史氏鲟免疫球蛋白重链可变区序列及多样性

为了研究史氏鲟免疫球蛋白重链可变区基因的组织结构和多样性,采用RTPCR技术从史氏鲟(Acipenserschrenckii)脾脏总RNA中获得了免疫球蛋白重链可变区cDNA克隆,随机挑取31个阳性克隆进行测序。结果表明:所有序列相同率高于75%,前导肽相同率高于90%,应属于同1个VH家族。其变异主要存在于互补性决定区,特别是CDR3区。在D片段序列中发现大量保守的基因序列(motif)。并发现多个VH基因片段可以共用一个J片段的现象。在基因组DNA重排过程中,VH片段可以与任意的D和J片段结合。此外,史氏鲟免疫球蛋白重链可变区的VH,D和J片段的随机重排外,外切核酸酶作用,以及在重排位点大量N,P片段的插入现象,都大大增加了鲟鱼免疫球蛋白的多样性。

Ig heavy chain of Amur sturgeon Acipenser schrenskii:cDNA sequence and diversity

To investigate the VH gene organization and diversity of the Amur sturgeon IgH gene locus,total RNA was extracted from the spleen,and the cDNA of the immunoglobulin heavy chain variable regions were obtained by RT-PCR with a pair of specific primers.31 clones were isolated and completely sequenced in double-strand sequenced over the variable regions.The erythrocyte DNA was cut with restriction endonuclease for Southern blotting to define the distribution in the sturgeon genome and whether they belong to the same family.Except for the leader,D and JH segments translation sequence,the aa sequence comparison results of the variable domain showed that the identity of all the clones was higher than 75%,subsequently those clones were separated into two clusters.The identity of the leader peptides was over 90%.According to the result of Southern blotting,we suspected that the 31 clones belong to the same IgH variable region family.The complementary determining regions(CDRs),especially the CDR3,have a number of variations.But the range of sturgeon CDR3 was shorter than other vertebrates.This indicates that the sturgeon is lower in the development of immune system.Some conservative motifs were found in the diversity segments.During the rearrangement,more than one VH gene segments could share the same JH segment.In another word,the variable segments can be associated with any of the D or JH segments in the rearrangement.Amur sturgeon,in addition to the random rearrangement of VH,D,and JH segments,has exonuclease activity and an introduction of N and probably P nucleotides at the site of rearrangement all of which could enhance the diversity of Amur sturgeon IgH variable region.