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??南方医科大学学报??2019, Vol. 39Issue (9): 1017-1022??DOI: 10.12122/j.issn.1673-4254.2019.09.03.
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郭睿, 田怡, 金雪媛, 陈海燕, 王贵虎, 黄小钟, 李步荣, 李宗芳, 杨军. 采用CRISPR/Cas9技术构建新品系HBeAg转基因小鼠[J]. 南方医科大学学报, 2019, 39(9): 1017-1022. DOI: 10.12122/j.issn.1673-4254.2019.09.03.
GUO Rui, TIAN Yi, JIN Xueyuan, CHEN Haiyan, WANG Guihu, HUANG Xiaozhong, LI Burong, LI Zongfang, YANG Jun. Generation of a novel HBeAg transgenic mice using CRISPR/Cas9 technique[J]. Journal of Southern Medical University, 2019, 39(9): 1017-1022. DOI: 10.12122/j.issn.1673-4254.2019.09.03.

基金项目

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)

作者简介

郭睿,硕士,主治医师,E-mail:25815166@qq.com

通信作者

杨军,博士,主任医师,博士导师,E-mail:yangjundr@163.com

文章历史

收稿日期:2019-02-08
采用CRISPR/Cas9技术构建新品系HBeAg转基因小鼠
郭睿 1, 田怡 1, 金雪媛 1, 陈海燕 2, 王贵虎 2, 黄小钟 1, 李步荣 3, 李宗芳 2, 杨军 1 ????
1. 西安交通大学第二附属医院 病理科,陕西 西安 710004;
2. 西安交通大学第二附属医院 生物诊断治疗国家地方联合工程研究中心,陕西 西安 710004;
3. 西安交通大学第二附属医院 检验科,陕西 西安 710004
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Cas9 mRNA、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。
关键词: 转基因小鼠????乙肝病毒????HBeAg????CRISPR/Cas9????C57BL/6J小鼠????
Generation of a novel HBeAg transgenic mice using CRISPR/Cas9 technique
GUO Rui 1, TIAN Yi 1, JIN Xueyuan 1, CHEN Haiyan 2, WANG Guihu 2, HUANG Xiaozhong 1, LI Burong 3, LI Zongfang 2, YANG Jun 1 ????
1. Department of Pathology, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China;
2. National & Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China;
3. Clinical Laboratory, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China
Supported by National Natural Science Foundation of China (NSFC30872403)
Abstract: 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.
Keywords: transgenic mice????hepatitis B virus????HBeAg????CRISPR/Cas9????C57BL/6J mice????

乙肝e抗原(HBeAg)是乙型肝炎病毒(HBV)源性免疫抑制因子,在HBV免疫逃逸和不良病程转归中发挥重要作用[1-3]。HBeAg血清转换是判断病情发展趋势和药物疗效的重要指标,也是慢性乙肝治疗的中期目标[4-5]。HBeAg有可能成为慢性乙肝治疗的潜在新靶点。因此,研究HBeAg的作用机制具有重要的临床价值。但由于HBV具有严格的种属限制性和嗜肝细胞特性,缺乏理想的HBV动物模型一直是严重制约HBV基础研究和药物研发的共同瓶颈[6-8]。因此,构建能在肝脏特异性稳定表达HBeAg的转基因动物对于深入研究HBeAg的作用机制、评估以HBeAg为治疗靶点的相关药物/疫苗疗效具有重要意义。已有多种能表达HBV不同组份的HBV转基因(HBV-Tg)小鼠相继问世[9-14],采用HBeAg和HBc/HBeAg转基因小鼠已证实HBeAg比HBcg能诱导更强的T细胞免疫耐受。但现有模型仍旧存在制备技术复杂、繁育困难、遗传稳定性差、目标蛋白表达不稳定等缺点[15-17],并不能满足深入研究的需要[18-20]

为此,本研究拟采用CRISPR/Cas9技术[21-22]构建一种新品系的HBeAg转基因小鼠,为深入研究HBeAg的生物学功HBeAg为靶点的乙肝治疗药物/疫苗等的临床疗效提供适宜的实验动物模型。

1 材料和方法 1.1 实验动物和材料

本实验选用SPF级C57BL/6J小鼠(上海南方模式生物科技股份有限公司)。实验严格遵守国家实验动物福利伦理相关规定,实验中对动物处置符合科技部颁行的《关于善待实验动物的指导性意见》,满足动物保护、动物福利以及伦理原则的相关要求,严格遵守动物使用的3R原则。

sgRNA载体、pliver-HBeAg质粒(上海南方模式生物科技股份有限公司构建并完成)。DNA提取试剂盒(康为世纪生物有限公司),引物合成(上海博锐生物技术公司)。I2000SR化学发光免疫分析仪(美国雅培)。HBV相关抗原检测试剂盒(胶体金法)(厦门波生生物技术有限公司)。

