【作者】 张莉；

【导师】 李庆章；

【作者基本信息】 东北农业大学 ， 动物生物化学与分子生物学， 2008， 博士

【摘要】 牛奶及其乳清中含有大量的乳糖,世界上平均70%～90%的成年人(尤其是亚洲和非洲人)喝牛奶后会产生乳糖不耐受,这在很大程度上限制了人们对乳及乳制品的摄入。β-半乳糖苷酶可将乳及乳清中的乳糖水解,降低乳糖含量,提高乳及乳制品的营养价值和利用率,解决乳糖不耐受患者的乳品消费问题,同时还可去除乳清浓缩时乳糖结晶析出给乳制品加工带来的不便。另外,牛乳和人乳在组成上是有差别的。人乳蛋白主要是乳清蛋白,约占总蛋白的70%,其他的为酪蛋白。而牛乳蛋白主要是酪蛋白,约占总蛋白的78.8%,乳清蛋白含量低,约占总蛋白的21.2%。所以,改良牛乳的蛋白组成和含量,使之更接近于人乳,会提高牛乳的营养价值。用转基因技术改变乳成分来提高乳的营养价值和加工特性,包括提高乳中高价值成分的浓度、消除乳中不理想成分、生产人乳中一些成分以及利用乳腺生物反应器来生产药物蛋白,将为整个乳业生产带来一场变革。α-乳白蛋白是一种主要的乳清蛋白,具有调节产乳的功能,是必需氨基酸和支链氨基酸的极好来源,也是唯一能与金属包括钙结合的乳清蛋白成分。另外,它还有抑菌、抑制细胞凋亡、增强大脑反应能力等多种功能。本研究旨在通过基因重组技术构建真核表达载体,使人α-乳白蛋白基因和β-半乳糖苷酶基因在奶牛乳腺上皮细胞中高效表达,一方面降低乳糖含量,另一方面产生活性人α-乳白蛋白。目的在于进一步使人α-乳白蛋白基因和β-半乳糖苷酶基因在奶牛乳腺中高水平表达,为今后制备转基因动物生产转基因牛奶、改良牛奶品质提供可行的技术手段和可信的理论支持。本研究PCR扩增了牛αS1-酪蛋白5′调控序列约1.2kb的片段,合成了0.76kb的人α-乳白蛋白的基因(α-LA)cDNA序列。应用基因重组技术,以含SV40启动子调控下β-半乳糖苷酶基因(LacZ)的PSV为载体,连接牛αS1-酪蛋白5′调控序列约1.2kb的片段为启动子,在其后连接0.76kb的人α-乳白蛋白基因cDNA序列,构建了双基因真核表达载体αS1-LA-psv。将人α-乳白蛋白(α-LA)0.76kb的cDNA序列连接到PSV载体SV40启动子后(去LacZ基因),构建了SV40启动子调控下人α-乳白蛋白单基因真核表达载体LA-cDNA-psv;培养了牛乳腺上皮细胞,纯化后,用免疫荧光细胞染色法对纯化的牛乳腺上皮细胞进行了角蛋白18鉴定,从而确定培养纯化的细胞为奶牛乳腺上皮细胞。本研究用阳离子脂质体将构建的双基因真核表达载体αS1-LA-psv转染奶牛乳腺上皮细胞。应用两种方法检测出了β-半乳糖苷酶在奶牛乳腺上皮细胞中的表达。一种方法是细胞原位染色法,表达的β-半乳糖苷酶催化X-Gal分解,在显微镜下观察到培养的细胞中生成了深蓝色产物。另一种方法是用Promega生产的Beta-Glo E2000检测系统,检测转染后不同时期细胞培养液和细胞裂解液中β-半乳糖苷酶的表达情况,结果显示转染24h～120h的细胞培养液和细胞裂解液中均检测到了β-半乳糖苷酶的表达。其中在细胞破碎液中,转染后24h就可以检测到lacZ基因的表达,转染72h表达量最高,之后表达水平有逐渐降低的趋势,144h降到最低,基本检测不到酶活性。在细胞培养液中,转染后24h也可以检测到lacZ基因的表达,但表达量明显低于细胞破碎液,之后表达水平有逐渐升高的趋势,48h达到最高,随后开始降低,144h也基本检测不到酶活性。将转染细胞传代后检测β-半乳糖苷酶的表达情况,结果传到第三代时基本检测不到酶活性。应用高效液相色谱(HLPC)检测了转染后不同时期细胞破碎液中β-半乳糖苷酶的表达所引起的细胞中乳糖含量的变化,结果显示转染24h乳糖含量变化不大,转染48～144h乳糖含量显著减少,且在48h至72h乳糖的减少量最多,与β-半乳糖苷酶的含量检测结果相符。转染后,应用Western Blot方法检测了人α-乳白蛋白的表达,结果显示转染72h的细胞裂解液中检测到了人α-乳白蛋白的表达,表达量约为0.64mg/mL。本研究还将构建的SV40启动子调控下人α-乳白蛋白基因的单基因真核表达载体LA-cDNA-psv用阳离子脂质体转染法转染奶牛乳腺上皮细胞,也应用Western Blot方法检测到了转染72h人α-乳白蛋白的表达,表达量约为0.85mg/mL。实验结果表明培养的牛乳腺上皮细胞具有外源基因表达活性;得到的牛αS1-酪蛋白5′调控序列能作为启动子指导外源基因在牛乳腺上皮细胞高效表达;构建的双基因真核表达载体αS1-LA-psv能在体外培养的牛乳腺上皮细胞中高效表达人α-乳白蛋白和β-半乳糖苷酶;构建的单基因真核表达载体LA-cDNA-psv能在体外培养的牛乳腺上皮细胞中高效表达人α-乳白蛋白。本研究还用阳离子脂质体将构建的双基因真核表达载体αS1-LA-psv通过体外多点注射的方法转染健康泌乳奶牛乳腺,检测转染后乳产量和乳糖含量变化。结果显示转染后24h～120h均检测到了牛乳中乳糖含量的显著降低,乳产量变化不明显,表明构建的双基因真核表达载体αS1-LA-psv能够在牛乳腺组织细胞中表达目的基因,产生低乳糖乳,且外源基因的转染不会影响奶牛的正常泌乳。更多还原

