转人胰岛素基因银耳的表达分析
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摘要
在实验室利用农杆菌介导法转化人胰岛素BCA基因成功的基础上，根据潮霉素抗性实验和PCR检测结果选取5个银耳转基因菌株进行人胰岛素BCA基因的表达分析，并对转基因菌株的配对出耳和发酵条件进行研究。实验结果如下：
采用qRT-PCR绝对定量法，以3-磷酸甘油醛脱氢酶基因（GPD）作为内参基因，构建外源目的基因BCA和内参基因GPD的标准质粒并绘制标准曲线。其中，内参基因GPD标准曲线为y=-3.349x+40.25，R~2=0.998，PCR扩增效率为98.89%；外源目的基因BCA标准曲线为y=-3.379x+37.75，R~2=0.995，PCR扩增效率为97.67%。根据标准曲线和各样品Ct值可计算出5个银耳转基因菌株中人胰岛素BCA基因的拷贝数：T20为非转基因菌株；T26、T56和T65的拷贝数为1；T53的拷贝数为2。
再选取银耳转基因菌株T26分别与从不同银耳菌种分离到的单孢菌株进行可亲和配对，得到T26+T_(4(3))和T26+T_(14)这两个成功出耳的组合。经RT-PCR和ELISA检测可知，人胰岛素BCA基因在这两个组合出耳后的子实体中成功表达。
随后，采用qRT-PCR相对定量法，对4个银耳转基因菌株中人胰岛素BCA基因在转录水平的表达量进行分析。根据外源目的基因BCA和内参基因GPD的扩增效率一致可知，实时荧光定量PCR实验结果能使用2-(Ct)法进行相对定量分析。其中，各银耳转基因菌株T53、T26、T56和T65中人胰岛素BCA基因的表达水平存在一定差异。但人胰岛素BCA基因在银耳芽孢中的表达量高于子实体。
经以上实时荧光定量PCR和表达产物的ELISA检测结果可知人胰岛素BCA基因在银耳转基因菌株的芽孢和子实体中均已成功表达。基于银耳作为生物反应器具有既可低成本人工种植又可大规模发酵培养的特性，选取银耳转基因菌株T65为研究材料，以发酵培养液的OD值、菌体湿重及表达产物的ELISA检测结果这三个方面作为筛选指标，对银耳转化子菌株的碳源、氮源和pH这3个发酵培养条件进行初步优化。实验结果表明：碳源应选择甘露醇；氮源应选择酵母粉和牛肉浸膏；pH条件应选择pH6.0和7.0。这些将为进一步研究银耳生物反应器表达人胰岛素BCA基因奠定基础。
Since Agrobacterium tumefaciens-mediated transformation system wassuccessfully for the first time applied to transfer the human insulin BCA gene intoyeast-like conidia of Tremella fuciformis in our lab, five randomly transgenic Tremellafuciformis strains have been selected to carry out research on the fruiting bodies andoptimization of fermentation conditions of transgenic Tremella fuciformis as well asanalysis of expression of the human insulin BCA gene with Real-timefluorescence-based quantitative PCR according to the results of hygromycin Bresistance and PCR assays. The experimental conclusions were as follows:
Before using the absolute method to estimate the human insulin BCA gene copynumbers from five transformants of transgenic Tremella fuciformis, plasmid standardsand standard curves were prepared for the transgene (BCA) and the endogenous GPDgene. The standard curves for the GPD and BCA genes, were y=-3.349x+40.25andy=-3.379x+37.75, respectively. Both curves were highly linear (R~2=0.998andR~2=0.995) in the range. From the slopes, amplification efficiencies of98.89%and97.67%were determined for the GPD and BCA genes, respectively, in theinvestigated range, indicating that our method was applicable to estimation oftransgene copy number. According to the results of standard curves and the Ct values,five transgenic Tremella fuciformis DNA samples were tested, and the resultsindicated that T26, T56and T65had single copy, T53had two copies of the transgene,while T20was a non-transgenic strain.
Moreover, the transformant strain T26was used to do further research on theformation of fruiting bodies cultured with various non-transgenic single-spore strainsfor the production of Tremella fuciformis. The results demonstrated that T26+T_(4(3))andT26+T_(14)could form transgenic fruiting bodies of Tremella fuciformis. RT-PCR andELISA experiments verified that the human insulin BCA gene had been expressed infruiting bodies of transgenic Tremella fuciformis.
Subsequently, the expression of the human insulin BCA gene at the level of transcription was determined by the relative method. As the absolute value of theslope was close to zero, the efficiencies of the GPD and BCA genes were similar, and2-(Ct)method calculation for the relative quantification of the human insulin BCAgene can be used. The results showed that the expression of the BCA gene had variedgreatly among the four transformants of transgenic Tremella fuciformis (T53, T26,T56and T65). However，the expression level of the human insulin gene in yeast-likeconidia was higher than in the fruiting bodies.
Based on the results of Real-time fluorescence-based quantitative PCR andenzyme-linked immunosorbent assay (ELISA), the human insulin BCA gene wassuccessfully expressed in both yeast-like conidia and fruiting bodies of transgenicTremella fuciformis. By using the dimorphism character (yeast-like conidia andmycelium; both are edible, nontoxic, and harmless) of Tremella fuciformis, thetransgene product can be produced by both conidia fermentation and large-scaleartificial cultivation of fruiting bodies. Considering the optical density and wet weightof fermentation culture in shaking flask, as well as the results of protein products ofthe human insulin BCA gene detected by sandwich ELISA as screening markers offermentation conditions for yeast-like conidia of transgenic Tremella fuciformis(taking the transformant strain T65for example), the optimal fermentation conditionswere as follows: cultured with mannitol as carbon sources, beef extract and yeastextract as sources of nitrogen, pH6.0and7.0as the optimum pH for growth. Thesefindings will lay a good foundation for further development of Tremella fuciformisbioreactor to be a new efficient expression system for human insulin.
