Sun J, Kacskovics I, Brown W R, Butler J E
Department of Microbiology, College of Medicine, University of Iowa, Iowa City 52242.
J Immunol. 1994 Dec 15;153(12):5618-27.
The sequences of 34 swine H chain V-regions expressed with either IgG, IgA, or IgM C regions in adult swine and newborn piglets are described and compared. Sixteen of these V-regions were cloned by using a specific leader primer whereas 18 were cloned by anchored PCR. According to the operational criterion of VH family classification, i.e., > 80% DNA sequence similarity, all sequences belong to a homogeneous VH gene family. A total of 31 additional VH-bearing C mu clones obtained by anchored PCR from colostrum-deprived newborn piglets hybridized equally with a probe for C mu and a pan-VH probe. When the consensus sequence of the expressed swine VH genes were compared with those of VH gene for humans (VHI, II, III), mouse (VHI, II, III), rabbit, and chicken, swine VH genes seem to have common ancestry with the human VHIII family, rabbit VH genes, and the single functional VH gene of the chicken. The leader peptides of all clones obtained by anchored PCR showed < 1% variability, and the deduced amino acid sequences from aa4 to aa25 in all 34 clones are identical. Framework (FR)1 and FR2 are conserved whereas FR3 shows greater variability. A total of 23 of 30 JH sequences were identical, suggesting preferential use of one JH. This frequent, putative JH sequence is not similar to any JH gene in humans. Identical genomic Southern hybridization patterns, each with 13 bands of differing intensity and regardless of stringency, were obtained when either a leader or a pan-specific VH probe was used. Analyses of individual genomic bands by single strand conformational polymorphism and sequence analysis suggest that the number of VHIII-related genes in the swine genome is < 20.
本文描述并比较了成年猪和新生仔猪中34个与IgG、IgA或IgM重链恒定区一起表达的猪重链可变区(H链V区)的序列。其中16个V区通过使用特异性前导引物进行克隆,而另外18个则通过锚定PCR进行克隆。根据VH家族分类的操作标准,即DNA序列相似度>80%,所有序列均属于一个同质的VH基因家族。通过锚定PCR从初乳缺乏的新生仔猪中获得的另外31个携带VH的Cμ克隆,与Cμ探针和泛VH探针的杂交效果相同。当将表达的猪VH基因的共有序列与人类(VHI、II、III)、小鼠(VHI、II、III)、兔和鸡的VH基因序列进行比较时,猪VH基因似乎与人类VHIII家族、兔VH基因以及鸡的单个功能性VH基因具有共同的祖先。通过锚定PCR获得的所有克隆的前导肽显示出<1%的变异性,并且所有34个克隆中从第4位氨基酸到第25位氨基酸的推导氨基酸序列是相同的。构架(FR)1和FR2是保守的,而FR3显示出更大的变异性。30个JH序列中有23个是相同的,表明优先使用一个JH。这个常见的推定JH序列与人类中的任何JH基因都不相似。当使用前导探针或泛特异性VH探针时,获得了相同的基因组Southern杂交模式,每种模式都有13条强度不同的条带,且与严格度无关。通过单链构象多态性和序列分析对单个基因组条带进行分析表明,猪基因组中与VHIII相关的基因数量<20。