组织特异性表达1，3-1，4-β-葡聚糖酶转基因鼠及猪的制备
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摘要
1,3-1,4-β-葡聚糖，简称β-葡聚糖是谷类植物饲料中含有的一种抗营养因子，对动物的消化吸收产生不良影响。虽然1,3-1,4-β-葡聚糖酶，简称β-葡聚糖酶能促进β-葡聚糖的分解，但在单胃动物的胃肠道中却不能产生β-葡聚糖酶，无法分解谷类植物饲料中的β-葡聚糖。而利用转基因技术在动物消化道内生产外源酶是提高饲料利用率新的研究策略和有效途径。本研究通过筛选出β-葡聚糖酶基因片段，构建β-葡聚糖酶基因腮腺组织特异性表达载体和β-葡聚糖酶基因肠道组织特异性表达载体，之后成功制备了腮腺和肠道组织特异性表达β-葡聚糖酶的转基因小鼠。通过对转基因小鼠进行基因水平、转录水平及酶活的检测，全面评估在转基因猪中生产β-葡聚糖酶来降低饲料中β-葡聚糖的抗营养作用的可行性。将线性化的β-葡聚糖酶基因腮腺组织特异性表达载体和β-葡聚糖酶基因肠道组织特异性表达载体分别转染猪成纤维细胞，获得稳定的整合有β-葡聚糖酶基因的转基因细胞系，并以这两种细胞为核供体利用核移植克隆技术制备转基因猪，最终获得腮腺组织特异性表达β-葡聚糖酶转基因猪3头，肠道组织特异表达β-葡聚糖酶转基因猪3头。结果如下：
1、成功构建了β-葡聚糖酶基因腮腺组织特异性表达载体（pPSPBGPneo-GLU），并通过原核注射法制备了转基因小鼠。PCR和Southern blotting分析表明β-葡聚糖酶基因成功的整合进小鼠的基因组中。RT-PCR和Northern blotting显示β-葡聚糖酶基因在小鼠的腮腺组织中进行了特异性表达。酶活检测唾液中β-葡聚糖酶活性为0.18±0.02U/mL。在饲料中添加2%β-葡聚糖进行营养代谢试验研究，结果发现转基因鼠和非转基因鼠的平均日增重分别为0.0024g/d和-0.0346g/d。与非转基因鼠相比，转基因鼠的粗蛋白和粗脂肪的消化率分别显著提高了12.15%和5.11%（p<0.05），而粪便湿度二者差异不显著（p>0.05）。表明β-葡聚糖酶基因可在小鼠腮腺中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
2、利用pPSPBGPneo-GLU载体成功转染长白猪成纤维细胞，经G418筛选获得带有腮腺组织特异性表达β-葡聚糖酶转基因细胞系。以该细胞系为核供体，采用核移植技术，获得腮腺组织特异性表达β-葡聚糖酶长白转基因猪3头。PCR和Southern blotting结果显示β-葡聚糖酶基因成功整合到猪基因组中。3头转基因猪唾液中酶活分别为为3.2U/mL、0.07U/mL和0.03U/mL。与非转基因猪相比，转基因猪的能量、粗蛋白和粗脂肪的消化率分别提高了（15.09%、6.20%和9.09%）、（20.73%、5.41%和1.76%）和（39.53%、5.99%和4.19%），而粪便湿度分别降低了16.10%、6.74%和2.08%。表明β-葡聚糖酶基因可在猪腮腺中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
3、采用人的粘蛋白启动子（MUC）成功构建了β-葡聚糖酶基因肠道特异性表达载体（MUC2-GLU-LV），利用原核注射法制备了转基因小鼠模型。PCR和Southern blotting结果表明β-葡聚糖酶基因成功整合到小鼠的基因组中。RT-PCR和Northern blotting结果显示β-葡聚糖酶基因在小鼠的肠道中进行了特异性表达。肠液中β-葡聚糖酶的活性为1.23±0.12U/mL。在饲料中添加2%β-葡聚糖进行营养代谢试验研究，结果发现转基因鼠和非转基因鼠的平均日增重分别为0.058g/d和-0.024g/d。与非转基因鼠相比，转基因鼠粗蛋白和粗脂肪的消化率分别显著提高了9.32%和5.09%（p<0.05），而粪便湿度显著降低了12.16%。结果表明β-葡聚糖酶基因在小鼠的肠道组织中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
4、利用MUC2-GLU-LV载体成功转染长白猪成纤维细胞，经G418筛选获得带有肠道组织特异性表达β-葡聚糖酶转基因细胞系，并以该细胞系为核供体，利用核移植技术共获得肠道组织特异表达β-葡聚糖酶长白转基因猪3头。经PCR检测为阳性，进一步的检测尚在进行。
本研究首次制备了腮腺及肠道转β-葡聚糖酶基因小鼠，证明可以有效的降低葡聚糖的抗营养作用；获得了组织特异性表达β-葡聚糖酶的转基因猪，为提高消化率，建立环境友好型转基因猪打下了基础。
1,3-1,4-β-glucan, referred to as β-glucan, is an anti-nutritional factor in cerealplant material, and has bad effects on animal digestive absorption. Although the1,3-1,4-β-glucanase, referred to as β-glucanase, is able to the decompose β-glucan,β-glucanase dose not exist in monogastric animal gastrointestinal tract, and so β-glucan would not be digested in them. Therefore, the new