恒河猴(Macaca mulatta)肝脏cDNA文库的构建

Construction of a cDNA Library from Liver Tissue of Rhesus Monkey, Macaca mulatta

目的构建正常恒河猴的肝脏组织cDNA表达文库,以筛选恒河猴的相关基因并提供其作为医学研究动物模型的遗传学依据。方法提取正常恒河猴肝脏组织总RNA,纯化的mRNA反转录合成cDNA,连接EcoR接头,经Xho酶切并用CHROMA SPIN-400分离cDNA,将大于500bp的cDNA片段连接于λZAP表达载体,体外包装后转染至XL1-BlueMRF’宿主菌中,再扩增文库。对cDNA文库的滴度、重组率和插入片段的大小进行检测。结果初始文库的库容量为1.2×106pfu,初始文库和扩增文库的滴度分别为1.1×106pfu/mL、7.7×109pfu/mL,重组率分别为99.3%、98.2%,插入片段平均长度分别为2.0kb、2.3kb。结论成功构建了恒河猴肝脏组织的cDNA表达文库,为同种和异种移植以及移植相关药物临床前研究奠定了良好的基础。

Objective To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a eDNA expression library from liver tissue of a healthy rhesus monkey. Methods With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase （MMLV-RT）, the firststrand eDNA was synthesized from purified mRNA, and subsequently the second-strand eDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand eDNA, which was subsequently digested by Xho I restriction enzyme and fraetionated with CHROMA APIN-400 column. The fraetionated eDNA fragments to be longer than 0. 5 kb were ligated into λ ZAP express vector to form the phagemid eDNA reeombinants, which were further packaged into the λ ZAP cDNA library according to the standard protocol with phage λ Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF＇. Then, the library titre, recombinant rate and length of inserted eDNA were measured, respectively. Results The capacity of the primary or unamplified library was 1.2 × 10^6 pfu. The titers of the unamplified library or the amplified library was 1.1 × 10^6 pfu/mL or 7. 7 × 10^9 pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%,and the average lengths of the inserts were 2.0 kb and 2.3 kb, respectively. Conclusion An excellent eDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.