| Hepatitis B, caused by hepatitis B virus (HBV), is an infectious disease with wide prevalence, vast transmission and great harm. HBV belongs to hepadnaviridae with high specificity. It infects specifically human and chimpanzee, having no threat to mammalian laboratory animals. The lack of ideal animal models for experiment and methods of in vitro culture limited the research on HBV replication, its pathogenic mechanism, induction of tumor and treatment .The construction of transgenic animal has brought hope to the research of HBV. The technology of transgenic animal is an important method to study and research in life territory. The technology is used widely in the territory of medicine, agriculture and biology. Since Gordon made use of the microinjection in the zygote of the mice for the first time in 1980, people have never stopped researching and exploring the technology of transgenic animal. The HBV transgenic mice is a more ideal animal model for the research of HBV. Many foreign scholars constructed many HBV transgenic mice models one after another,according to genotype and serosubtype of their countries. Many foreign scholars had gained great achievement in constructing transgenic mice of HBV. However, in order to prevent and cure HBV patients in our country effectively, it is important for us to construct a HBV transgenic mice, according to genotype and serosubtype of our own country. Because adr is the most prevailing subtype in China, so many scholars of our country are dedicated to constructing transgenic mice of HBV (adr). At present, pronucleus microinjection and the transfection of stem spermatogonium are commonly used in the transgenic mice. But recently, some reports considered that the pronucleus microinjection and the transfetion of stem spermatogonium are the main reason which lead to the malformation and death of mice and embryo even though it could obtain high copy integration.Retrovirus-mediated gene transfer technique is one of the highly efficient gene transfer methods established in 1980. By recombination with exogenous gene and retrovirus vector and package into cell line, a defected recombinant retrovirus was produced and target cell could be transfected successfully, exogenous gene could be integrated into the genome of target cell and expressed in it.This experiment was to obtain the transgenic mouse with full-length HBV (adr) genome of single copy in order to decrease the malformation and death of mice and embroyo and offer the ideal animal model for HBV (adr) research in our country, by injecting zona pellucida of mouse zygote with recombinant HBV (adr) retrovirus.Full-length HBV (adr) genome is cloned into the skeleton of leukemia virus (gene deletion group) of Moloney mice and the retroviral vector pLNCL carrying the NeoR, to establish recombinant retroviral vector pLNCB with positive strand containing full-length HBV genome and negative strand pLNCBR. Using liposome as the transfection agent, we transfected packaging cell PA317, packaged pLNCB and pLNCBR plasmids.After G418 selection, the positive strands of three strains (P1 ,P2 ,P3) and negative strands of four strains (P4, P5 ,P6 ,P7) which persistently produced virus particles were obtained and amplified. Then after concentration, the virus infection titre of every cell strain producing toxin is determined through in vitro infection of NIH3T3 cell. In comparison, P2 showed the highest infection titre of 1×106 CFU /ml in positive strands ,while in negative strands, P4 got the highest infection titre of 1×105 CFU /ml.On the basis of the above experiment, the recombinant retroviruses of both the positive and negative strands are injected into the zona pellucida of mice zygote after several times of optimization of injection condition and parameters. Then we transplanted the injected zygotes into 65 pseudo-pregnant mother mice bodies (34 of positive strands, 31 of negative strands). When the fetal mice delivered by pseudo-pregnant mother mice grew up to about 40 days , PCR test was performed on their gene group with HBV specific primers. At the same time, the mice that grew from the transplanted normal mice zygote and normal mice are to be compared. The result showed that among the survived 46 mice (34 of positive strands, 12 of negative strands), positive mice were four (3 of positive strands, 1 of negative strands) which were all mother mice ,and which meant HBV gene was integrated into fetal mice gene group. The positive ratio of F0 generation was 8.7%. PCR test-positive mother mice is multiplied with normal father mice, producing 15 of F1 generation mice.Using the same primer described above we performed PCR and Southern blotting test on the filial generation mice gene group which were extracted from mice tails of late generation. The result showed that the number of positive mice of F1 generation was 5,with 3 positive strands (1 of father mice and 2 of mother mice), 2 negative strands (mother mice). The rest of F1 generation mice were all negative. The results of the mice that grow from the transplanted normal mice zygote and normal mice are negative.We did ELISA , pathology and immunohistochemistry on 5 transgenic mice of F1 generation mice ,the mice that grew from the transplanted normal mice zygote and normal mice. Results: ELISA test- the transgenic mice of F1generation mice were HBsAg positive; Pathology test- transgenic mice of F1generation mice all showed spotty necrosis or focal necrosis of liver ; Immunohistochemistry test–liver and kidney cells of transgenic mice of F1generation mice showed HBsAg and HBcAg positive expression in liver (All the results of the mice that grew from the transplanted normal mice zygote and normal mice are negative). The results proved that the HBV (adr) transgenic mice were constructed successfully, and the HBV (adr) not only expressed but also replicated in the HBV (adr) transgenic mice.The creative points of this research:(1) Full-length HBV genome (adr) is cloned into the skeleton of leukemia virus (gene deletion group) of Moloney mice and the retroviral vector pLNCL carrying the NeoR, to establish recombinant retroviral vector pLNCB with positive strand containing full-length HBV genome and negative strand pLNCBR. (2) Using liposome as the transfection agent, we transfected packaging cell PA317,packaged pLNCB and pLNCBR plasmids, after G418 selection, the positive strands of three strains (P1 ,P2 ,P3) and negative strands of four strains (P4, P5 ,P6 ,P7) which persistently produced virus particles were obtained. (3) The recombinant retroviruses of both the positive and negative strands are injected into the zona pellucida of mice. Then we transplanted the injected zygotes into pseudo-pregnant mother mice bodies to construct transgenic mice (single copy). |
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