【Abstract】 Milk and whey contained large amounts of lactose.There was 70%-90%adult,especial the Asian and African in the world who had lactose intolerance.Lactose intolerance largely limited the intake of milk and dairy products.β-galactosidase can hydrolyze the lactose of milk and whey.The reduction of lactose can solve the problem of the people who had lactose intolerance and improve the nutritional value of milk and dairy products.At the same time,the reduction of lactose can get rid of the inconvenience of lactose crystallization and separation when whey condensed in the dairy processing.The milk content between cow and human is different.The most proteins in human milk are whey proteins,about 70%of total proteins and the others are caseins.The most proteins in cow milk are caseins,about 78.8%of total proteins,and whey proteins are fewer,about 21.2%of total proteins.So the nutritional value of cow milk can be improved if the composition and content of milk protein can be reformed to be closed to human milk.It can bring a reformation to use the transgenic technology to change the milk composition to improve the nutritional value and fabrication character.Those include content improvement of some high value ingredient, elimination of no-wanted ingredient,producing of some ingredient of human milk and producing protein drugs by mammary gland bioreactor.Alpha lactalbumin is the main protein of whey proteins.It is the good source of essential amino-acid and branched-chain amino acid.It can regulate the producing of milk.And it is the only protein of whey proteins which can bind metals including calcium.It also has the abilities,such as bacteriostasis,apoptosis suppression and cerebrum reaction ability enhancement.This study was designed to construct the eukaryotic expression vector by gene recombination technology and express human alpha lactalbumin gene andβ-galactosidase gene in the bovine mammary epithelial cells to produce active human alpha lactalbumin andβ-galactosidase.Theβ-galactosidase can reduce the lactose content in milk.It aimed at the expression of human alpha lactalbumin gene andβ-galactosidase gene at a high level in the mammary glands of cows further.That would provide a viable technical means and credible theories on production of transgenic animal milk and improvement of the milk quality.In this study,the 5’ flanking regulatory regions of bovineαS1 casein gene(1.2kb) was amplified by PCR and human alpha lactalbumin gene cDNA(0.76kb) was synthetized.The 1.2kb region of 5’ flanking sequence of bovine casein gene and human alpha lactalbumin gene was combined with the PSV vector which has SV40 promotor andβ-galactosidase gene(LacZ gene) to construct the expression vectorαS1-LA-psv.At the same time,human alpha lactalbumin gene cDNA(0.76kb) was combined with the PSV vector behind the SV40 promotor(without LacZ gene) to construct the expression vector LA-cDNA-psv.Bovine mammary epithelial cell was cultured, purified and identified by the tissue-special expression of cytokeratin 18.Afterwards,the eukaryotic expression vectorαS1-LA-psv was transfered into bovine mammary epithelial cells by cationic liposome vector.Then the expression ofβ-galactosidase was detected by two ways in bovine mammary epithelial cells.The first way is by situ staining in the cells,and in which theβ-galactosidase could catalyze the disassociation of X-gal.The dark blue production in the cells was observed by optical microscope,which is the expression ofβ-galactosidase in the cells.Another way is by the Beta-Glo E2000 detection system produced by Promega.The activity ofβ-galactosidase from 24h to 120h in cell lysate and culture medium were detected.The results showed that in the cell lysate,the expression of lacZ gene could be detected at 24h after transfection,and had a gradually upward trend which declined at 72h,reached the minimum at 144h.In the cell culture medium,the expression of lacZ gene can be detected also at 24h after transfection,and had a gradually upward trend which declined at 48h,reached the minimum also at 144h.The cells which were transfected were passaged,the expression ofβ-galactosidase was also detected at the first and second generation,but from the third generation, the expression could not be detected again.The lactose content in the transfected cells were detected by HLPC.The results showed there was no change at 24h,and notable reduction appeared from 24h to 144h,the most reduction appeared from 48h to 72h.The expression of human alpha lactalbumin was detected at 72 hour by Western Blot after transfected The production of human alpha lactalbumin was 0.64mg/mL approximately.The eukaryotic expression vector LA-cDNA-psv was transfered into bovine mammary epithelial cells by cationic liposome vector too.The expression of human alpha lactalbumin was detected at 72 hour by Western Blot after transfected.The production of human alpha lactalbumin was 0.85 mg/mL approximately.All these results showed bovine mammary epithelial cell which was cultured and purified have the ability to express the exogenous genes,the 5’ flanking regulatory regions of bovineαS1 casein gene(1.2kb) which was cloned can direct the expression of exogenous genes highly,and the constructed expression vector can expressβ-galactosidase and alpha lactalbumin in bovine mammary epithelial cells.In this study,the eukaryotic expression vectorαS1-LA-psv was also injected to normal bovine mammary gland.After transfected,the lactose content and milk production were detected from 24h to 120h The notable reduction of lactose content appeared from 24h to 120h,and there was no notable change in milk production.These results showed the constructed expression vector also can express the exogenous genes in bovine mammary gland and produce the diabetic milk without effect on the normal lactation.更多还原