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为筛选罗非鱼链球菌疫苗免疫前后白细胞表达变化基因及探讨鱼类白细胞免疫机理,利用抑制性差减杂交(SSH)技术,以链球菌疫苗免疫前后罗非鱼白细胞cDNA为材料,构建了罗非鱼免疫前后正、负2个cDNA差减文库,并采用地高辛(DIG)标记差减文库cDNA作为探针,进行差异表达片段的筛选鉴定。结果显示:2个文库重组率均在90%以上,插入片段在300～900 bp,接头连接效率超过50%,差减效率非常高效,阳性率均超过70%。杂交筛选后正、负文库各挑选30个阳性克隆进行测序组装聚类,正、负文库各获得16和18条非冗余EST(unigene),所得序列经GenBank同源性分析,正、负文库各有10和11条unigene获得功能注释,且各有6和7条unigene没有同源性匹配,为未知新基因。
In order to screen the gene expression of leukocyte before and after immunization of Streptococcus tilapia, and to explore the mechanism of leucocyte immunity in fish, we use suppression subtractive hybridization (SSH) Positive and negative fish immunization two cDNA subtractive library, and the use of digoxin (DIG) subtracted cDNA library as a probe, the differential expression fragment screening identification. The results showed that the recombination rates of the two libraries were more than 90%, the insert size ranged from 300 to 900 bp, the efficiency of adapter ligation was over 50%, and the efficiency of subtractive efficiency was very high. The positive rates were over 70%. After hybridization screening, positive and negative libraries were selected 30 positive clones for sequencing assembly clustering, positive and negative libraries were obtained 16 and 18 non-redundant EST (unigene), the obtained sequence by GenBank homology analysis, positive and negative libraries Each of 10 and 11 unigene was functionally annotated, and 6 and 7 unigene each had no homology match for unknown new genes.