CRISPR/Cas9系统敲除山羊TLR4基因载体构建及检测  

Construction and Evaluation of Goat TLR4 Gene Plasmid by CRISPR/Cas9 System

作　　者：惠文巧[1] 汤继顺[1] 章薇[1] 班谦 朱德建[1] 陈胜[1] HUI Wenqiao;TANG Jishun;ZHANG Wei;BAN Qian;ZHU Dejian;CHEN Sheng(Anhui Province Key Laboratory of Livestock and Poultry Product Safety Engineering,Institute of Animal Husbandry and Veterinary Medicine,Anhui Academy of Agriculture Sciences,Hefei 230031,China;Center for Stem Cell and Translational Medicine,School of Life Sciences,Anhui University,Hefei 230601,China)

机构地区：[1]安徽省农业科学院畜牧兽医研究所,畜禽产品安全工程安徽省重点实验室,合肥230031 [2]安徽大学生命科学学院,干细胞及转化医学研究中心,合肥230031

出　　处：《中国草食动物科学》2021年第3期26-30,共5页China Herbivore Science

基　　金：国家自然科学基金项目(31402048);安徽省重点研究和开发计划(1804a07020115,1804e03020322,202004a06020043);安徽省农业科学院创新团队项目(2020YL033)。

摘　　要：TLR4是天然免疫系统中的一种重要模式识别受体,可选择性地识别病原微生物而启动天然免疫,在宿主天然免疫和获得性免疫中具有重要作用。山羊TLR4基因与多种疾病相关,然而,目前在山羊上关于TLR4基因敲除及相关功能的研究尚未见报道。采用CRISPR/cas9系统,首先设计TLR4基因的sgRNA片段,合成sgRNA核苷酸序列,构建同时表达sgRNA和Cas9 D10A的质粒PYSY-sgRNA,连接并转化至大肠杆菌DH5α感受态细胞,最后对转化子进行验证,提取质粒并转染山羊成纤维细胞,通过测序进行敲除检测。结果表明:酶切和测序鉴定证明TLR4基因敲除载体构建正确。说明采用CRISPR/Cas9构建的载体,可为后续研究TLR4在支原体肺炎感染的免疫应答分子机制提供理论依据。It has been demonstrated that TLR4 is an important pattern recognition receptor in the innate immune system,which can selectively recognize pathogenic microorganisms and initiate innate immunity,thereby plays an important role in the host’s innate and acquired immunity.Goat TLR4 gene is associated with a variety of diseases,however,few reports on TLR4 gene knockout and related functions in goats have been published. In the present study,the CRISPR/cas9 system was applied.First,the sgRNA fragment of TLR4 gene was designed,the sgRNA nucleotide sequence was synthesized,and the plasmid PYSY-sgRNA expressing both sgRNA and Cas9 D10A was constructed,connected and transformed into E.coli DH5α competent cells.Finally,the plasmid was isolated,transfected into goat muscle fibroblast cells,and TLR4 was sequenced in different cells.Restriction digestion and sequencing proved that the TLR4 gene knockout vector was constructed correctly.By constructing the vector by CRISPR/Cas9 system,it would provide a theoretical basis for the molecular mechanism of TLR4 in the immune response to mycoplasma pneumonia infection.

关 键 词：CRISPR/Cas9 山羊 TOLL样受体4 基因敲除 

分 类 号：S827.2[农业科学—畜牧学]