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为大规模快速克隆罗非鱼细胞免疫功能基因,采用SMART技术,构建了尼罗罗非鱼链球菌疫苗免疫后外周血白细胞全长cDNA文库。提取免疫后第3、5和7天外周血白细胞总RNA,用PowerscripTM反转录酶逆转录合成第一链cDNA,LD-PCR扩增获得双链cDNA,经SfiI酶切和CHROMASPIN-400TM柱分级分离,收集500 bp以上的片段重组于改造的pBluescript II SK载体并转化大肠杆菌DH5α,测定文库滴度、重组率及库容量。结果表明,构建的cDNA文库原始库容量1.021×106克隆,文库滴度1.078×106pfu/mL,重组率94.71%,插入片段大小0.75~3.0 kb,平均长度约为1.5 kb。随机对24个克隆测序所获得的18条Contigs进行BLASTx分析,发现有15条Contigs有相关同源信息,其中10条为全长cDNA,全长率为66.7%。说明所构建文库的各项指标均达到要求,可为筛选罗非鱼免疫功能相关基因和进一步研究基因的结构和功能奠定基础。
To rapidly clone the cellular immune function genes of tilapia, a full-length cDNA library of peripheral blood leukocytes after immunization with Nile tilapia non-streptococcal vaccine was constructed by using SMART technology. Total RNA was extracted from peripheral blood leukocytes on the 3rd, 5th and 7th day after immunization. The first strand cDNA was reverse transcribed by Powerscrip ™ reverse transcriptase. The double-stranded cDNA was amplified by LD-PCR and was digested by SfiI and CHROMASPIN-400TM The fragments above 500 bp were isolated and cloned into the modified pBluescript II SK vector and transformed into E. coli DH5α. The library titer, recombination rate and library volume were determined. The results showed that the constructed cDNA library had a capacity of 1.021 × 106 clones, a library titer of 1.078 × 106pfu / mL and a recombination rate of 94.71%. The inserted fragment size was 0.75-3.0 kb with an average length of 1.5 kb. A total of 18 Contigs obtained from 24 clones were randomly analyzed by BLASTX. Fifteen Contigs shared homologous information, of which 10 were full-length cDNA with a full-length of 66.7%. The results showed that all the indexes of the constructed library met the requirements and laid the foundation for screening the genes related to immune function of tilapia and further studying the structure and function of the gene.