1.2 HBeAg基因小鼠的构建及鉴定 1.2.1 HBeAg基因的表达载体的构建及鉴定

常规方法克隆乙型肝炎病毒HBeAg基因,采用CRISPR/Cas9技术[21-22],通过同源重组的方式将HBeAg基因在Rosa26基因位点定点插入pliver-HBeAg表达框(图 1),获得含有HBeAg基因的重组R26-e(Alb-HBeAg)1打靶载体。采用ScaⅠ酶切技术对该打靶载体中目的基因进行酶切电泳鉴定。

图 1 Rosa26基因位点定点插入相应表达框结构示意图 Fig.1 Map of the ROSA26 genomic region. The line indicates the genomic DNA drawn with the insertion position of pliver-HBeAg integration.
1.2.2 HBeAg转基因小鼠的构建和鉴定

将重组R26-e(Alb-HBeAg)1打靶载体进行酶切,得到含有HBeAg基因的线性DNA片段,并进行PCR和电泳鉴定,并获取想用目的片段。再采用显微注射技术将Cas9 mRNA、gRNA和重组R26-e(Alb-HBeAg)1打靶载体注射到C57BL/6J小鼠的受精卵中,然后将受精卵随后移植入C57BL/6J雌性代孕小鼠子宫腔内。采用长片段PCR技术,分别利用5'和3'同源臂PCR鉴定引物对出生的F0代幼鼠进行基因型鉴定。引物如下:

5'同源臂重组阳性F0代小鼠PCR鉴定引物:

Forward1: 5'-GCCGGGCCTCGTCGTCT-3'

Reverse2: 5'-TTTTTGGGGGTGATGGTGGTC-3'

3'同源臂重组阳性F0代小鼠PCR鉴定引物:

Forward3: 5'-TGCCCCTATCCTATCAACACTTCC-3'

Reverse4: 5'-IVGATCCATTGCCACCTTTCACTTAG-3'

1.3 HBeAg纯合子转基因小鼠的繁育和鉴定

HBeAg转基因小鼠繁殖,将建系的F0代小鼠与野生型C57BL/6J小鼠交配,繁育获得F1代小鼠,并对F1代小鼠5'和3'同源臂进行PCR鉴定(方法同上),并采用sanger基因测序对鉴定阳性的F1代小鼠进行序列分析。

1.4 BeAg纯合子转基因小鼠的繁育和鉴定

再次将携带HBeAg基因的F1代转基因小鼠(基因敲入杂合子)进行交配,获得子代(杂合子和纯合子)HBeAg转基因小鼠。并采用短片段PCR对其基因型(纯合子、杂合子和野生型)进行鉴定,将携带HBeAg基因的F1代转基因小鼠再次回交,逐步繁育纯化,直至获得纯合子子代转基因小鼠。用于小鼠基因型鉴定的PCR引物序列和P1、P2、P3、P4引物位置如下(图 2)。

图 2 小鼠基因型鉴定PCR引物位置示意图 Fig.2 Position of PCR primers for identification of the genotype of the mice.

P1-F:5'-TCAGATTCTTTTATAGGGGACACA-3'

P2-R:5'-TAAAGGCCACTCAATGCTCACTAA-3'

P3-F:5'-CTTCTAGATACCGCCTCAGC-3'

P4-R:5'-AGCCATGTTTTATATTCCTTACC-3'

1.5 HBeAg转基因小鼠中HBeAg和HBeAb的检测

小鼠尾静脉和球后采血,以野生型C57BL/6J小鼠为对照,采用雅培i2000SR全自动化学发光免疫分析仪及配套的HBeAg、HBeAb试剂检测1、2、6、10、11、14号F1代小鼠中HBeAg、HBeAb的表达;采用胶体金法HBV检测试剂盒(厦门市波生生物技术有限公司),检测HBeAg转基因小鼠中HBsAg、HBsAb、HBeAg(双抗体夹心法)和HBeAb、HBcAb(竞争法)的表达;采用Ventana Bench Mark XT全自动免疫组化染色仪检测HBeAg转基因小鼠各脏器组织中HBeAg的表达。具体操作和结果判定参照仪器和试剂说明书。

