不同转染介质对狂犬病病毒糖蛋白基因免疫的效应

Effects of Different Transfection Reagents on Genetic Immunization of Rabies Virus Glycoprotein cDNA

将狂犬病病毒糖蛋白 ( RGP) c DNA Bgl 片段 ( 1 .6 7kb)分别克隆进质粒 p GFP-Cl、p SV2 -dhfr和pc DNA3 ,构建了重组质粒 p GFP-C1 -RGP、p SV2 -RGP和 pc DNA3 -RGP,通过脂质体和聚乙烯亚胺 ( PEI)包裹后分别转染 BHK-2 1细胞和乳鼠脑内接种 ,3种重组质粒均能表达出 RGP。表达水平高低依次为 p GFP-C1 -RGP>pc DNA3 -RGP>p SV2 -RGP。 3种重组质粒分别以全裸质粒、质粒 -脂质体、质粒 -PEI形式 ,经骨骼肌免疫小鼠 ,间接 ELISA检测。结果显示 ,免疫鼠血清中特异性抗体水平明显高于对照组 ;80 %免疫小鼠能抵抗狂犬病病毒强毒的攻击。全裸质粒免疫诱生的抗体水平同其剂量相关 ;而以 PEI或脂质体作为转染介质、质粒接种量超过 2 0μg时 ,诱生抗体水平不再随质粒剂量增大而显著变化。质粒免疫后 1 50 d采用 PCR仍然能从注射部位检测到

Three rabies virus glycoprotein expressing vectors, pGFP Cl RGP, pSV 2 RGP and pcDNA 3 RGP were constructed by cloning rabies virus glycoprotein cDNA into pGFP Cl,pSV 2 dhfr and pcDNA 3,respectively.Expression of all three vectors was confirmed on cells and in newborn mouse brains. The highest expression level was achieved when the rabies virus glycoprotein gene was regulated by CMV promoter/enhancer. After 3 times of inoculations at intervals of 2 weeks in the form of naked DNA, DNA lipofectamine and DNA PEI complex, specific antibodies against rabies virus were detected in sera of mice by means of ELISA. The antibody titer went up with the increase of the amount of plasmids injected. However, when the amount of the plasmid went beyond 20 μg/mouse, there was no positive correlation between the dose of DNA injected and the level of immune response when PEI and lipofectamine were used as transfection reagents. The plasmid vaccination could protect mice from the challenge of CVS. Long lasting humoral immune responses were proved with ELISA and PCR amplification 6 months after the primary inoculation.