副溶血弧菌感染12h和24h杂色鲍肝脏全长cDNA文库的构建

Construction of liver full-length cDNA libraries of abalone Haliotis diversicolor infected with Vibrio parahaemolyticus for 12 h and 24 h

采用RDP试剂提取杂色鲍(Haliotisdiversicolor)不同免疫时相肝脏的总RNA,经Oligotex试剂盒纯化得到mR NA。根据SMART(switchingmechanismat5′endofRNAtranscript)原理,利用含sfiIB酶切位点的Oligo(dT)引物合成cDNA第1条链,利用含sfiIA酶切位点的SMARTⅢ寡核苷酸作为cDNA第1条链在mRNA5′端延伸出去的模板,采用LD-PCR引物合成双链cDNA。双链cDNA用sfiI酶切和过柱分级分离后,与λTripEx2两臂进行连接,随后进行λ噬菌体体外包装反应,构建成杂色鲍不同免疫时相(12h和24h)全长cDNA文库。构建好的文库分别含有约5.04×106和2.95×106个重组子。插入cDNA长度分布在500bp～3kb,说明文库具有良好的质量,为进一步筛选、克隆杂色鲍免疫调控基因奠定了基础。

To construct different immunological phase full-length cDNA libraries of Haliotis diversicolor, RDP reagent was used to isolate total RNA from liver of Haliotis diversicolor which had been exposed to V. paralaemolyticus at different time courses. Oligotex kit of QIAGEN was used to separate mRNA from the total RNA. With the SMART technology, SMART III oligo and CDS Oligo(dT) primers containing sfiIA and sfiIB restriction enzyme digested sites respectively were used to synthesize the first-strand cDNA. And the double-strand cDNA which has the sfiIA and sfiIB restriction enzyme digested sites at its two ends was synthesized by LD-PCR. After digested with sfiI, cDNA were fractionated by using CHROMA^(TM) SPIN-400. The full-length cDNAs (longer than 500 bp) were pooled and ligated into sfiI-digested λTripEx2 Vector. Ligation product was packaged into lambda capsids using the MaxPlax^(TM) Lambda Packaging Extracts (EPICENTRE) to generate the Haliotis diversicolor different immunological phase full-length cDNA libraries. The titer of unamplified cDNA libraries were 9.01×10~6 for 12 h library and 4.4 ×10~6 for 24 h library, respectively. And the containing capacities of these two libraries were 5.4×10~6 and 2.6×10~6 cDNA clones, respectively. The length of cDNA inserts ranges from 500 bp to 3 kb,indicating that the quality of these two cDNA libraries is very good. And it will form a fundamental base for further screen and clone of the genes related to immunology.