【作者】 李敏；

【导师】 陈永福； 林爱星；

【作者基本信息】 中国农业大学 ， 生理学， 2005， 博士

【摘要】 抗体在临床医学中具有强大的应用价值,其最大的来源是从人血液提取或应用鼠源性单克隆抗体。由于爱滋病、肝炎等流行病的广泛传播,血液提取物的应用越来越不安全;而鼠源性单抗由于易引发免疫反应等原因不能大量使用;基因工程技术改造的人源化抗体,由于亲和力低、产量低、制备费事和纯化费用高等原因难以取得良好的疗效。近年来,利用转基因小鼠表达人类抗体成为生产全人抗体最有效的方法之一。为了取得更大的经济效益,研制能表达全人抗体的转基因克隆牛引起人们的强烈关注,然而与转基因小鼠的制备一样,制备内源性抗体基因被敲除的克隆牛,是最终获得能正常表达人类抗体的转基因克隆牛的基础。本实验综合考虑基因小鼠抗体基因成功敲除的经验和牛抗体重链基因的特点,选择牛抗体重链基因中JH区的4,5,6外显子及Eμ片段作为目的基因,构建了基因敲除打靶载体。 根据已发表的牛抗体重链基因序列设计特异性引物,以Holstein牛肝脏基因组DNA为模板,扩增1.11kb(J),3.63kb(M1)以及1.12kb(M2)片段。其中J片段包含JH区第1,2,3无功能外显子序列,M1片段包括Cμ基因的CH1、CH2、CH3、CH4和TM1外显子,M2片段是Cμ-Cσ内含子序列的一部分。 以J片段作为短同源臂,M1、M2作为长同源臂,分别通过酶切位点KpnⅠ-HindⅢ和EcoRⅠ-EcoT22Ⅰ定向克隆至载体pGTN29-TK,成功构建了能够定点敲除牛JH区有功能外显子及其3’端增强子序列的打靶载体pGTN29-TK-J-M2-M1,其中正筛选标记基因neo放在长臂与短臂之间,负筛选标记tk位于长臂外侧。 为提高打靶效率,转染前首先线性化打靶载体pGTN29-TK-J-M2-M1,通过脂质体和磷酸钙法转染牛胎儿成纤维细胞,用G418和GANC两种药物进行正负筛选,最后获得123个抗药性细胞克隆,挑选单克隆扩大培养后,经微量PCR鉴定法证明获得了6个阳性细胞克隆。 由于抗体可变区基因数目较少且只优先表达其中一个基因家族,牛抗体多样化的产生似乎受到了限制,但它可能还有其他的抗体产生机制。本实验通过PCR、RT-PCR和Southern blot分析,证明序列号为AY158087和AY149283的两个JH和序列号为AY230207和U63637的两个Cμ基因以双拷贝基因形式存在于牛基因组中,它们都是功能基因,但表达水平相差悬殊,NCBIGeneBankTM上登录的cDNA序列中主要表达AY158087JH基因和AY230207 Cμ基因。进一步分析发现,以上两种基因分别以JH(AY158087)—Cμ(AY230207),JH(AY149283)—Cμ(U63637)的组合形式存在,后者比前者缺少1.6kb的Sμ序列,这可能是其表达水平低的原因之一。双拷贝JH,Cμ基因的存在可能是牛抗体多样性产生的新的基因来源。更多还原

【Abstract】 Antibodies were widely used in the clinical medicine. Most sources of them are extracted from the human blood and mouse monoclonal antibodies. But even with the most vigilant of preventive measures, an arrays of the potentially deadly infections agents such AIDS or hepatitis continuous threaten to contaminate the plasma products of serum, and the mouse monoclonal antibodies application was limited as their immune reaction. To reduce these risks, the humanized monoclonal antibodies were carried out using combination of recombinant DNA technique, but they cannot be applied extensive due to their low yield and affinity, difficult to purification and expensive. At the beginning of 1990s, the mice in which the endogenous heavy and kappa light chain loci have been knock out and can generate repertoires of fully human antibodies was obtained. Now the cloned cattle expressed human antibody have been produced with the development of large-animal transfer technology, but the presence of active endogenous bovine Ig loci may interfere with the expression of the human antibodies, hence, knocking out or in some other way inactivating IgH or Igλ. loci was required first. Based on the experience of creation of mice perform a human repertoire and the characteristic of bovine Ig heavy chain, we construct a gene targeting vector to knock out the bovine JH functional exons and E μ gene.In this experiment, genomic DNA was extracted from Holstein bovine liver tissue, PCR primers were designed based on the genes, which have been deposited on NCBI GenBankTM. Three fragments: 1.12kb (J), 3.63kb (Ml), 1.12kb (M2) were amplified from bovine genomic DNA. The J fragment including the first, second and third exons (unfunctional) of JH locus; Ml fragment including the Cμ CH1, CH2, CH3, CH4; TM1 exons; M2 including partial sequence of Cμ-Cσ intron. Comparison these products sequences with the corresponding gene submitted on NCBI GenBank? showed that the homologous of them exceeding 99%. It is demonstrated that they are all conserved genes and adapt to use as homologous arm in the targeting vectors.Using J as short arm, Ml, M2 as long arm, the targeting vector pGTN29-TK-J-M 1-M2 was constructed. The positive selection marker neo was placed in the middle of short and long arm and the negative selection marker tk was just outside the long arm.The linearized targeting vector DNAs were introduced to the bovine fetal fibroblast cells by Lipofectamine or alcium phosphate methods, 123 clones were picked up after cultured in the G418 and Gancyclovir for two weeks. The clones were amplified and identified by PCR and six positive clones, which may take place correct homologous recombination evens, were obtained.The limited germline sequence diversity both at the heavy and light chain loci, especially only one VH family was used preferential imposes constrains on generation of combinatorial diversity in cattle, the cattle thus must employ other strategies during evolution for antibody diversification.Using PCR, RT-PCR and Southern analysis, we found the two JH genes (accession no. AY 158087, AY 149283) and two C μ genes (accession no.AY230207, U63637) all exit as two copies genes in the bovine germline. Although they are functional genes, the expression levels between them are distinctlydifferent. A BLAST search of the NCBI GenBankTM and bovine EST data bases suggested that the JH gene (accession no. AY 158087) and C μ gene (accession no. AY230207) are the only used genes in the reported cDNA sequences. The PCR results also indicated that the JH gene (accession no. AY 158087) is in series with C μ gene (accession no. AY230207) and the JH gene (accession no. AY 149283) is in series with C μ gene (accession no.U63637). Interestingly, there is a shortened lack of 1.5kb S μ sequence occurred in the last combination. This may be the reason that it expressed at low level. Two copies of JH and C μ genes in the bovine germline may contribute to the antibody diversification as well.更多还原