哲罗鱼免疫球蛋白轻链可变区序列及其多样性

Ig VL cDNA sequence and diversity analysis of Hucho taimen (Pallas)

采用RT-PCR技术从哲罗鱼主要免疫器官脾脏总RNA中获得免疫球蛋白(immunoglobulin,Ig)轻链可变区cDNA克隆,随机挑取51个阳性克隆菌落进行测序,得到46条完整的不重复序列,用以确定哲罗鱼IgL的家族种类,为哲罗鱼病毒性疾病的疫苗研制提供理论依据。经过BLUST网络对比,结果显示:其与GenBank报导的虹鳟(序列号为X68517.1和X68519.1)序列的相似性均达90%以上,46条序列的核苷酸相同率分别为最低86.1%(P4与P43)至最高的99.8%(P21与P38)之间不等;经DNAstar5.0软件包MegAling中JotunHein方法对所得序列进行氨基酸同源性比较,得到两个不同的家族;通过可变性参数计算方法可知哲罗鱼IgL的可变性主要集中在CDRs区,可变性最高的区域为CDR3区;对可变区中CDR3序列进行分析后,得出哲罗鱼Ig轻链可变区的重排方式是Jκ基因片段位于Vκ基因片段的3′端的结论,这种重排方式增加了核苷酸不同的连接方式,使哲罗鱼抗体具有更多的可变性空间;对照其亲水性分布图可知:可变区的亲水性氨基酸主要分布于LP前端,FR1末端,完整的FR2,FR3两端,以及CDR1,CDR2和CDR3前端。

To provide data for immunoglobulin （Ig） phyletic evolution,isotypes classification and vaccine used in Hucho taimen （Pallas） cultivation,cDNA sequence,gene diversity and physico-chemical property on Ig VL （variable region of light chain） of Hucho taimen were studied. The cDNA clones of the immunoglobulin light chain variable regions were first time obtained by RT-PCR with a pair of specific primers after total RNA was extracted from the spleen of Hucho taimen （Pallas）. 51 positive clones were isolated and double-strand sequenced over the variable regions. 46 complete sequences were obtained without duplicating to determine variety of Ig VL genes families. After comparing with other vertebrate Ig sequences in BLUST,the results show that the 46 sequences obtained are similar to the sequences of rainbow trout Oncorhynchus mykiss Walbaum,no. X68517.1 and no. X68519.1,reported in GenBank,with the similarity rate from the lowest 86.1% （that of P4 and P43） to the highest 99.8% （that of P21 and P38）. Jotun Hein method from MegAling of DNAstar soft package was used to analyze amino acid homology of Ig VL and the VL genes were classified into two families. After computing method of variability parameters,it was found that CDRs （complementary determining regions） are the main variable regions,and hyper variability was showed in CDR3. By analysis of CDR3 sequences,a conclusion was drawn that character of rearrangement style of Ig VL of Hucho taimen is that Jκ genes are located at 3′side,and Vκ genes are followed. The rearranging way makes nucleotides link styles variable,and thus causes more diversity of the Ig. The hydropathic analysis showed that hydrophilic amino acids of VL are mainly distributed at forepart of LP,terminal of FR1,the whole FR2,amphi-sides of FR3 and foreparts of CDR1,CDR2 and CDR3.