菌寄生真菌纤细齿梗孢蛋白酶基因克隆与表达
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摘要
生物之间的相互识别及信号传导一直以来都是生命科学研究的重要领域。菌寄生真菌与其寄主之间的相互作用是自然界普遍存在的一种生命现象。我们可以通过研究菌寄生真菌与寄主之间的相互作用来揭示生物之间的相互作用的分子机制。纤细齿梗孢(Olpitrichum tenellum)是一种活体营养接触型菌寄生真菌,其寄主包括Fusarium moniliforme, Alternaria alternata等。我们实验室一直在努力以其为基础研究菌寄生真菌与寄主真菌之间的相互作用的分子机制。细胞壁是细胞和细胞之间相互作用的第一道屏障,细胞壁蛋白研究已经成为生物间相互识别机制研究的热点。近年来,菌寄生真菌蛋白酶在菌寄生过程中的作用被人们逐渐重视起来。克隆和表达菌寄生真菌的蛋白酶编码基因变得很有意义。
丝氨酸蛋白酶是一类以丝氨酸为活性中心的重要的蛋白水解酶。在生物体中丝氨酸蛋白酶超家族成员执行多种多样的生理功能,在很多致病过程以及细胞内的信号转导等方面起着重要作用。通过分析丝状真菌丝氨酸蛋白酶氨基酸序列,我们发现丝状真菌丝氨酸蛋白酶催化区的氨基酸序列非常保守。根据丝状真菌丝氨酸蛋白酶的同源保守序列设计兼并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该蛋白酶的编码基因mppro,全长cDNA包含3’、5’非编码区为2024bp,包含一个1554bp的ORF,编码517个氨基酸,该基因在GenBank中注册的登记号为EU368754。
通过生物信息学的方法对获得的蛋白酶的编码基因mppro的核苷酸序列及推导的氨基酸序列进行了序列分析,通过分析发现mppro编码的蛋白是一种分泌型的丝氨酸蛋白酶,它属于与丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin,具有典型的信号肽-前肽-成熟肽的结构,并且和菌寄生真菌较多的木霉属真菌丝氨酸蛋白酶相似性很高,这可能是因为其都为菌寄生真菌有关。
将经EcoRⅠ和NotⅠ双酶切的mppro基因和原核表达载体pET-22b(+)进行连接,构建成原核表达载体pET/mppro,并转化大肠杆菌E. coli BL21。经IPTG诱导培养4~5h,目的蛋白得到大量表达,SDS-PAGE电泳检测,在约31kDa处有一条明显的蛋白带;而空质粒pET-22b(+)转化BL21经诱导培养后无相应蛋白产生。原核表达后的蛋白没有活性,可能由于表达的蛋白以包涵体形式出现。
mppro基因和酵母分泌型表达载体pPIC9K双酶切后体外连接,构建酵母重组表达载体pPIC9K/mppro并测序,保证正确的阅读框。将重组表达质粒pPIC9K/mppro和pPIC9K空质粒分别用限制性内切酶XbaⅠ(位于HIS4区内)线性化后,采用电击法转化Pichia pastoris GS115酵母感受态细胞,于MD/MM平板上筛选His+Mut+表型的酵母转化子。经过PCR检测筛选整合子,进行甲醇诱导培养,甲醇诱导5d酶活性达到最高,达153U/mL。SDS-PAGE电泳检测,在约39kDa处有一条明显的蛋白带,蛋白大小与从该蛋白酶氨基酸序列估计的蛋白分子量相符;对转化子遗传稳定性进行了测定。对表达的蛋白产物进行了纯化并测定表达蛋白酶的酶学性质。
Cell to cell recognition and signal transduction is an important area of life sciences research.The parasite relationship between mycoparasites and their host fungi exists in nature universally,makes it one of the best models to research the mechanism of interaction and signal transduction between two organisms. Olpitrichum tenellum is a biotrophic contact mycoparasite fungus.In last several years,our lab has establised a molecular interaction reaserch platform with it as a foundment.Cell wall is the first screen in cell to cell interacton.In recent years,reaserchers found the cell wall proteins play important roles in interaction of organisms,so the research of fungi cell wall proteins becames a hot spot and more and more attention was paid to the proteases of mycoparasitic fungi.In conclusion,cloning and expression of protease genes of mycoparasite fungi has a great significance.
The serine protease is a group of proteolytic enzymes that are important for their potential applications. The members in the serine protease super family serve a wide range of physiological functions, especially in the pathogenesis of many diseases and intracellular signal transduction. Degenerate primers based on the conserved domain of other reported proteases were designed, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate the full-length cDNA clone. The full length of mppro cDNA gene is 2024bp, which contained an ORF of 1554bp encoding 517amino acids. The cDNA sequence of gene mppro has been registered in Genbank with accession number EU368754.
We have analysed the cDNA sequence and amino acid sequnce of mppro with bioinformation technique.Through analysis,we found that mppro is a secretory protein,belongs to the serine protease, peptidase S8,subtilisin,has typical stucture of this family-a signal peptide,the propeptide,the catalytic domain,the P/middle or homoB domain and the C-terminus.We found that it shares high homology with Trichoderma ,which contains a lot of mycoparasitic fungi and maybe that is because they are all mycoparasitic fungi.
The mppro gene and expression vector pET-22b(+) were digested with EcoRⅠa nd NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/mppro was generated and transformed into E. coli BL21. The recombinant protein was produced in large amount after IPTG induction, approximately 31kDa protein was detected by SDS-PAGE. No interest protein was produced by inducing with IPTG after the pET-22b(+) was transformed into BL21.But the expressed protein has no protease activity,maybe it is because the recombinant protein was presented in a fusion form.
