摘要
以绵羊金属硫蛋白基因（ｏＭＴ）为启动子，构建了２种结构的ＨＳＡ质粒：ｐｏＭＴＨＳＡ和ｐｏＭＴＨＳＡＵＤ。其中ｐｏＭＴＨＳＡ是将ＨＳＡｃＤＮＡ直接置于ｏＭＴ启动子的控制下，以ｐＳＰＯＲＴ１为载体构建而成；而ｐｏＭＴＨＳＡＵＤ另融有ＨＳＡ基因５′，３′侧翼序列（包括内含子１，２，１３，１４）。将这２种结构的质粒用ＮｏｔⅠ线化后，注入昆明种小鼠受精卵的雄原核，再移植到假孕受体鼠的输卵管。共注射５７７枚受精卵，有４０６枚形态完好的注射卵被移植到１９只假孕受体，其中８只妊娠，产仔３５只。经ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交检测，获得了４只转基因小鼠，其中１只来自于注射ｐｏＭＴＨＳＡ，３只来自于注射ｐｏＭＴＨＳＡＵＤ。转基因总阳性率和总效率分别为１１．４％和０．７％。用ＲＴ－ＰＣＲ技术在转录水平检测４只转基因鼠外源基因的表达情况，结果２只鼠（均来自于注射ｐｏＭＴＨＳＡＵＤ）在肝脏中表达有ＨＳＡｍＲＮＡ，免疫琼脂试验发现，这２只小鼠的血液中均有ＨＳＡ的表达产物，与ＲＴ－ＰＣＲ检测结果一致。ｐｏＭＴＨＳＡＵＤ在小鼠体内得到了整合和表达；而ｐｏＭＴＨＳＡ只有整合而未得到表达，故认为含有侧翼序列及部分内含子的基因ｐｏＭＴＨＳＡＵＤ优?
Using ovine metallothionein (oMT) as promoter, two structural HSA fused genes (poMTHSA and poMTHSAUD) were constructed. poMTHSA consists of oMT promoter and HSA cDNA. poMTHSAUD fused 5′,3′ flanking sequences (included intron 1, 2, 13, 14) additionally compared with poMTHSA. The two recombinant genes in linear form (Not Ⅰ cut), were microinjected into the male pronuclei of fertillized egg through Leitz microinjector. During this study, 577 fertillized eggs from donor mice were injected, and 406 normal eggs injected were transferred to the oviduct of 19 pseudopregnant recipient mice, 8 of 19 recipient mice became gestation and gave birth to 35 F 1 offspring mice. The zygote′s survival rate and birth rate were 70.4% and 8.6%, respectively. 4 of 35 mice integerated the foreign HSA gene detected by PCR and Southern hybridization, one from poMTHSA, the others from poMTHSAUD. Total integration rates and efficiency of transgene were 11.4% and 0.7%, respectively. Transgene expression was analyzed to the transcriptional level by RT PCR. The results showed that 2 transgenic mice expressed HSA mRNA in their livers. To detect the expression products of HSA, serum samples from 4 transgenic mice were analyzed by immunoassay of agarose expansion experiment. It was proved that 2 transgenic mice expressed HSA. There was a complete correlation between immunoassay and RT PCR. The results demonstrated that the foreign poMTHSAUD gene was expressed in the body of transgenic mice, but poMTHSA gene was integrated only. It is suggested that the poMTHSAUD fused 5′, 3′ flanking sequences and some introns seems to be more suitable to transgene than the poMTHSA.
出处
《中国兽医学报》
CAS
CSCD
1996年第6期570-575,共6页
Chinese Journal of Veterinary Science
基金
国家"863"资助项目
湖北省自然科学基金
关键词
人血清白蛋白
转基因构建
转基因小鼠
HSA
transgene construct
transgenic mice * Inst of Anim Husbandry and Vet Sci, Hubei Academy of Agri Sci ** The First Hospital, Hubei Medical University