research strategies whichsolve the problem are to produce heterologous enzymes in the animal digestive tractto improve feed utilization rate by generating transgenic animal. In this study,β-glucanase gene fragment was amplified using PCR, and the parotid gland andintestine tissue-specific expressing β-glucanase vector were constructed. Thetransgenic mice expressing β-glucanase in parotid gland and intestine tissue weresuccessful prepared by microinjection method. The transgenic mice were detected atthe DNA, mRNA levels and enzyme activity in order to comprehensively assess thefeasibility of reducing feed β-glucan anti nutritional effect by producing β-glucanasetransgenic pigs. The linearized β-glucanase gene parotid gland and intestinetissue-specific expressing vector were transfected into the porcine fibroblasts andobtained the transgenic cell lines stably expressing β-glucanase. The two kinds of celllines were used as nuclear donors for nuclear transfer technology, and transgenic pigswere produced using cloning method. Finally,3transgenic cloned pigs expressingspecifically β-glucanase in parotid gland tissue and3transgenic cloned pigsexpressing specifically β-glucanase in the intestinal tissue were obtained, respectively.The results were as follows:
1. The parotid gland tissue-specific expressing β-glucanase vector was successfullyconstructed. The transgenic mice were produced by the pronuclear microinjection.Both PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene and the β-glucanase gene would be stably inherited. RT-PCR andNorthern blotting analysis indicated that β-glucanase gene was specifically expressedin the parotid gland tissue. The β-glucanase activity in the saliva was found to be0.18±0.02U/mL. Mice were fed a diet containing2%β-glucan for nutrition metabolism test. The results showed that the average daily gain of transgenic and non-trangenicmice was0.0024g/d and-0.0346g/d, repectively. The crude protein, crude fatdegestibility of transgenic mice were increased by12.15%and5.11%(p<0.05),respectively, compared with that of the non-transgenic mice. While the moisture infeces showed no significant difference between transgenic and non-transgenicmice(p>0.05). These results suggested that foreign β-glucanase were successfullyexpressed in the animal parotid of mice and could reduce the anti-nutritional effect ofβ-glucans in feed.
