CN109652452A - 长期携带乙型肝炎病毒的实验动物模型构建方法 - Google Patents
长期携带乙型肝炎病毒的实验动物模型构建方法 Download PDFInfo
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Abstract
本发明公开了一种长期携带乙型肝炎病毒的实验动物模型构建方法,包括如下步骤:1)提取乙型肝炎患者血清病毒DNA,使用引物对HBVF/HBVR进行PCR扩增;2)将PCR产物克隆到pEASY‑HBV载体中,挑选阳性克隆;3)提取阳性克隆的质粒,将提取到的质粒用BspQI内切酶酶切,回收酶切片段并纯化,用T4连接酶将其环化并纯化,得到HBV cccDNA;4)将cccDNA注射入小鼠中,构建乙型肝炎e抗原阴性小鼠模型。本发明通过一种简单、快速和方便的方法,成功的构建了一个能有效模拟HBV慢性感染的具有免疫活性的CBA/CaJ小鼠模型,HBsAg、HBeAg和HBcAg的表达均可长达26周。
Description
技术领域
本发明属于基因工程领域,具体涉及一种长期携带乙型肝炎病毒的实验动物模型构建方法。
背景技术
乙型病毒性肝炎由乙型肝炎病毒(hepatitis B virus,HBV)感染引起,该疾病是一个严重的全球公共卫生问题,可造成的临床症状多种多样,包括慢性乙型肝炎(CHB)、肝硬化(LC)和肝细胞癌(HCC)等。其中,慢性乙型肝炎,其乙肝病毒检测为阳性,病程超过半年或发病日期不明确而临床有慢性肝炎表现。据最新报道,世界上约有2.48亿人患有慢性乙肝。HBV在患者体内不易消除,导致病人对病毒有较高的发展成HCC的风险。基于HBV动物模型在乙型肝炎的发病机理和药物筛选研发中扮演中非常重要的角色。传统的HBV动物模型有黑猩猩、土拨鼠、树鼩、鸭等,在HBV感染研究中发挥着重要作用,传统模型有黑猩猩、土拨鼠、树鼩、鸭、树鼩等,他们在乙型肝炎病毒感染研究中发挥着重要作用。但是,黑猩猩模型成本较高,并且受伦理限制;土拨鼠和树鼩则表现为一过性的急性肝炎;虽然鸭肝炎产生的病理过程和愈后与人的乙型肝炎相似,现在广泛用于人的乙型肝炎研究的动物模型,但是鸭是鸭型肝炎病毒,与人的乙型肝炎病毒相差甚远,因而限制了其应用价值。所以,现有模型对于乙型肝炎的治疗研究都存在一定的局限性,特别是导致其无法用于CHB的治疗研究。因此,目前需要建立一个能够携带HBV时间长达26周(半年)的实验动物模型。
发明内容
本发明的目的针对上述问题提供一种长期携带乙型肝炎病毒的实验动物模型构建方法。
本发明实现其目的采用的技术方案是:
一种长期携带乙型肝炎病毒的实验动物模型构建方法,包括如下步骤:
1)提取乙型肝炎患者血清病毒DNA,使用引物对HBVF/HBVR进行PCR扩增,引物序列为:
HBVF:
5’-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3’,
HBVR:
5’-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGT-3’;
2)将获得的PCR产物克隆到pEASY-HBV载体中,挑选阳性克隆;
3)提取步骤2)中阳性克隆的质粒,将提取到的质粒用BspQI内切酶酶切,回收酶切片段并纯化,用T4连接酶将其环化并纯化,得到HBV cccDNA;
4)将步骤3)得到的HBV cccDNA注射入小鼠中,即构建了乙型肝炎e抗原阴性小鼠模型。
步骤4)中采用水动力法将HBV cccDNA注射入小鼠中。
每只小鼠的HBV cccDNA注射量为2μg/2mL/只。
步骤1)中的PCR扩增体系是:5×GC Buffer 10μL,2.5mM dNTPs 4μL,5μM前引物HBVF 1μL,5μM后引物HBVF 1μL,Pusion酶0.5μL,血清DNA模版2μL,水补足50μL;PCR扩增条件是:(1)98℃,30s;(2)98℃,10s,65℃,20s,72℃,1min 45s;此步骤循环25次;(3)72℃,10min。
本发明的有益效果是:通过一种简单、快速和方便的方法,成功的构建了一个能有效模拟HBV慢性感染的具有免疫活性的CBA/CaJ小鼠模型。这种小鼠模型的HBV标记物,包括HBsAg、HBeAg和HBcAg的表达均可长达26周(即半年)。该模型为研究慢性乙肝的持久性感染,临床治疗评价及筛选新的抗病毒药物提供了一个重要工具。该模型的建立方法为使用患者血清DNA建立个体HBV感染模型奠定了基础,从而为个体化治疗提供工具。
