转人血清白蛋白延边奶山羊—绵羊体细胞克隆胚胎生产体系的优化
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摘要
人血清白蛋白(Human serum albumin, HSA)是迄今为止产量最大、用量最大的蛋白质药物。可是现在的生产方式主要是人的血液通过分级过滤获得,产量有限,而且因为血浆来源复杂,容易导致污染。因此,通过转基因技术构建乳腺表达载体,利用体细胞克隆技术得到分泌含人血清白蛋白乳汁的转基因克隆动物,并从动物乳汁中提取人血清白蛋白,是解决人血清白蛋白供应短缺的有效方法。乳腺生物反应器生产人血清白蛋白具有生产成本低、产品产量高、活性高、易于纯化等优点。制约转基因乳腺生物反应制药的主要因素是体细胞核移植技术效率低下。因此,本研究以延边奶山羊和本地绵羊为研究对象,对卵母细胞体外成熟、延边奶山羊-绵羊重构胚胎的构建及体外发育进行了研究,以期提高核移植效率,为以后得到表达人血清白蛋白的转基因延边奶山羊奠定坚实的基础。
本研究共分为三个部分,第一部分进行了不同核成熟抑制剂及抗氧化剂对绵羊卵母细胞体外成熟的影响的研究；第二部分进行了转人血清白蛋白延边奶山羊-绵羊重构胚的构建研究；第三部分是表没食子儿茶素没食子酸酯(Epi-gallocatechin-3-gallate, EGCG)对转人血清白蛋白延边奶山羊-绵羊重构胚体外发育质量的影响。主要研究结果如下：
1.在绵羊卵母细胞体外成熟培养过程中,核成熟抑制剂HX、IBMX最适宜的添加浓度分别为2mM和0.2mM,抗氧化剂最佳添加量,除了半胱氨酸为0.6mM、 EGCG为5μM之外,其余三种β-ME、VC、VE最佳添加量均为100μM；
2.盲吸去核法或化学辅助去核法去核,重构胚的卵裂率及囊胚率没有显著差异,但化学辅助去核法(0.2μg/mL的Deme处理0.5h)去核率为100%,可以高效快速地去核,且不影响重构胚的早期发育；
3.延边奶山羊-绵羊重构胚的适宜电融合参数为2个120V,40μs次的电脉冲,适宜激活方法为Ion预激活5分钟,6-DMAP激活2h；
4.重构胚体外培养时EGCG的适宜添加浓度为15μM,适宜添加时间为IVM和重构胚体外培养1-2天。
Human Serum Albumin (Human serum albumin, HSA) is a protein drugs that needed largely by recently. Now, however, the mode of production is in the person's blood which is obtained by fractional filter, production is limited, and because of the complexity of the plasma source, easily lead to contamination.
Accordingly, the mammary gland expression vector was constructed by transgenic technology, the use of somatic cell cloning techniques to obtain the secretion of HSA-containing milk of transgenic cloned animals, and from animal milk extraction HSA, is an effective method to solve the shortage of supply of HSA. Mammary gland bioreactor production has the advantages of low production cost, high yield, high activity, easy to purify. The main factors restricting transgenic mammary gland bioreactor pharmaceutical somatic cell nuclear transfer is inefficient.
In this study, oocyte maturation rate, enucleation, turn HSA the Yanbian milk goat-sheep reconstructed embryos (hereinafter referred to as the reconstructed embryos) integration, activation and in vitro development of the quality of research in order to improve the efficiency of nuclear transfer, transgenic the Yanbian milk goat after expression of HSA to lay a solid foundation.
Our study is divided into three parts, the first part is about oocyte maturation in vitro studies of different nuclear maturation inhibitors and antioxidants; Second part is about the construction of turn HSA Yanbian dairy goat-sheep reconstructed embryos; And the third is about the effect of epigallocatechin gallate (Epi-gallocatechin-3-gallate, EGCG) on the quality of transfer HSA goat-sheep reconstruction embryos.
