利用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠的研究

目的 应用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠。方法 针对miRNA-29b1基因设计一段sgRNA,sgRNA和Cas9体外转录后显微注射至C57BL/6小鼠受精卵细胞。小鼠出生后取其基因组DNA进行测序以鉴定基因型,同时取小鼠心、肝、脾、肺、肾等脏器研磨后提取总RNA,通过real-time PCR分析miRNA-29b1在这些脏器中的表达。结果 设计了20 bp的miRNA-29b1sgRNA并与Cas9一起进行了体外转录,显微注射小鼠受精卵细胞后获得miRNA-29b1基因突变小鼠。测序结果表明突变小鼠有两种基因型,一种为10 bp的缺失突变;另一种为22 bp的缺失突变,同时伴有3 bp的插入突变。与野生型小鼠相比,基因突变小鼠心、肝、脾、肺、肾等组织中miRNA-29b1表达量下降明显。结论 应用CRISPR/Cas9技术成功构建miRNA-29b1基因敲除小鼠。     

Rapid Detection of Shigella and Salmonella in Rhesus monkeys Using Loop-mediated Isothermal Amplification Assay

To establish a loop-mediated isothermal amplification (LAMP) method for detecting diarrhea pathogens (Shigella and Salmonella) in Rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in non-human primate laboratory animals. Total 205 fecal samples of Rhesus monkeys were detected in LAMP assay, the specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed, and real-time polymerase chain reaction (REAL-TIME PCR) method was employed as control. The LAMP method established here needed only 45 min to complete the reaction at 63癈. Its detection limit was 10pg/μl and it had high specificity. The positive rate of Shigella and Salmonella was 1.5% and 6.3% respectively. Here we established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for the rapid detection of bacterial disease of macaques. The rapid detection kit is underway, which will be helpful to the diarrhea detection.     

真菌免疫调节蛋白在大肠杆菌中的表达及其抗体制备

目的通过在大肠杆菌中表达真菌免疫调节蛋白LZ-8,以制备高效价的抗体。方法将所克隆的LZ-8基因构建成原核表达载体pET-30a-lz8,转化入大肠杆菌BL21感受态细胞中,经IPTG诱导其表达；原核表达产物经金属镍离子螯合柱纯化后,采用常规方法制备家兔多克隆抗体,并用间接ELISA法测定阳性血清的效价。结果大肠杆菌表达产物经SDS-PAGE电泳检测到相对分子量约为14ku的目的蛋白；用考马斯亮兰法测定所表达的外源蛋白表达量为34．46mg/L；纯化后的目的蛋白免疫家兔后,经间接ELISA检测阳性血清的稀释比例达到了10~4,证明该蛋白具有良好的抗原性。结论真菌免疫调节蛋白LZ-8能够在大肠杆菌中获得高水平的表达,且获得了高效价的家兔多克隆抗体,为以后对该蛋白的功能研究打下了基础。     

Ⅰ型牛疱疹病毒TK-/gE- 基因双缺失突变株生物学特性

目的构建Ⅰ型牛疱疹病毒(BHV-1)的TK-/gE-双缺失突变株,检测其生物学功能,为治疗牛传染性鼻气管炎提供参考。方法构建了带有EGFP表达盒的BHV-1 TK-/gE-双缺失突变株。通过southern杂交、蛋白质斑点印迹、空斑试验、MDBK细胞增殖实验等技术方法,对重组基因所构成病毒突变株的生物学特性进行鉴定。结果 Southern杂交及蛋白斑点印迹表明,课题组成功构建了带有EGFP表达盒的双缺失突变株;该突变株具有与野生株相当的繁殖力,但TK-/gE-成功缺失,毒力大幅减弱;BHV-1 TK-/gE-遗传稳定,不会返强。结论本项目组成功构建了Ⅰ型牛疱疹病毒(BHV-1)的TK-/gE-双缺失突变株,该缺失株具有良好的安全性和稳定性,为该突变株进一步作为生物安全疫苗和多价病毒载体提供了依据,为预防和治疗牛传染性鼻气管炎提供了技术支持。