Goto Y, Kondo T, Ide T, Yasueda H, Kuramoto N, Yamamoto K
Forest Tree Breeding Center, Taga, Ibaraki, Japan.
Clin Exp Allergy. 2004 Nov;34(11):1754-61. doi: 10.1111/j.1365-2222.2004.02108.x.
We identified five Cryptomeria japonica trees producing Cry j 1 isoforms that cannot be detected in a sandwich ELISA using two monoclonal antibodies, J1B01 and J1B07, suggesting that the binding affinity of these isoforms for both monoclonal antibodies is low.
The binding properties of the Cry j 1 isoforms produced by five trees to J1B07 and J1B01 were examined. The complementary DNA (cDNA) sequences of the Cry j 1 isoforms were also determined.
To clarify the binding properties of these Cry j 1 isoforms to J1B01 and J1B07, Cry j 1 was quantified in pollen samples collected from each of the five trees, by sandwich ELISAs using polyclonal antibodies and either J1B01 or J1B07. The cDNA sequences of isoforms with different binding properties were determined. To test the assumption that amino acid substitutions affect the binding affinities of Cry j 1 isoforms for monoclonal antibodies, cleaved amplified polymorphic sequences (CAPS) markers representing the putative polymorphisms were used to analyse additional trees.
Four of the five trees produced Cry j 1 isoforms with extremely low binding affinity for J1B07, whereas the other tree produced two different isoforms with low binding affinity for either J1B01 or J1B07. Cry j 1-encoding cDNA sequences for one of the four trees and for the exceptional fifth tree indicate that amino acid substitutions at positions 55 and 352 in mature Cry j 1 affect its binding to J1B01 and J1B07, respectively. This was supported by the results of CAPS analysis.
The existence of Cry j 1 isoforms with low binding affinity for either J1B01 or J1B07 was established. Furthermore, a single amino acid substitution is involved in this difference in binding affinity for each monoclonal antibody.
我们鉴定出五棵日本柳杉，它们产生的Cry j 1亚型在使用两种单克隆抗体J1B01和J1B07的夹心酶联免疫吸附测定（ELISA）中无法检测到，这表明这些亚型与两种单克隆抗体的结合亲和力较低。
检测五棵树产生的Cry j 1亚型与J1B07和J1B01的结合特性。还测定了Cry j 1亚型的互补DNA（cDNA）序列。
为了阐明这些Cry j 1亚型与J1B01和J1B07的结合特性，通过使用多克隆抗体和J1B01或J1B07的夹心ELISA对从五棵树中各自采集的花粉样本中的Cry j 1进行定量。测定了具有不同结合特性的亚型的cDNA序列。为了检验氨基酸取代影响Cry j 1亚型对单克隆抗体结合亲和力的假设，使用代表推定多态性的酶切扩增多态性序列（CAPS）标记分析其他树木。
五棵树中有四棵产生的Cry j 1亚型与J1B07的结合亲和力极低，而另一棵树产生了两种对J1B01或J1B07结合亲和力较低的不同亚型。四棵树之一和特殊的第五棵树的Cry j 1编码cDNA序列表明，成熟Cry j 1中第55位和第352位的氨基酸取代分别影响其与J1B01和J1B07的结合。CAPS分析结果支持了这一点。
确定了存在对J1B01或J1B07结合亲和力较低的Cry j 1亚型。此外，每种单克隆抗体结合亲和力的这种差异涉及单个氨基酸取代。