The mppro gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/mppro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/mppro was linearized with a restriction enzyme XbaI (insertion at HIS4),transformed into Pichia pastoris GS115 competent cell by electroporation methods and screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed into GS115 as a control. P. pastoris integrants,selected by PCR analysis, are induced by methanol.After 5 days methanol inducing, the protease activity of P. pastoris integrants increased to 153U/mL.Just as the result of prediction by bioinformation technique,the protein’s MW is 39kDa, determined by SDS-PAGE. The genetic stability of P. pastoris recombinant was tested.The recombinant protein is purified and characterized.
丝氨酸蛋白酶是一类以丝氨酸为活性中心的重要的蛋白水解酶。在生物体中丝氨酸蛋白酶超家族成员执行多种多样的生理功能,在很多致病过程以及细胞内的信号转导等方面起着重要作用。通过分析丝状真菌丝氨酸蛋白酶氨基酸序列,我们发现丝状真菌丝氨酸蛋白酶催化区的氨基酸序列非常保守。根据丝状真菌丝氨酸蛋白酶的同源保守序列设计兼并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该蛋白酶的编码基因mppro,全长cDNA包含3’、5’非编码区为2024bp,包含一个1554bp的ORF,编码517个氨基酸,该基因在GenBank中注册的登记号为EU368754。
通过生物信息学的方法对获得的蛋白酶的编码基因mppro的核苷酸序列及推导的氨基酸序列进行了序列分析,通过分析发现mppro编码的蛋白是一种分泌型的丝氨酸蛋白酶,它属于与丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin,具有典型的信号肽-前肽-成熟肽的结构,并且和菌寄生真菌较多的木霉属真菌丝氨酸蛋白酶相似性很高,这可能是因为其都为菌寄生真菌有关。
将经EcoRⅠ和NotⅠ双酶切的mppro基因和原核表达载体pET-22b(+)进行连接,构建成原核表达载体pET/mppro,并转化大肠杆菌E. coli BL21。经IPTG诱导培养4~5h,目的蛋白得到大量表达,SDS-PAGE电泳检测,在约31kDa处有一条明显的蛋白带;而空质粒pET-22b(+)转化BL21经诱导培养后无相应蛋白产生。原核表达后的蛋白没有活性,可能由于表达的蛋白以包涵体形式出现。
mppro基因和酵母分泌型表达载体pPIC9K双酶切后体外连接,构建酵母重组表达载体pPIC9K/mppro并测序,保证正确的阅读框。将重组表达质粒pPIC9K/mppro和pPIC9K空质粒分别用限制性内切酶XbaⅠ(位于HIS4区内)线性化后,采用电击法转化Pichia pastoris GS115酵母感受态细胞,于MD/MM平板上筛选His+Mut+表型的酵母转化子。经过PCR检测筛选整合子,进行甲醇诱导培养,甲醇诱导5d酶活性达到最高,达153U/mL。SDS-PAGE电泳检测,在约39kDa处有一条明显的蛋白带,蛋白大小与从该蛋白酶氨基酸序列估计的蛋白分子量相符;对转化子遗传稳定性进行了测定。对表达的蛋白产物进行了纯化并测定表达蛋白酶的酶学性质。
Cell to cell recognition and signal transduction is an important area of life sciences research.The parasite relationship between mycoparasites and their host fungi exists in nature universally,makes it one of the best models to research the mechanism of interaction and signal transduction between two organisms. Olpitrichum tenellum is a biotrophic contact mycoparasite fungus.In last several years,our lab has establised a molecular interaction reaserch platform with it as a foundment.Cell wall is the first screen in cell to cell interacton.In recent years,reaserchers found the cell wall proteins play important roles in interaction of organisms,so the research of fungi cell wall proteins becames a hot spot and more and more attention was paid to the proteases of mycoparasitic fungi.In conclusion,cloning and expression of protease genes of mycoparasite fungi has a great significance.
The serine protease is a group of proteolytic enzymes that are important for their potential applications. The members in the serine protease super family serve a wide range of physiological functions, especially in the pathogenesis of many diseases and intracellular signal transduction. Degenerate primers based on the conserved domain of other reported proteases were designed, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate the full-length cDNA clone. The full length of mppro cDNA gene is 2024bp, which contained an ORF of 1554bp encoding 517amino acids. The cDNA sequence of gene mppro has been registered in Genbank with accession number EU368754.
We have analysed the cDNA sequence and amino acid sequnce of mppro with bioinformation technique.Through analysis,we found that mppro is a secretory protein,belongs to the serine protease, peptidase S8,subtilisin,has typical stucture of this family-a signal peptide,the propeptide,the catalytic domain,the P/middle or homoB domain and the C-terminus.We found that it shares high homology with Trichoderma ,which contains a lot of mycoparasitic fungi and maybe that is because they are all mycoparasitic fungi.
The mppro gene and expression vector pET-22b(+) were digested with EcoRⅠa nd NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/mppro was generated and transformed into E. coli BL21. The recombinant protein was produced in large amount after IPTG induction, approximately 31kDa protein was detected by SDS-PAGE. No interest protein was produced by inducing with IPTG after the pET-22b(+) was transformed into BL21.But the expressed protein has no protease activity,maybe it is because the recombinant protein was presented in a fusion form.
The mppro gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/mppro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/mppro was linearized with a restriction enzyme XbaI (insertion at HIS4),transformed into Pichia pastoris GS115 competent cell by electroporation methods and screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed into GS115 as a control. P. pastoris integrants,selected by PCR analysis, are induced by methanol.After 5 days methanol inducing, the protease activity of P. pastoris integrants increased to 153U/mL.Just as the result of prediction by bioinformation technique,the protein’s MW is 39kDa, determined by SDS-PAGE. The genetic stability of P. pastoris recombinant was tested.The recombinant protein is purified and characterized.
引文
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