The main results are as follows:
1. In sheep oocytes matured in vitro nuclear maturation, the most suitable concentrations of the inhibitor HX, IBMX were2mM and0.2mM and the most suitable concentrations of cysteine is0.6mM; EGCG is5μM and the remaining three amount of the β-ME, VC, VE are100mM;
2. There is no significant differences between the two methods of nuclear transfer in the cleacage rate of reconstructed embryos and the enucleation rate can reach100%when we used Chemical auxiliary enucleation method(0.2μg/mL, Deme processing0.5h), it is quickly and efficiently, and does not affect the early development of the reconstructed embryos;
3. The most suitable fusion parameters of reconstructed embryos is120-130V,40μs pulses of electricity and the most suitable activation method for reconstructed embryos is Ion pre-active for5minutes,6-DMAP activation2h;
4.The most suitable concentration of EGCG for reconstructed embryos culture in vitro is15μM and EGCG can be used in the IVM period and reconstructed embryos were cultured in vitro for1-2days.
本研究共分为三个部分,第一部分进行了不同核成熟抑制剂及抗氧化剂对绵羊卵母细胞体外成熟的影响的研究；第二部分进行了转人血清白蛋白延边奶山羊-绵羊重构胚的构建研究；第三部分是表没食子儿茶素没食子酸酯(Epi-gallocatechin-3-gallate, EGCG)对转人血清白蛋白延边奶山羊-绵羊重构胚体外发育质量的影响。主要研究结果如下：
1.在绵羊卵母细胞体外成熟培养过程中,核成熟抑制剂HX、IBMX最适宜的添加浓度分别为2mM和0.2mM,抗氧化剂最佳添加量,除了半胱氨酸为0.6mM、 EGCG为5μM之外,其余三种β-ME、VC、VE最佳添加量均为100μM；
2.盲吸去核法或化学辅助去核法去核,重构胚的卵裂率及囊胚率没有显著差异,但化学辅助去核法(0.2μg/mL的Deme处理0.5h)去核率为100%,可以高效快速地去核,且不影响重构胚的早期发育；
3.延边奶山羊-绵羊重构胚的适宜电融合参数为2个120V,40μs次的电脉冲,适宜激活方法为Ion预激活5分钟,6-DMAP激活2h；
4.重构胚体外培养时EGCG的适宜添加浓度为15μM,适宜添加时间为IVM和重构胚体外培养1-2天。
Human Serum Albumin (Human serum albumin, HSA) is a protein drugs that needed largely by recently. Now, however, the mode of production is in the person's blood which is obtained by fractional filter, production is limited, and because of the complexity of the plasma source, easily lead to contamination.
Accordingly, the mammary gland expression vector was constructed by transgenic technology, the use of somatic cell cloning techniques to obtain the secretion of HSA-containing milk of transgenic cloned animals, and from animal milk extraction HSA, is an effective method to solve the shortage of supply of HSA. Mammary gland bioreactor production has the advantages of low production cost, high yield, high activity, easy to purify. The main factors restricting transgenic mammary gland bioreactor pharmaceutical somatic cell nuclear transfer is inefficient.
In this study, oocyte maturation rate, enucleation, turn HSA the Yanbian milk goat-sheep reconstructed embryos (hereinafter referred to as the reconstructed embryos) integration, activation and in vitro development of the quality of research in order to improve the efficiency of nuclear transfer, transgenic the Yanbian milk goat after expression of HSA to lay a solid foundation.
Our study is divided into three parts, the first part is about oocyte maturation in vitro studies of different nuclear maturation inhibitors and antioxidants; Second part is about the construction of turn HSA Yanbian dairy goat-sheep reconstructed embryos; And the third is about the effect of epigallocatechin gallate (Epi-gallocatechin-3-gallate, EGCG) on the quality of transfer HSA goat-sheep reconstruction embryos.
The main results are as follows:
1. In sheep oocytes matured in vitro nuclear maturation, the most suitable concentrations of the inhibitor HX, IBMX were2mM and0.2mM and the most suitable concentrations of cysteine is0.6mM; EGCG is5μM and the remaining three amount of the β-ME, VC, VE are100mM;
2. There is no significant differences between the two methods of nuclear transfer in the cleacage rate of reconstructed embryos and the enucleation rate can reach100%when we used Chemical auxiliary enucleation method(0.2μg/mL, Deme processing0.5h), it is quickly and efficiently, and does not affect the early development of the reconstructed embryos;
3. The most suitable fusion parameters of reconstructed embryos is120-130V,40μs pulses of electricity and the most suitable activation method for reconstructed embryos is Ion pre-active for5minutes,6-DMAP activation2h;
4.The most suitable concentration of EGCG for reconstructed embryos culture in vitro is15μM and EGCG can be used in the IVM period and reconstructed embryos were cultured in vitro for1-2days.
引文
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