附图说明
图1为小鼠血清标志物HBsAg和HBeAg检测结果图,其中,(A)为小鼠血清HBsAg含量;(B)为小鼠血清HBeAg含量。
图2为IHC检测HBsAg和HBcAg在小鼠肝脏的表达结果图,其中,A为HBsAg在肝脏的表达,a、b、c依次是4w、12w和26w时对照组的结果,d、e、f依次是4w、12w和26w实验组的结果;B为HBcAg在肝脏的表达,a、b、c依次是4w、12w和26w对照组的结果,d、e、f依次是4w、12w和26w实验组的结果。
图3为小鼠肝脏组织病理分析结果图,其中,a、b、c依次是4w、12w和26w时对照组的结果,d、e、f依次是4w、12w和26w实验组的结果。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
主要试剂及生产者:
pEASY-BluntZero载体(全式金公司,中国)
High-Fidelity DNAPolymerases&MasterMixes(Thermo Scientific,美国)
质粒提取试剂盒(QIAGEN公司,德国)
BspQI内切酶(NEB公司,美国)
胶回收试剂盒(Takara公司,日本)
T4连接酶(Takara公司,日本)
放射免疫鉴定试剂盒(北京北方生物技术研究所,中国)
Pusion酶(NEB公司,美国)
实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
实施例1
一、HBV病毒基因组克隆
乙型肝炎e抗原阳性患者血清DNA样本由重庆医科大学附属第二医院提供,患者编号为11061012。用Phusion聚合酶(High-Fidelity DNAPolymerases&MasterMixes),血清DNA模板,前引物HBVF:5’-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3’,后引物HBVR:5’-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGT-3’,通过PCR扩增获得长度为3.2kb的HBV全长序列。PCR扩增体系是:5×GC Buffer 10μL,2.5mM dNTPs 4μL,5μM前引物HBVF 1μL,5μM后引物HBVF 1μL,Pusion酶0.5μL,血清DNA模版2μL,水补足50μL。
PCR扩增条件是:(1)98℃,30s;(2)98℃,10s,65℃,20s,72℃,1min45s;此步骤循环25次;(3)72℃,10min;(4)4℃保存。
随后用PCR扩增产物将HBV克隆至pEASY-Blunt Zero载体获得pEASY-HBV阳性质粒,具体步骤为:在1.5mL离心管中加入3μL PCR扩增产物和1μL pEASY-Blunt Zero室温连接30min。加入100μL DH5α于连接产物中,置于冰上冰浴20min,42℃水浴热激30s,转至冰上2min,加入800μL SOC培养基于热激产物中,置于摇床200rpm/min、37℃恢复生长30min,10000rpm离心1min弃大部分上清,吹打重悬沉淀,将重悬的细胞涂布于预先涂有IPTG和X-Gal的LBA平板上;置于37℃培养箱倒置培养过夜。挑取10个白斑单克隆进行LBA液体培养,12h后提取质粒,进行BspQI酶切验证,电泳结果显示已成功得到了3个阳性克隆(即pEASY-HBV质粒),经Sanger测序分析检验,我们成功获得了HBV DNA全长序列。
二、HBV cccDNA体外环化
将前述阳性克隆的菌株进行扩大培养,用质粒提取试剂盒提取前述得到的阳性pEASY-HBV质粒,保存。将提取到的pEASY-HBV质粒替换血清DNA,作为HBV DNA扩增的模版,使用的引物和PCR扩增体系以及PCR扩增条件和前述的一样,大量扩增获得HBV DNA全长PCR产物。纯化后的PCR产物用BspQI酶切,纯化后得到带有粘性末端的HBV DNA,随后通过T4连接酶的作用,使HBV DNA发生环化,纯化后所得产物即为HBV cccDNA。
三、实验动物模型构建
