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目的制备能用于人乙型肝炎研究的HBV转基因小鼠模型。方法将首尾相连的adr型HBV全基因组克隆至pBR322质粒,经扩增纯化后,用显微注射法导入C57BL/6小鼠受精卵原核,让外源HBV自然整合至小鼠受精卵基因组。用PCR、DNA测序、放免及免疫组化等方法分析HBV在小鼠体内的整合、复制、表达情况。结果显微注射1712枚卵,产仔128只,其中有14只鼠尾HBV PCR检测阳性,12只在血清中检测到HBV DNA及HBsAg,对其中10只HBV DNA阳性鼠用基因分析仪测序,9只与所转基因序列完全相同,1只于1739、1740位缺失2个碱基。待这批首建鼠性成熟后,交配产仔,分别在其子代血清中检测到HBV DNA及HBsAg;肝组织切片免疫组化分析,发现散在点状HBsAg表达。结论本实验结果表明,HBV DNA已整合至小鼠基因组,并能复制、表达。证明我们已获得HBV转基因小鼠模型。
Objective To prepare a HBV transgenic mouse model that can be used in human hepatitis B research. Methods The full-length adr HBV genome was cloned into pBR322 plasmid. After amplification and purification, the prokaryotic nuclei of C57BL / 6 mice were induced by microinjection and the exogenous HBV was naturally integrated into the genome of mouse fertilized eggs. The integration, replication and expression of HBV in mice were analyzed by PCR, DNA sequencing, radioimmunoassay and immunohistochemistry. Results 1712 eggs were microinjected, 128 were born, of which 14 mice were positive for HBV PCR detection, 12 were detected in serum HBV DNA and HBsAg, of which 10 HBV DNA positive mice sequenced by gene analyzer, Nine identical with the transgene sequence, one at 1739,1740 missing two bases. After the first mice were mature, the mating litters were used to detect HBV DNA and HBsAg in their offspring serum respectively. Immunohistochemical analysis of liver tissue sections showed that scattered HBsAg was found. Conclusion The results of this experiment show that HBV DNA has been integrated into the mouse genome and can be replicated and expressed. Prove that we have obtained the HBV transgenic mouse model.