生长激素释放因子肝脏定向表达复合物在家兔体内的表达

外源基因在体内的转染及表达存在转染效率低、表达时间短等缺点，利用受体介导基因转移技术，可以将基因定向转入到特定组织，并且能提高转染效率和表达时间。在本试验中，利用肝脏去唾液酸糖蛋白受体(ASGR)将生长激素释放因子(GRF)基因定向转入到家兔肝脏组织并得到了表达。首先通过DNA阻滞试验确定质粒DNA与多聚赖氨酸(poly—L—lysine，PLL)的结合比例及反应液的最佳NaCl浓度，然后将PLL与α—D-吡喃半乳糖苯基异硫氰酸盐(α—D—galactopyranosylphenyl isotlhiocyanate)(物质的量之比为8：1)在pH9．0的Na2CO3溶液中进行糖基化反应，得到糖基化的PLL(galPLL)后透析除去未反应的糖并测定反应物糖含量。将糖基化的PLL与克隆有GRF基因的pcDNA3-GRF质粒(质量比为3．16：1)在1．031mol／L的NaCl溶液中进行连接，最终得到DNA—galPLL复合物，电镜观察其结构。耳缘静脉注射质粒复合物到家兔体内(1．5mg／只)，用RT—PCR和ELISA检测不同组织的表达情况。结果注射后7d家兔的肝脏RT—PCR结果呈阳性，27d仍能检测到GRF蛋白，但同时在其他的组织也检测到表达。并且进行了增重试验，发现定向转移GRF基因到家兔的肝脏对家兔的生长有一定的促进作用。

Directed Delivery Growth Hormone Releasing Factor Gene to Rabbit′s Liver and Its Expression

Exogenous gene transfection and expression in vivo have some shortcomings,such as low transfection efficiency and short expression time.Receptor mediated gene transfer can directly deliver gene to specific tissues and enhance its expression.In our experiment,pcDNA3 1 plasmid with GRF gene was condensed with galactosylated PLL(poly L lysine) in 1 031 mol/L NaCL solution.This process resulted in complex of defined size(80 100 nm in diameter) and shape.The pcDNA3 1 GRF galPLL complex was injected into the ear margin vein of adult rabbit.We detected its expression on mRNA and functional protein levels by RT PCR and ELISA.27 days after injection,RT PCR result of liver was positive.And ELISA result of liver was positive 27 days after administration.But at the same time,we detected expression in other tissue.The investigation showed that the GRF expression in rabbit liver by this way can improve growth rate.