采用qRT-PCR绝对定量法，以3-磷酸甘油醛脱氢酶基因（GPD）作为内参基因，构建外源目的基因BCA和内参基因GPD的标准质粒并绘制标准曲线。其中，内参基因GPD标准曲线为y=-3.349x+40.25，R~2=0.998，PCR扩增效率为98.89%；外源目的基因BCA标准曲线为y=-3.379x+37.75，R~2=0.995，PCR扩增效率为97.67%。根据标准曲线和各样品Ct值可计算出5个银耳转基因菌株中人胰岛素BCA基因的拷贝数：T20为非转基因菌株；T26、T56和T65的拷贝数为1；T53的拷贝数为2。
再选取银耳转基因菌株T26分别与从不同银耳菌种分离到的单孢菌株进行可亲和配对，得到T26+T_(4(3))和T26+T_(14)这两个成功出耳的组合。经RT-PCR和ELISA检测可知，人胰岛素BCA基因在这两个组合出耳后的子实体中成功表达。
随后，采用qRT-PCR相对定量法，对4个银耳转基因菌株中人胰岛素BCA基因在转录水平的表达量进行分析。根据外源目的基因BCA和内参基因GPD的扩增效率一致可知，实时荧光定量PCR实验结果能使用2-(Ct)法进行相对定量分析。其中，各银耳转基因菌株T53、T26、T56和T65中人胰岛素BCA基因的表达水平存在一定差异。但人胰岛素BCA基因在银耳芽孢中的表达量高于子实体。
经以上实时荧光定量PCR和表达产物的ELISA检测结果可知人胰岛素BCA基因在银耳转基因菌株的芽孢和子实体中均已成功表达。基于银耳作为生物反应器具有既可低成本人工种植又可大规模发酵培养的特性，选取银耳转基因菌株T65为研究材料，以发酵培养液的OD值、菌体湿重及表达产物的ELISA检测结果这三个方面作为筛选指标，对银耳转化子菌株的碳源、氮源和pH这3个发酵培养条件进行初步优化。实验结果表明：碳源应选择甘露醇；氮源应选择酵母粉和牛肉浸膏；pH条件应选择pH6.0和7.0。这些将为进一步研究银耳生物反应器表达人胰岛素BCA基因奠定基础。
Since Agrobacterium tumefaciens-mediated transformation system wassuccessfully for the first time applied to transfer the human insulin BCA gene intoyeast-like conidia of Tremella fuciformis in our lab, five randomly transgenic Tremellafuciformis strains have been selected to carry out research on the fruiting bodies andoptimization of fermentation conditions of transgenic Tremella fuciformis as well asanalysis of expression of the human insulin BCA gene with Real-timefluorescence-based quantitative PCR according to the results of hygromycin Bresistance and PCR assays. The experimental conclusions were as follows:
Before using the absolute method to estimate the human insulin BCA gene copynumbers from five transformants of transgenic Tremella fuciformis, plasmid standardsand standard curves were prepared for the transgene (BCA) and the endogenous GPDgene. The standard curves for the GPD and BCA genes, were y=-3.349x+40.25andy=-3.379x+37.75, respectively. Both curves were highly linear (R~2=0.998andR~2=0.995) in the range. From the slopes, amplification efficiencies of98.89%and97.67%were determined for the GPD and BCA genes, respectively, in theinvestigated range, indicating that our method was applicable to estimation oftransgene copy number. According to the results of standard curves and the Ct values,five transgenic Tremella fuciformis DNA samples were tested, and the resultsindicated that T26, T56and T65had single copy, T53had two copies of the transgene,while T20was a non-transgenic strain.
Moreover, the transformant strain T26was used to do further research on theformation of fruiting bodies cultured with various non-transgenic single-spore strainsfor the production of Tremella fuciformis. The results demonstrated that T26+T_(4(3))andT26+T_(14)could form transgenic fruiting bodies of Tremella fuciformis. RT-PCR andELISA experiments verified that the human insulin BCA gene had been expressed infruiting bodies of transgenic Tremella fuciformis.
Subsequently, the expression of the human insulin BCA gene at the level of transcription was determined by the relative method. As the absolute value of theslope was close to zero, the efficiencies of the GPD and BCA genes were similar, and2-(Ct)method calculation for the relative quantification of the human insulin BCAgene can be used. The results showed that the expression of the BCA gene had variedgreatly among the four transformants of transgenic Tremella fuciformis (T53, T26,T56and T65). However，the expression level of the human insulin gene in yeast-likeconidia was higher than in the fruiting bodies.
Based on the results of Real-time fluorescence-based quantitative PCR andenzyme-linked immunosorbent assay (ELISA), the human insulin BCA gene wassuccessfully expressed in both yeast-like conidia and fruiting bodies of transgenicTremella fuciformis. By using the dimorphism character (yeast-like conidia andmycelium; both are edible, nontoxic, and harmless) of Tremella fuciformis, thetransgene product can be produced by both conidia fermentation and large-scaleartificial cultivation of fruiting bodies. Considering the optical density and wet weightof fermentation culture in shaking flask, as well as the results of protein products ofthe human insulin BCA gene detected by sandwich ELISA as screening markers offermentation conditions for yeast-like conidia of transgenic Tremella fuciformis(taking the transformant strain T65for example), the optimal fermentation conditionswere as follows: cultured with mannitol as carbon sources, beef extract and yeastextract as sources of nitrogen, pH6.0and7.0as the optimum pH for growth. Thesefindings will lay a good foundation for further development of Tremella fuciformisbioreactor to be a new efficient expression system for human insulin.
引文
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