Development of monoclonal antibodies against bovine mucin core 2 β6 N-acetylglucosaminyltransferase
Development of monoclonal antibodies against bovine mucin core 2 β6 N-acetylglucosaminyltransferase
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DOI：
10.1023/a:1007030223118
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发表时间：
1999-09-01
影响因子：
3
通讯作者：
Cheng, PW
中科院分区：
文献类型：
--
作者：
Li, CM;Joshee, N;Cheng, PW
分子克隆技术已被用来生产大量的重组糖基转移酶的生化研究。我们最近克隆了一个编码牛粘蛋白核心2 β 6 N-乙酰葡糖胺基转移酶（C2 TF）的cDNA。将克隆的cDNA在E. coliXpress系统，并用于产生单克隆抗体（MAbs）。我们在小鼠腹水中获得了7株分泌抗牛C2 TF单克隆抗体的杂交瘤，通过免疫荧光测定（IF）评估滴度范围为1：1280至1：40960。结果表明，7株单抗均为IgG型（4株IgG 1，2株IgG 2b，1株IgG 2a）。这些单克隆抗体的亲和力常数（M（-）1）范围为5.4 x 10（7）至1.2 x 10（9）。这些单克隆抗体在组织切片和蛋白质印迹中识别牛C2 TF。在Western印迹上，这些单克隆抗体中的六个与人核心2-M酶反应，一个与核心2-L和核心2-M酶反应。因此，这些抗体对牛和人的核心2酶的进一步研究是有用的.
Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 beta 6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M(-)1) for these MAbs range from 5.4 x 10(7) to 1.2 x 10(9). These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, These antibodies should be useful for further study of bovine and human core 2 enzymes.