cDNA cloning of milk fat globule membrane-glycoprotein antigens recognized by the monoclonal antibodies

乳脂球膜(MFGM)起源于哺乳期乳腺上皮细胞的质膜，含有多种膜糖蛋白。这些MFGM糖蛋白的结构和生物学功能尚未得到很好的表征。本研究的目的是分离MFGM糖蛋白的克隆并测定其核苷酸序列，以确定MFGM糖蛋白抗原的蛋白质结构，并推测其未知的功能。本研究以奶牛乳腺组织的mRNA为模板，以单抗为探针，构建了MFGM糖蛋白基因文库。筛选出几个阳性克隆作为模板进行PCR扩增。对部分克隆进行了测序，推导的氨基酸序列表明其中一个克隆编码的基因与从小鼠乳腺中克隆的小鼠基因MFG-E8相似。用RT-PCR方法克隆了该基因的5‘和3’端，并测定了其全长的核苷酸序列。推导的氨基酸序列表明，MFGM糖蛋白抗原MGP53/57的单抗3F12识别的一个克隆与小鼠MFG-E8相似，并含有MGP53/57的一些氨基酸序列。因此，该基因编码MFGM的主要糖蛋白MGP53/57。MGP53/57也具有EGF样结构域和与第VIII因子C区同源的重复结构，但缺少MFG-E8序列中的富含脯氨酸区域。MGP53/57推导的氨基酸序列中有三个潜在的N-糖基化位点。MGP53/57有两个53 kDa和57 kDa的异构体，这可能是由于单一多肽的每个糖基化位点的糖基化作用不同所致。

Milk fat globule membrane (MFGM) is originated from plasma membrane of lactating mammary epithelial cells, and contains a variety of membrane glycoproteins. Structure and biological functions of these MFGM glycoproteins have not yet been well characterized. The objective of this research was to isolate cDNA clones and determine their nucleotide sequences to characterize protein structure and presume unknown functions of the MFGM glycoprotein antigens recognized by the monoclonal antibodies raised against MFGM.A cDNA library was constructed from mRNA of mammary gland of a lactating cow, and screened by using monoclonal antibodies as probes. Several positive clones were selected and used as template for PCR amplification. Some clones were sequenced and the deduced amino acid sequences indicated that one of these clones encoded cDNA similar to the mouse cDNA MFG-E8 cloned from mouse mammary gland. The 5' and 3' ends of the cDNA were cloned by RT-PCR using the cDNA fragment, and the nucleotide sequence for a full lenght cDNA was determined. The deduced amino acid sequence showed that one clone recognized by the monoclonal antibody 3F12, which is specific to MFGM glycoprotein antigen MGP53/57, was similar to mouse MFG-E8, and contained some amino acid sequences determined for MGP53/57. Thus, this cDNA was found to encode the major MFGM glycoprotein MGP53/57. MGP53/57 also has a EGF-like domain and a repeat-sturucture homologous to the C region of Factor VIII,but lacks the proline-rich region which was found in the MFG-E8 sequence.There were three potential N-glycosylation sites in the deduced amino acid sequence of MGP53/57. Two isoforms with 53kDa and 57kDa of MGP53/57 was suggested to be due to differential glycosylation to each glycosylation site fo the single polypeptide.