五指山小型猪外周血白细胞cDNA文库的构建与鉴定

Construction and Identification of cDNA Library from the Peripheral Blood Leucocytes in Wuzhishan Miniature Pigs

目的运用SMART技术,构建了五指山小型猪外周血白细胞cDNA文库。方法用RNAiso reagent(TaKaRa)处理新鲜血液,以总RNA抽提试剂盒(上海华舜)制备总RNA。用Powerscript TM反转录酶逆转录合成第一链cDNA,LD-PCR扩增获得cDNA双链,Sfi I酶切和CHROMASPIN-400TM柱分离后,500 bp以上的片段与pDNR-LIB载体连接,电转化入E.coli.DH5α,建成原始文库。然后,扩增文库并随机挑取单菌落,PCR鉴定重组子插入片段大小。结果经鉴定,库容量达到1.5×106克隆,原始文库滴度为9.7×109cfu/mL,扩增后的文库滴度为6.4×1010cfu/mL。重组率接近100%,插入片段大小为0.5～2 kb,平均长度约为1.1 kb。结论构建的五指山小型猪外周血白细胞cDNA文库符合建库要求,可以用于全长cDNA的筛选。为五指山小型猪新基因的获得及结构和功能研究提供了可靠材料。

Objective A cDNA library from the peripheral blood leucocytes in Wuzhishan miniature pigs was constructed with SMART （switching mechanism at 5＇ end of RNA transcript） technique. Methods Total RNA was extracted from the peripheral blood leucocytes and reversely transcripted into full-length cDNA using PowerseriptTM reverse transcriptase, and the double strand cDNA was synthesized and amplified by long-distance PCR （LD-PCR）. The PCR products were digested by proteinase K. After digestion with SfiI and size fractionation using CHROMA SPIN-400TM columns, cDNAs （ 〉 500 bp） were ligated to the Sill digested, dephosphorylated pDNR-LIB vector. The ligation mixture was electroporated into E. coli. DH5a by electroporation. Then, the library was amplified, and clones were picked randomly from the library for screening size of cDNA inserts by PCR. Results The constructed primary cDNA library contained 1.5 × 10^6 independent clones. The titer of the cDNA library was estimated as 9.7 × 10^9 cfu/mL with the percentage of recombinant clones almost 100%, and that of the amplified library was 6.4 × 10^10 cfu/mL. PCR results showed that the inserts varied from 0.5 to 2.0 kb with an average size larger than 1100 bp, which indicated that the library built a strong basis for screening the full-length cDNA. Conclusion These data show that this library meets the requirement of a standard cDNA library. The Wuzhishan miniature pig cDNA library from the peripheral blood leucocytes constructed in this study may lay a basis for subsequent work of clone and selection of some kinds of new functional genes.