2. Landrace porcine fibroblasts were successfully transfected bypPSPBGPneo-GLU vector harboring β-glucanase gene. By G418screening, thistransgenic cell lines were finally obtained and could specifically expressedβ-glucanase gene in parotid gland tissue and this kind of cell line were used as nucleardonors for nuclear transfer technology. Finally,3landrace transgenic cloned pigsexpressing specifically β-glucanase in parotid gland tissue were obtained. PCR andSouthern blotting analysis showed that the pigs carried the β-glucanase gene. Theβ-glucanase activity in the saliva was found to be3.2U/mL、0.07U/mL and0.03U/mL in the pigs. The metabolism test showed the energy, crude protein, crude fatdegestibility of transgenic pigs were increased by (15.09%,6.20%and9.09%),(20.73%,5.41%and1.76%) and (39.53%,5.99%and4.19%)compared with that ofnon-transgenic pigs, respectively. While the moisture in feces were reduced by16.10%,6.74%and2.08%, respectively. These results suggested that foreignβ-glucanase were successfully expressed in the animal parotid of pig and could reducethe anti-nutritional effect of β-glucans in feed.
3. The β-glucanase gene was cloned into a intestine specific expressing vector withMUC2promoter (MUC2-GLU-LV). The transgenic mice were prepared bymicroinjection. PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene. Northern blotting analysis indicated that β-glucanase wasspecifically expressed in the intestine of the transgenic mice. The β-glucanase activityin the intestinal juice was found to be1.23±0.12U/mL. Mice were fed a dietcontaining2%β-glucan for nutrition metabolism test. The results showed that theaverage daily gain of transgenic and non-transgenic mice was0.058g/d and-0.024g/d, respectively. The crude protein, crude fat degestibility of transgenic mice wereincreased by9.32%and5.09%(p<0.05), respectively, compared with that of the non-transgenic mice. While the moisture in feces were reduced by12.16%(p<0.05).These results suggested that foreign β-glucanase were successfully expressed in theintesteine of mice and could reduce the anti-nutritional effect of β-glucans in feed.
4. Landrace porcine fibroblasts were successfully transfected by MUC2-GLU-LVvector harboring β-glucanase gene. By G418screening, this kind of transgenic celllines were finally obtained and could specifically expressed β-glucanase gene inintestine tissue, and this kind of cells were used as nuclear donors for nuclear transfertechnology. Finally,3landrace transgenic cloned pigs expressing specificallyβ-glucanase in the intestine tissue were obtained by PCR analysis. Additionaldetection is under way.
It is the first to produce the transgenic mice expressing β-glucanase gene inparotid gland and intestine tissue in the study. The results demonstrated that theanti-nutritional effects of β-glucan could be effectively reduced. Transgenic pigstissue-specifically expressing β-glucanase were obtained, which provided thefoundation for improving the rate of digestion and the establishment ofenvironment-friendly transgenic pigs.
1、成功构建了β-葡聚糖酶基因腮腺组织特异性表达载体（pPSPBGPneo-GLU），并通过原核注射法制备了转基因小鼠。PCR和Southern blotting分析表明β-葡聚糖酶基因成功的整合进小鼠的基因组中。RT-PCR和Northern blotting显示β-葡聚糖酶基因在小鼠的腮腺组织中进行了特异性表达。酶活检测唾液中β-葡聚糖酶活性为0.18±0.02U/mL。在饲料中添加2%β-葡聚糖进行营养代谢试验研究，结果发现转基因鼠和非转基因鼠的平均日增重分别为0.0024g/d和-0.0346g/d。与非转基因鼠相比，转基因鼠的粗蛋白和粗脂肪的消化率分别显著提高了12.15%和5.11%（p<0.05），而粪便湿度二者差异不显著（p>0.05）。表明β-葡聚糖酶基因可在小鼠腮腺中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