取54只10周龄SPF级雄性CBA/CaJ小鼠,随机分为实验组和空白组。27只为实验组:采用水动力注射体外环化所制备的HBV cccDNA,2μg/2mL/只(即每只小鼠注射2μg cccDNA,cccDNA的浓度为1μg/mL);27只为空白对照组:注射生理盐水,2mL/只。两组分别于注射后第1w、2w、4w、8w、12W、16W、20W、24w和26w尾静脉采血,同时,取肝脏组织。
实施例2
一、小鼠血清标志物HBsAg和HBeAg检测
在实施例1设定时间点采取的血样,采用放射免疫法(使用放射免疫鉴定试剂盒)检测小鼠血清中HBV血清标志物HBsAg和HBeAg的含量。
由图1可知,实验组在注射HBV cccDNA后第1周开始就可以检测到HBsAg和HBeAg。图1A,HBsAg的含量在1-12周呈下降趋势,第12周后,HBsAg含量趋于平稳,一直持续到26周。图1B,HBeAg的含量在第2周有下降趋势,第2周至第16周略微上升,随后又呈现下降趋势,一直持续至第26周。HBsAg和HBeAg含量均明显高于空白对照组。证明HBV的血清标志物能够持续在小鼠体内持续26周表达。
二、HBsAg和HBcAg在小鼠肝脏中的表达
将水动力注射HBV cccDNA后收集的第4周、第12周和第26周的小鼠肝脏组织用石蜡包埋,切片,采用免疫组织化学法(immunohistochemistry,IHC)检测HBV表面抗原HBsAg和核心抗原HBcAg的在肝脏组织的定位与表达,一抗分别为HBsAg(马抗,1:1000),HBcAg(兔抗,1:1000),二抗分别为马anti-HBsAg(1:1000)和兔anti-HBcAg(1:1000),随后进行染色,封片和采图。
IHC检测小鼠肝脏组织中HBsAg和HBcAg的表达,如图2所示,HBsAg和HBcAg在注射HBVcccDNA后的第4周、第12周和第26周的小鼠肝组织中分布表达,证明HBV可在小鼠体内持续携带长达26周。
三、小鼠肝脏组织病理变化
将水动力注射HBV cccDNA后收集的第4周、第12周和第26周的小鼠肝脏组织用石蜡包埋,切片,采用苏木素伊红(hematoxylin and eosin,HE)染色法,将切片脱水、染色、复水、封片、镜检、采图,对肝脏进行病理学分析。
经HE染色分析发现,结果如图3所示,这些长期表达HBsAg、HBeAg和HBcAg的小鼠,无论是第4周、第12周还是第26周,都并没有造成严重的肝脏组织损伤,表明该模型引起的肝脏损伤是较轻微的。
Claims (4)
1.一种长期携带乙型肝炎病毒的实验动物模型构建方法,其特征在于:包括如下步骤:
1)提取乙型肝炎患者血清病毒DNA,使用引物对HBVF/HBVR进行PCR扩增,引物序列为:
HBVF:
5’-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3’,
HBVR:
5’-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGT-3’;
2)将获得的PCR产物克隆到pEASY-HBV载体中,挑选阳性克隆;
3)提取步骤2)中阳性克隆的质粒,将提取到的质粒用BspQI内切酶酶切,回收酶切片段并纯化,用T4连接酶将其环化并纯化,得到HBV cccDNA;
4)将步骤3)得到的HBV cccDNA注射入小鼠中,即构建了乙型肝炎e抗原阴性小鼠模型。
2.如权利要求1所述的长期携带乙型肝炎病毒的实验动物模型构建方法,其特征在于:步骤4)中采用水动力法将HBV cccDNA注射入小鼠中。
3.如权利要求2所述的长期携带乙型肝炎病毒的实验动物模型构建方法,其特征在于:每只小鼠的HBV cccDNA注射量为2μg/2mL/只。
4.如权利要求1所述的长期携带乙型肝炎病毒的实验动物模型构建方法,其特征在于:步骤1)中的PCR扩增体系是:5×GC Buffer 10μL,2.5mM dNTPs 4μL,5μM前引物HBVF 1μL,5μM后引物HBVF 1μL,Pusion酶0.5μL,血清DNA模版2μL,水补足50μL;PCR扩增条件是:(1)98℃,30s;(2)98℃,10s,65℃,20s,72℃,1min 45s;此步骤循环25次;(3)72℃,10min。
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