Gao Li-Fen, Sun Wen-Sheng, Ma Chun-Hong, Liu Su-Xia, Wang Xiao-Yan, Zhang Li-Ning, Cao Ying-Lin, Zhu Fa-Liang, Liu Yu-Gang
Institute of Immunology, Medical College, Shandong University, Jinan 250012, Shandong Province, China.
World J Gastroenterol. 2004 Mar 15;10(6):841-6. doi: 10.3748/wjg.v10.i6.841.
To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA.
HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes.
By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage.
A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.
利用乙肝病毒转基因小鼠建立乙肝小鼠模型，并将由含乙肝病毒全基因组DNA的真核表达载体免疫同基因BALB/c小鼠诱导产生的乙肝病毒特异性细胞毒性T淋巴细胞（CTL）进行转移。
从经酶切的pBR322-2HBV中获取乙肝病毒DNA，并与载体pcDNA3连接。通过限制性内切酶分析鉴定重组pcDNA3-HBV，并用脂质体转染人肝癌细胞系HepG2。采用ELISA检测培养上清中乙肝表面抗原（HBsAg）的表达，用逆转录聚合酶链反应（RT-PCR）确定乙肝病毒前S1 mRNA的存在。通过肌肉注射用pcDNA3-HBV或pcDNA3免疫BALB/c小鼠。采用ELISA检测血清中乙肝表面抗体（HBsAb）的表达。采用MTT法检测脾淋巴细胞的非特异性或特异性增殖能力及特异性杀伤活性。将免疫小鼠的淋巴细胞转移至乙肝病毒转基因小鼠（每只小鼠2.5×10⁷个）。48小时后，用生化方法检测血清蛋白和转氨酶水平，取肝和肾组织切片，进行苏木精-伊红（HE）染色，观察病理变化。
经Eco RI、Xho I和Hind III酶切验证，重组pcDNA3-HBV含有单拷贝乙肝病毒基因组，且以正向插入。转染重组体的HepG2细胞能稳定表达前S1 mRNA和HBsAg。用pcDNA3-HBV免疫4周后，在BALB/c小鼠血清中检测到HBsAb。脾淋巴细胞的非特异性和特异性增殖潜力以及对靶细胞的特异性杀伤活性均增强。模型组转基因小鼠血清蛋白水平无明显变化，但谷丙转氨酶（ALT）和谷草转氨酶（AST）明显升高。肝脏有明显病理变化，而肾脏无明显损伤。
成功构建了含乙肝病毒全基因组的真核表达载体pcDNA3-HBV。转染重组体的HepG2细胞能稳定表达前S1 mRNA和HBsAg。用pcDNA3-HBV免疫小鼠可诱导特异性细胞免疫反应。建立了乙肝病毒急性肝炎小鼠模型。
