Establishment of mice model with human viral hepatitis B.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Gao
Li-Fen
LF
Institute of Immunology, Medical College, Shandong University, Jinan 250012, Shandong Province, China.
Sun
Wen-Sheng
WS
Ma
Chun-Hong
CH
Liu
Su-Xia
SX
Wang
Xiao-Yan
XY
Zhang
Li-Ning
LN
Cao
Ying-Lin
YL
Zhu
Fa-Liang
FL
Liu
Yu-Gang
YG
D000818:Animals; D045744:Cell Line, Tumor; D004279:DNA, Viral; D004195:Disease Models, Animal; D016679:Genome, Viral; D006509:Hepatitis B / Q000276:immunology; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000637:transplantation*; D014162:Transfection
D004279:DNA, Viral