Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.
Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.
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DOI：
10.4049/jimmunol.140.10.3654
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发表时间：
1988-05
影响因子：
4.4
通讯作者：
K. Knight;M. Suter;R. Becker
中科院分区：
文献类型：
--
作者：
K. Knight;M. Suter;R. Becker
构建了牛重组噬菌体文库，并用兔S母细胞和人C-γ探针进行了筛选。用兔S木探针对IgM、Ig A和Ig E H链恒定区(CH)基因进行分离，用C Gamma探针法分离到3个Ig G CH基因。将CH基因分别克隆到含有小鼠VDJ基因的表达载体中，该基因是从一株产生抗Dansyl半抗原抗体的杂交瘤细胞中克隆出来的。将构建的重组表达载体导入小鼠杂交瘤细胞，获得分泌嵌合的牛-鼠Ig A、Ig E或Ig G亚类抗Dansyl抗体的细胞株，获得稳定的细胞株。用免疫沉淀法和抗牛H链抗体共沉淀的嵌合抗体经SDSPAGE分析，均含有预期大小的H链和L链。3株来源于C-γ基因的嵌合抗体中，1株与抗IgG1抗体反应，1株与抗IgG2抗体反应，3株既不与抗IgG1抗体反应，也不与抗IgG2抗体反应。这第三个免疫球蛋白似乎代表了牛免疫球蛋白的一个“新”亚类，即免疫球蛋白3。Southern杂交分析表明，牛基因组中含有第四个C-伽马基因。这些实验证明了分子遗传学技术在分离和鉴定免疫球蛋白方面的有效性，这些免疫球蛋白不容易从生物液中纯化。这些技术将有助于从其他近交系哺乳动物中分离和鉴定Ig基因。
A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.