2 结果 2.1 HBeAg基因的表达载体的构建和酶切鉴定

对重组R26-e(Alb-HBeAg)1打靶载体进行酶切和PCR电泳鉴定,结果显示目的片段与理论条带大小(1445、3206、10 305 bp)一致(图 3~4),其中:gRNA:5'ggggacacactaagggagct-3'。

图 3 重组R26-e(Alb-HBeAg)1打靶载体酶切鉴定电泳图 Fig.3 Agarose gel electrophoresis of enzyme digested recombinant r26-e (alb-hbeag)1 plasmid. Lane1: Recombinant r26-e (alb-hbeag)1 plasmid; M: 1kD marker.
图 4 体外转录gRNA、Cas9电泳结果 Fig.4 Agarose gel electrophoresis of the PCR products for gRNA and Cas9 plasmid in vitro. Left: gRNA (Lanes 1-4: Guide RNA1; Lane 5: 1 kB DNA marker); Right: t7-Cas 9 plasmid (lane 1), lined-t7-Cas 9 plasmid (lane 2), Cas 9 (lane 3), and 1 kB marker (lane 4).
2.2 F0代HBeAg转基因小鼠的鉴定

采用长片段PCR结果显示,2,9号为双臂同源重组阳性的F0代小鼠,已经获得HBeAg基因正确同源重组的阳性F0代建系小鼠(图 5)。

图 5 同源重组F0代小鼠PCR鉴定电泳图 Fig.5 Agarose gel electrophoresis of the PCR products from F0 offspring homologous recombinant HBeAg transgenic mice. Left: 3.4 kb and 6.8 kb fragments amplified from the 5'-arm homologous recombinant positive genome (Lanes 1-4 are No. 2, 9, 3 and 8 HBeAg transgenic mice, respectively); Right: 4.7 kb fragment amplified from 3 'homologous arm of HBeAg transgenic mice (Lanes 1 and 2 are No. 2 and 9 HBeAg transgenic mice, respectively. M: 1 kb DNAmarker).
2.3 HBeAg转基因小鼠的纯化

采用长片段PCR技术对携带HBeAg基因的F1代转基因小鼠(图 6),结果显示编号为1、2、6、10、11、14号的F1代小鼠阳性,且基因测序结果显示HBeAg基因序列正确。

图 6 F1代HBeAg转基因小鼠PCR鉴定电泳图 Fig.6 Agarose gel electrophoresis of the PCR products for F1 offspring homologous recombinant HBeAg transgenic mice. Left: 3.4 kb fragments amplified from the 5'-arm homologous recombinant positive genome (Lanes 1-6 are No. 1, 2, 6, 10, 11 and 14 HBeAg transgenic mice, respectively); Right: 4.7 kb fragment amplified from 3' homologous arm of HBeAg transgenic mice (Lanes 1-14 are No, 1-14 HBeAg transgenic mice, respectively. M: 1 kb DNAmarker).

采用短片段PCR对F1代转基因小鼠交配所获得子代(杂合HBeAg转基因小鼠的基因型进行鉴定,结果显示其中P1、P2、P3、P4为所使用的引物。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(图 7)。

图 7 PCR鉴定HBeAg转基因小鼠基因型 Fig.7 PCR for identification of the genotype of HBeAg transgenic mice. Left: 994 bp fragments amplified from heterozygous (He) HBeAg transgenic mice and wild-type (WT) mice by PCR usingP1/P2 primer; Right: 1280 bp fragments amplified from heterozygous HBeAg transgenic mice by PCR usingP3/P4 primer. M: 1 kb DNAmarker.
2.4 HBeAg、HBeAb在HBeAg转基因小鼠中的表达

化学发光免疫分析和胶体金法检测,雅培i2000SR全自动化学发光定量分析结果显示杂合子和纯合子HBeAg转基因小鼠静脉血浆中HBeAg效价均大于1300 PEIU/mL;胶体金法检测结果显示HBeAg阳性,HBsAg、HBsAb、和HBeAb、HBcAb均呈阴性(图 8)。免疫组化方法检测结果证实杂合子和纯合子HBeAg转基因小鼠肝脏肝细胞可高表达HBeAg蛋白(图 9)。

