋白基因表达片段定向插入到表达载体启动子下并形成正确的开放阅读框(ORF)。
2、用salⅠ酶切pPIC9K-PoIFN-γ重组质粒，采用原生质体法转化毕赤酵母，通过MD平板和G418抗性筛选，得到重组转化子53个。用特异性引物，在提取质粒之后，对转化菌株进行PCR鉴定，结果显示扩增出了特异条带，表明外源基因整合进了宿主细胞。同时SDS-PAGE鉴定、效价测定等实验结果也表明重组菌株得以成功转化。
3、重组酵母菌株通过MGY培养基培养，对其上清液直接进行SDS-PAGE分析，没有发现17KD左右的蛋白质条带，但对其进行超声裂解之后，得到了预期的17KD目的蛋白带，并对其超声裂解上清液通过细胞病变抑制法测定蛋白活性。结果表明，重组猪干扰素-γ蛋白能够很好地保护WISH细胞免受VSV水泡性口炎病毒的感染。
Porcine interferon-gamma (PoIFN-γ),produced by activated cytotoxic T cells and natural killer cells, plays important roles on anti-infectious, Antitumor Activity and immunomoduLatory effects.PoIFN-γhave capacious applied foreground as biological fungicide and Vaccine Adjuvant.
In order to develop the application of recombinant PoIFN-γ, a recombinant expression vector was constructed in this study.The porcine interferon-gamma gene, in which the sequence encoding signal peptide was cutted then followed that of the a-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. in order to secrete express the PoIFN-γ.The recombinant plasmid pPIC9K-PoIFN-γwas constructed. The objective of this research is to transform the cloned PoIFN-γgene into Pichia pastoris. Then culture the Pichia pastori, expressing the PoIFN-γ, determining the activity. which provides a good foundation for the research and development of PoIFN-γ.Main resuLts of this research are as followed:
1、Expression fragment of mature protein of PoIFN-γabout 441bp was obtained from recombinant plasmid of PMD-18T-PoIFN-γcontaining complete PoIFN-γby PCR amplification with primers designed according to the sequence PoIFN-γ. Desired fragments obtained from double-digestion of plasmid PoIFN-γand pPIC9K with EcoRI and NotI were restored and linked together. thus a recombinant plasmid vector pPIC9K-PoIFN-γwas constructed. Sequencing resuLts of plasmid pPIC9K-PoIFN-γindicated that structural PoIFN-γgene was integrated into the correct reading frame.
2、salⅠ-linized recombinant plasmid pPIC9K-PoIFN-γwas transformed into Pichia pastoris by produce plasmatosome.53 positive transformants were screened on MD plates containing G418. Spcific Pichia clony PCR product showed that foreign PoIFN-γgene was integrated into the host cell. The experimental resuLts from SDS-PAGE and protien of PoIFN-γactivity assay showed recombined pichia was successfuLly transformed into by the PoIFN-γgene.
3、combined pichia cell was cuLtured in the MGY.The experimental resuLts of SDS-PAGE assay of cuLture medium showed protein was not successfuLly secreted expression. But the 17KD target proteins can be found in the uLtrasonic lysate. The specific activity of uLtrasonic lysate was assayed by the standard atuviral activity test on WISH cells challenged with VSV virus.resuLt showed.The recombinant PoIFN-γcan well protect WISH cells against viruLent vesicuLar stomatitis virus (VSV) infection in vitro.
引文
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