2、利用pPSPBGPneo-GLU载体成功转染长白猪成纤维细胞，经G418筛选获得带有腮腺组织特异性表达β-葡聚糖酶转基因细胞系。以该细胞系为核供体，采用核移植技术，获得腮腺组织特异性表达β-葡聚糖酶长白转基因猪3头。PCR和Southern blotting结果显示β-葡聚糖酶基因成功整合到猪基因组中。3头转基因猪唾液中酶活分别为为3.2U/mL、0.07U/mL和0.03U/mL。与非转基因猪相比，转基因猪的能量、粗蛋白和粗脂肪的消化率分别提高了（15.09%、6.20%和9.09%）、（20.73%、5.41%和1.76%）和（39.53%、5.99%和4.19%），而粪便湿度分别降低了16.10%、6.74%和2.08%。表明β-葡聚糖酶基因可在猪腮腺中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
3、采用人的粘蛋白启动子（MUC）成功构建了β-葡聚糖酶基因肠道特异性表达载体（MUC2-GLU-LV），利用原核注射法制备了转基因小鼠模型。PCR和Southern blotting结果表明β-葡聚糖酶基因成功整合到小鼠的基因组中。RT-PCR和Northern blotting结果显示β-葡聚糖酶基因在小鼠的肠道中进行了特异性表达。肠液中β-葡聚糖酶的活性为1.23±0.12U/mL。在饲料中添加2%β-葡聚糖进行营养代谢试验研究，结果发现转基因鼠和非转基因鼠的平均日增重分别为0.058g/d和-0.024g/d。与非转基因鼠相比，转基因鼠粗蛋白和粗脂肪的消化率分别显著提高了9.32%和5.09%（p<0.05），而粪便湿度显著降低了12.16%。结果表明β-葡聚糖酶基因在小鼠的肠道组织中成功表达，并具有减弱饲料中β-葡聚糖抗营养的作用。
4、利用MUC2-GLU-LV载体成功转染长白猪成纤维细胞，经G418筛选获得带有肠道组织特异性表达β-葡聚糖酶转基因细胞系，并以该细胞系为核供体，利用核移植技术共获得肠道组织特异表达β-葡聚糖酶长白转基因猪3头。经PCR检测为阳性，进一步的检测尚在进行。
本研究首次制备了腮腺及肠道转β-葡聚糖酶基因小鼠，证明可以有效的降低葡聚糖的抗营养作用；获得了组织特异性表达β-葡聚糖酶的转基因猪，为提高消化率，建立环境友好型转基因猪打下了基础。
1,3-1,4-β-glucan, referred to as β-glucan, is an anti-nutritional factor in cerealplant material, and has bad effects on animal digestive absorption. Although the1,3-1,4-β-glucanase, referred to as β-glucanase, is able to the decompose β-glucan,β-glucanase dose not exist in monogastric animal gastrointestinal tract, and so β-glucan would not be digested in them. Therefore, the new research strategies whichsolve the problem are to produce heterologous enzymes in the animal digestive tractto improve feed utilization rate by generating transgenic animal. In this study,β-glucanase gene fragment was amplified using PCR, and the parotid gland andintestine tissue-specific expressing β-glucanase vector were constructed. Thetransgenic mice expressing β-glucanase in parotid gland and intestine tissue weresuccessful prepared by microinjection method. The transgenic mice were detected atthe DNA, mRNA levels and enzyme activity in order to comprehensively assess thefeasibility of reducing feed β-glucan anti nutritional effect by producing β-glucanasetransgenic pigs. The linearized β-glucanase gene parotid gland and intestinetissue-specific expressing vector were transfected into the porcine fibroblasts andobtained the transgenic cell lines stably expressing β-glucanase. The two kinds of celllines were used as nuclear donors for nuclear transfer technology, and transgenic pigswere produced using cloning method. Finally,3transgenic cloned pigs expressingspecifically β-glucanase in parotid gland tissue and3transgenic cloned pigsexpressing specifically β-glucanase in the intestinal tissue were obtained, respectively.The results were as follows:
1. The parotid gland tissue-specific expressing β-glucanase vector was successfullyconstructed. The transgenic mice were produced by the pronuclear microinjection.Both PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene and the β-glucanase gene would be stably inherited. RT-PCR andNorthern blotting analysis indicated that β-glucanase gene was specifically expressedin the parotid gland tissue. The β-glucanase activity in the saliva was found to be0.18±0.02U/mL. Mice were fed a diet containing2%β-glucan for nutrition metabolism test. The results showed that the average daily gain of transgenic and non-trangenicmice was0.0024g/d and-0.0346g/d, repectively. The crude protein, crude fatdegestibility of transgenic mice were increased by12.15%and5.11%(p<0.05),respectively, compared with that of the non-transgenic mice. While the moisture infeces showed no significant difference between transgenic and non-transgenicmice(p>0.05). These results suggested that foreign β-glucanase were successfullyexpressed in the animal parotid of mice and could reduce the anti-nutritional effect ofβ-glucans in feed.