图 8 胶体金法检测HBeAg转基因小鼠中HBeAg和HBeAb的表达 Fig.8 Expression of HBeAg and HBeAb in HBeAg transgenic mice detected by colloidal gold immunoassay method. A: HBeAg was positively expressed in peripheral blood of HE-F1, HE-F2, HO-F2, HO-F3, HO-F4 HBeAg transgenic mice, while HBeAg was negative in C56BL/6J mice; B: HBsAg, HBsAb, HBeAg, HBsAb and HBcAb were all negative in the plasma of C57BL/6J mice; C, D: HBeAg was positive in the plasma of heterozygous (C) and homozygous (D) HBeAg transgenic mice, while HBsAg, HBsAb, HBeAb and HBcAb were all negative.
图 9 免疫组织化学法检测HBeAg转基因小鼠中HBeAg的表达 Fig.9 Expression of HBeAg in HBeAg transgenic mice detected by immunohistochemistry. A: HE staining of the liver tissue of HBeAg transgenic mice (Original magnification: ×10); B: Negative HBeAg expression in the liver of wild- type C57BL/6J mice (× 20); C: High expression of HBeAg in the liver cells of homozygous HBeAg transgenic mice (× 20); D: High expression of HBeAg in the liver cells of heterozygous HBeAg transgenic mice (×20).
3 讨论

由于HBV的严格种属限制性和嗜肝细胞特性以及HBeAg在HBV感染和慢性乙肝进展中的重要作用,构建能在肝脏特异性稳定高表达HBeAg的转基因动物具有重要的临床价值,而近年来,CRISPR/Cas9技术凭借其高效的基因编辑能力已在细胞治疗中发挥出了重要作用,也为转基因动物的制备提供了新技术[23-24]。本研究采用CRISPR/Cas9技术将HBeAg基因定点插入带有白蛋白启动子的pliver-HBeAg表达框,以期实现HBeAg在肝细胞中特异性地高表达。长片段PCR鉴定结果证实共获得2只同源重组的F0代小鼠;再将F0代小鼠交配繁殖,采用短片段PCR对F1代转基因小鼠的基因型进行鉴定共获得6只阳性的F1代小鼠,利用F1代杂合子小鼠进行回交共获得22只F2代纯合子和29只杂合子HBeAg转基因小鼠。可见,与传统技术相比较采用CRISPR/Cas9技术构建转基因动物具有很好的优势。

同时,本研究还采用显微注射技术在C57BL/6J小鼠的受精卵环节进行基因编辑操作,从而降低HBeAg的免疫原性。而且采用免疫化学发光法和胶体金检测试剂盒检测杂合子和纯合子HBeAg转基因小鼠外周血中HBV相关抗原和抗体(包括HBsAg、HBsAb、HBeAg、HBeAb和HBcAb的表达,结果证实所有F1~F4代杂合子和纯合子HBeAg转基因小鼠外周中均能检出高滴度HBeAg蛋白,而HBsAg、HBsAb、HBeAb和HBcAb均未检出。同时,免疫组化染色结果证实杂合子和纯合子HBeAg转基因小鼠能在肝脏肝细胞中特异性高表达HBeAg蛋白,而在肝脏其他细胞和其他器官、组织中呈阴性表达。同时,HBeAg并非弥漫性表达于所有肝细胞中,而是特异性表达于肝小叶内部分肝细胞中,这种HBeAg表达的组织细胞学分布与人自然感染HBV后HBeAg蛋白在肝细胞中的表达模式完全一致。截至目前,该HBeAg转基因小鼠已经繁育到F4代,其遗传学特征稳定,仍能在肝细胞稳定高表达HBeAg蛋白且对HBeAg免疫耐受,并能在最大程度上接近并模拟人感染HBV后初期HBeAg的在体免疫耐受状态和生物功能。显然,该HBeAg转基因小鼠显然优于既往建立的HBeAg转基因小鼠[25-26],且尚未见文献报道,属于一种新品系的HBeAg转基因小鼠。

如前所述,由于HBeAg是HBV分泌性非颗粒状病毒衣壳蛋白,是HBV特有的免疫耐受因子,是引起HBV免疫耐受、慢性乙肝迁延不愈、并出现不良结局的重要原因之一[1-3]。而HBeAg血清转换(HBeAg转阴)已被认为是乙肝患者良好转归的里程碑事件,是判断病情发展趋势和药物疗效的一个重要指标,是最终彻底清除HBV、根治乙肝的基础,也是慢性乙肝治疗的中期目标[4-5]。因此,该新品系HBeAg转基因小鼠的构建无疑对于深入研究HBeAg的作用机制、评估以HBeAg为治疗靶点相关药物/疫苗的疗效具有重要的临床价值。

综上所述,该模型小鼠的建立为深入研究HBeAg感染机制、评价慢性乙型肝炎治疗性药物/疫苗疗效提供了新的适宜的实验动物模型,尤其适用于从原位细胞学和组织器官水平深入研究HBeAg的生物功能、HBeAg对肝脏局部器官免疫微环境作用及免疫耐受的分子机制研究。

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