摘要
目的培育一种HBeAg转基因小鼠新品系。方法克隆乙型肝炎病毒HBeAg基因;采用CRISPR/Cas9技术,通过同源重组的方式分别在Rosa26基因位点定点插入pliver-HBeAg表达框,获得含有HBeAg基因的表达载体pliver-HBeAg,经酶切得到含有HBeAg基因的线性DNA片段,将Cas9mRNA、gRNA和donor vector显微注射到C57BL/6J小鼠的受精卵中,再将其移植入C57BL/6J雌性代孕小鼠子宫,获得F0代建系小鼠;采用长片段PCR对出生小鼠进行鉴定,共获得HBeAg基因正确同源重组的F0代建系小鼠;F0代阳性小鼠与野生型C57BL/6J小鼠交配,繁育获得F1代小鼠,经PCR鉴定及测序确认阳性F1代小鼠;将携带HBeAg基因的F1代转基因小鼠再次回交,对子代小鼠进行PCR基因型鉴定,直至获得纯合子子代转基因小鼠;采用全自动化学发光免疫分析仪、胶体金法和免疫组织化学方法分别检测HBeAg转基因鼠血浆和肝组织中HBeAg和HBeAb的表达。结果采用CRISPR/Cas9技术共获得56只F0代小鼠,其中2只为正确同源重组的F0代小鼠;F1代小鼠中6只阳性F1代小鼠。截至目前,共获得22只F2代纯合子和29只杂合子HBeAg转基因小鼠。所有HBeAg转基因小鼠外周血中均可检出高浓度的HBeAg蛋白,但未检出HBeAb的表达。而且免疫组化结果显示HBeAg转基因小鼠肝脏肝细胞中可特异性表达HBeAg蛋白。结论采用CRISPR/Cas9技术成功构建了能在肝脏肝细胞中稳定表达HBeAg蛋白且对HBeAg免疫耐受的新品系HBeAg转基因小鼠,为HBV的研究提供了新的实验动物模型。
Objective To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique.Methods Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene.The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion.Cas9 mRNA,gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice,which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice.The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene.The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice,which were identified by PCR and sequencing.The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained.The genotypes of the offspring mice were identified.The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay,immune colloidal gold technique and immunohistochemistry method.Results A total of 56 F0 mice were obtained,and 2 of them carried homologous recombined HBeAg gene.Six positive F1 mice were obtained,from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained.High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice.Conclusion We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique,which provide a new animal model for studying HBV.
作者
郭睿
田怡
金雪媛
陈海燕
王贵虎
黄小钟
李步荣
李宗芳
杨军
GUO Rui;TIAN Yi;JIN Xueyuan;CHEN Haiyan;WANG Guihu;HUANG Xiaozhong;LI Burong;LI Zongfang;YANG Jun(Department of Pathology,Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China;National & Local Joint Engineering Research Center of Biodiagnostics and Biotherapy,Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China;Clinical Laboratory,Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2019年第9期1017-1022,共6页
Journal of Southern Medical University
基金
国家自然科学基金(NSFC30872403)
教育部新世纪优秀人才计划(NCET-10-0647)
陕西省科技统筹创新工程计划(2011KTCL03-14)~~
