C基因型HBV转基因小鼠的制备

Establishment of C genotype hepatitis B virus genome transgenic mice

目的:建立C型HBV转基因小鼠,为乙肝防治研究提供实验动物模型.方法:采用受精卵显微注射法,制备HBV转基因小鼠,用PCR,ELISA,荧光定量PCR和免疫组化的方法研究HBV基因在转基因小鼠体内的整合、复制和表达情况.结果:注射受精卵2840枚,筛选注射后成活的受精卵共2256枚,注射成活率79.4%.注射后受精卵共移植假孕雌鼠85只,其中有42只怀孕,假孕雌鼠妊娠率49%,共产下F0代小鼠168只,PCR检测共有14只小鼠阳性,其中ELISA检测血清HBsAg阳性5只,HBeAg均阴性.荧光定量PCR血清HBV DNA显示,2只小鼠的拷贝数分别为3.12×105copies/L,1.98×105copies/L,经传代产下F1代小鼠61只,PCR检测HBV DNA阳性5只,ELISA检测HBsAg阳性4只,其中肝脏HBsAg免疫组化阳性2只,HBcAg均阴性,且肝组织表达HB-sAg为胞浆型.结论:构建的C型HBV基因不但可以在转基因小鼠体内进行复制表达,而且可以遗传给下一代小鼠.

AIM： To generate HBV （ C ） genome transgenic mice to provide suitable animal models for the related researches in China. METHODS： The HBV transgenic mice were generated by microinjection of 1.3 copies HBV genome into the pronucleus of FVB/N zygotes. The PCR assay, ELISA, RT-PCR and immuno- histochemical methods were used to detect the HBV integration, replication and expression in the transgenic mice. RESULTS： In this study, 2840 zygotes were injected, the survived 2256 zygotes after microinjection were reimplanted into the oviducts of 85 psu- dopregnant KM female mice. After birth, 42 female mice were pregnant and 168 F0 offsprings were born. PCR analysis indicated that 14 out of 168 F0 were integrated with HBV genome. With ELISA,5 of them expressed HBsAg in serum. And two mice were positive for HBV DNA replication in serum with 3. 12 × 10^5copies/L and 1. 98 × 10^5 copies/L respectively. Then the transgenic mice were mated to normal mice to establish transgenic mouse strain. 61 F1 offsprings were born. Further study demonstrated that 5 of them were integrated with HBV genome by PCR, 4 of them were serum HBsAg positive by ELISA and 2 of them with the intracellular distribution of HBsAg in hepatocytes by immunohistochemistry. Both serum HBeAg and liver HBcAg immunohistochemistry were negative. CONCLUSION： The established HBV （C） genome could be not only replicated and expressed in transgenic mice but also inherited to the next generations.