Rapacz J, Hasler-Rapacz J, Rapacz J, McConathy W J
Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
J Lipid Res. 1989 Feb;30(2):199-206.
Studies were performed to investigate the separation of Lpb (lipoprotein B) species present in plasma of heterozygous swine bearing the Lpb2 and Lpb3 apoB mutant genes. Low density lipoprotein (LDL) fractions from Lpb2/2 and Lpb3/3 homozygotes were coupled to a matrix and used to isolate affinity-purified antibodies anti-Lpb2 and anti-Lpb3 from swine alloimmune sera, one with specificity for the Lpb2 epitope(s) and the other for Lpb3. These antibodies in turn were used to construct two immunosorbers, anti-Lpb2 and anti-Lpb3 Sepharose columns. To separate the two Lpb haplotype populations present in LDL, a density gradient ultracentrifuge subfraction (d 1.032-1.043 g/ml) obtained from Lpb2/3 heterozygous pigs was applied to the specific immunosorbers. The retained fraction from the anti-Lpb2 column reacted in the double immunodiffusion test with anti-Lpb2 and anti-Lpb13 immune sera but not with either anti-Lpb3 or anti-Lpb12, while the unretained fractions reacted with anti-Lpb3 and anti-Lpb12 but not with either anti-Lpb2 or anti-Lpb13. The reaction patterns obtained with the two sets of alloimmune sera indicate the existence of two separate lipoprotein populations in LDL: one lipoprotein carrying the Lpb2 and Lpb13 epitopes corresponding to the Lpb2 apoB allele, and the other carrying the Lpb3 and Lpb12 allotypes specified by the Lpb2 gene. Immunoblotting with anti-Lpb2 and anti-Lpb3 and silver staining showed that the epitopes of both isolated LDL subpopulations are associated with apoB-100. Neutral lipid analyses showed no differences between the isolated Lpb2 and Lpb3 lipoprotein species from the Lpb2/3 heterozygotes. These studies demonstrate that plasma LDL subfractions from Lpb heterozygous swine can be separated into two haplotype populations, each corresponding to the product of one apoB gene, and reveal a new insight into the phenotypic expression of plasma LDL, and the LDL phenotype-genotype relationship. Furthermore, this approach will facilitate studies on metabolic differences of two structurally distinct LDL, unaffected by in vitro manipulation, exposed to the metabolic milieu of one individual.
开展了多项研究，以探究携带Lpb2和Lpb3载脂蛋白B突变基因的杂合猪血浆中Lpb（脂蛋白B）种类的分离情况。来自Lpb2/2和Lpb3/3纯合子的低密度脂蛋白（LDL）组分与一种基质偶联，并用于从猪同种免疫血清中分离亲和纯化的抗Lpb2和抗Lpb3抗体，一种对Lpb2表位具有特异性，另一种对Lpb3具有特异性。这些抗体进而被用于构建两种免疫吸附剂，即抗Lpb2和抗Lpb3琼脂糖柱。为了分离LDL中存在的两种Lpb单倍型群体，将从Lpb2/3杂合猪获得的密度梯度超速离心亚组分（d 1.032 - 1.043 g/ml）应用于特异性免疫吸附剂。抗Lpb2柱的保留组分在双向免疫扩散试验中与抗Lpb2和抗Lpb13免疫血清发生反应，但不与抗Lpb3或抗Lpb12反应，而未保留组分与抗Lpb3和抗Lpb12反应，但不与抗Lpb2或抗Lpb13反应。用两组同种免疫血清获得的反应模式表明LDL中存在两种不同的脂蛋白群体：一种脂蛋白携带与Lpb2载脂蛋白B等位基因相对应的Lpb2和Lpb13表位，另一种携带由Lpb2基因指定的Lpb3和Lpb12同种异型。用抗Lpb2和抗Lpb3进行免疫印迹及银染显示，两种分离的LDL亚群的表位均与载脂蛋白B - 100相关。中性脂质分析表明，从Lpb2/3杂合子中分离出的Lpb2和Lpb3脂蛋白种类之间没有差异。这些研究表明，来自Lpb杂合猪的血浆LDL亚组分可被分离成两个单倍型群体，每个群体对应一个载脂蛋白B基因的产物，并揭示了对血浆LDL表型表达以及LDL表型 - 基因型关系的新见解。此外，这种方法将有助于研究两种结构不同的LDL的代谢差异，不受体外操作影响，且暴露于同一个体的代谢环境中。