2. Landrace porcine fibroblasts were successfully transfected bypPSPBGPneo-GLU vector harboring β-glucanase gene. By G418screening, thistransgenic cell lines were finally obtained and could specifically expressedβ-glucanase gene in parotid gland tissue and this kind of cell line were used as nucleardonors for nuclear transfer technology. Finally,3landrace transgenic cloned pigsexpressing specifically β-glucanase in parotid gland tissue were obtained. PCR andSouthern blotting analysis showed that the pigs carried the β-glucanase gene. Theβ-glucanase activity in the saliva was found to be3.2U/mL、0.07U/mL and0.03U/mL in the pigs. The metabolism test showed the energy, crude protein, crude fatdegestibility of transgenic pigs were increased by (15.09%,6.20%and9.09%),(20.73%,5.41%and1.76%) and (39.53%,5.99%and4.19%)compared with that ofnon-transgenic pigs, respectively. While the moisture in feces were reduced by16.10%,6.74%and2.08%, respectively. These results suggested that foreignβ-glucanase were successfully expressed in the animal parotid of pig and could reducethe anti-nutritional effect of β-glucans in feed.
3. The β-glucanase gene was cloned into a intestine specific expressing vector withMUC2promoter (MUC2-GLU-LV). The transgenic mice were prepared bymicroinjection. PCR and Southern blotting analysis showed that the mice carried theβ-glucanase gene. Northern blotting analysis indicated that β-glucanase wasspecifically expressed in the intestine of the transgenic mice. The β-glucanase activityin the intestinal juice was found to be1.23±0.12U/mL. Mice were fed a dietcontaining2%β-glucan for nutrition metabolism test. The results showed that theaverage daily gain of transgenic and non-transgenic mice was0.058g/d and-0.024g/d, respectively. The crude protein, crude fat degestibility of transgenic mice wereincreased by9.32%and5.09%(p<0.05), respectively, compared with that of the non-transgenic mice. While the moisture in feces were reduced by12.16%(p<0.05).These results suggested that foreign β-glucanase were successfully expressed in theintesteine of mice and could reduce the anti-nutritional effect of β-glucans in feed.
4. Landrace porcine fibroblasts were successfully transfected by MUC2-GLU-LVvector harboring β-glucanase gene. By G418screening, this kind of transgenic celllines were finally obtained and could specifically expressed β-glucanase gene inintestine tissue, and this kind of cells were used as nuclear donors for nuclear transfertechnology. Finally,3landrace transgenic cloned pigs expressing specificallyβ-glucanase in the intestine tissue were obtained by PCR analysis. Additionaldetection is under way.
It is the first to produce the transgenic mice expressing β-glucanase gene inparotid gland and intestine tissue in the study. The results demonstrated that theanti-nutritional effects of β-glucan could be effectively reduced. Transgenic pigstissue-specifically expressing β-glucanase were obtained, which provided thefoundation for improving the rate of digestion and the establishment ofenvironment-friendly transgenic pigs.
引文
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